B , HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22 . Cells were harvested , fractionated , and fractions were subjected to immunoblotting with the indicated antibodies .

O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O

B, HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were harvested, fractionated, and fractions were subjected to immunoblotting with the indicated antibodies.


C , In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24 . HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22 . Lysates were generated using buffer containing protease cocktail inhibitor , pan - DUB inhibitor PR619 , and the JAMM protease inhibitor o - phenanthroline . Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested ( lanes 1 and 2 ) or digested with USP2 to strip polyubiquitin ( lanes 3 and 4 ) and reduce target proteins to unit length . Digestion reactions were subjected to immunoblotting with the indicated antibodies .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O

C, In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24. HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22. Lysates were generated using buffer containing protease cocktail inhibitor, pan-DUB inhibitor PR619, and the JAMM protease inhibitor o-phenanthroline. Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested (lanes 1 and 2) or digested with USP2 to strip polyubiquitin (lanes 3 and 4) and reduce target proteins to unit length. Digestion reactions were subjected to immunoblotting with the indicated antibodies.


A , Genome browser image of ChIP - seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40 . H3K4me1 and H3K27ac tracks are from the ENCODE Consortium ( GEO accessions GSM945858 and GSM945853 ) . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 and GSE49952 ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O

A, Genome browser image of ChIP-seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40. H3K4me1 and H3K27ac tracks are from the ENCODE Consortium (GEO accessions GSM945858 and GSM945853). ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).


B - F Live confocal images of ddaC clones expressing mCD8 : : GFP driven by ppk - Gal4 at WP stage or 16 h APF . l ( 3 ) 810 and l ( 3 ) 924 mutant ddaC clones ( C , E ) displayed dendrite arborization defects at WP stage and pruning defects at 16 h APF . ( D , F ) Introduction of one genomic construct of msps , HN267 / g - msps , rescued dendrite pruning defects in msps810 and msps924 clones . Red arrowheads point to the ddaC somas .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE B-SUBCELLULAR O

B-F Live confocal images of ddaC clones expressing mCD8::GFP driven by ppk-Gal4 at WP stage or 16 h APF. l(3)810 and l(3)924 mutant ddaC clones (C, E) displayed dendrite arborization defects at WP stage and pruning defects at 16 h APF. (D, F) Introduction of one genomic construct of msps, HN267/g-msps, rescued dendrite pruning defects in msps810 and msps924 clones. Red arrowheads point to the ddaC somas.


G Percentages of ddaC clones showing severing defects at 16 h APF .

G Percentages of ddaC clones showing severing defects at 16 h APF.


H Quantitative analysis of unpruned dendrite lengths at 16 h APF .

O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O

H Quantitative analysis of unpruned dendrite lengths at 16 h APF.


A Pan - neuronal driver elav - Gal4 was used to co - express GFP - Msps and TACC in postmitotic neurons . elav - Gal4 driven mCD8 : : GFP expression was used as a control . TACC was pulled down by GFP - Msps but not by mCD8 : : GFP . Alpha - tubulin was used as a loading and probing control . Neither mCD8 : : GFP nor GFP - Msps could pull down alpha - tubulin .

O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O

A Pan-neuronal driver elav-Gal4 was used to co-express GFP-Msps and TACC in postmitotic neurons. elav-Gal4 driven mCD8::GFP expression was used as a control. TACC was pulled down by GFP-Msps but not by mCD8::GFP. Alpha-tubulin was used as a loading and probing control. Neither mCD8::GFP nor GFP-Msps could pull down alpha-tubulin.


B Endogenous Msps proteins were co - immunoprecipitated with Venus - TACC by GFP beads but not by control IgG beads . Alpha - tubulin was used as loading and probing control .

O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

B Endogenous Msps proteins were co-immunoprecipitated with Venus-TACC by GFP beads but not by control IgG beads. Alpha-tubulin was used as loading and probing control.


C Endogenous Msps or TACC was pulled down by their respective antibodies , and the other protein was simultaneously detected in the immunoprecipitated contents . Alpha - tubulin was used as loading and probing control .

O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

C Endogenous Msps or TACC was pulled down by their respective antibodies, and the other protein was simultaneously detected in the immunoprecipitated contents. Alpha-tubulin was used as loading and probing control.


D - G Confocal images of ddaC neurons expressing control RNAi ( D ) , msps RNAi ( E ) , tacc RNAi ( F ) and αTub84B RNAi ( G ) that were immunostained for TACC at wL3 stage . ddaC somas are labeled by dashed lines . ddaC neurons are identified by ppk - Gal4 driven mCD8 : : GFP ( green channel ) expression , as shown at the top right corner .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O

D-G Confocal images of ddaC neurons expressing control RNAi (D), msps RNAi (E), tacc RNAi (F) and αTub84B RNAi (G) that were immunostained for TACC at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons are identified by ppk-Gal4 driven mCD8::GFP (green channel) expression, as shown at the top right corner.


H Quantitative analysis of normalized TACC fluorescence intensity in ddaC somas .

O O O O O B-GENEPROD O O O B-CELL_TYPE B-SUBCELLULAR O

H Quantitative analysis of normalized TACC fluorescence intensity in ddaC somas.


I - L Confocal images of ddaC neurons expressing control RNAi ( I ) , msps RNAi ( J ) , tacc RNAi ( K ) and αTub84B RNAi ( L ) that were immunostained for Msps at wL3 stage . ddaC somas are labeled by dashed lines . ddaC neurons are identified by ppk - Gal4 driven mCD8 : : GFP ( green channel ) expression , as shown at the top right corner .

I-L Confocal images of ddaC neurons expressing control RNAi (I), msps RNAi (J), tacc RNAi (K) and αTub84B RNAi (L) that were immunostained for Msps at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons are identified by ppk-Gal4 driven mCD8::GFP (green channel) expression, as shown at the top right corner.


M Quantitative analysis of normalized Msps fluorescence intensity in ddaC somas .

M Quantitative analysis of normalized Msps fluorescence intensity in ddaC somas.


N , O Protein expression of Msps and TACC in brain tissues . ( N ) Larval brains of msps810 / mspsP transheterozygotes at early wL3 stage were immunoblotted and probed with α - Msps and α - TACC antibody . Actin blotting was used as loading control . The dot plot on the right depicts the quantitative analysis of total TACC levels in control and msps transheterozygous larvae .

O O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O B-ORGANISM B-TISSUE O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM O

N, O Protein expression of Msps and TACC in brain tissues. (N) Larval brains of msps810/mspsP transheterozygotes at early wL3 stage were immunoblotted and probed with α-Msps and α-TACC antibody. Actin blotting was used as loading control. The dot plot on the right depicts the quantitative analysis of total TACC levels in control and msps transheterozygous larvae.


O Protein expression of Msps and TACC in brain tissues . ( O ) Adult brains of tacc59 / tacc74 transheterozygotes were immunoblotted and probed with α - Msps and α - TACC antibody . Actin blotting was used as loading and probing control . The dot plot on the right displays the quantitative analysis of total Msps level in control and tacc transheterozygotes .

O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O B-TISSUE O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O

O Protein expression of Msps and TACC in brain tissues. (O) Adult brains of tacc59/ tacc74 transheterozygotes were immunoblotted and probed with α-Msps and α-TACC antibody. Actin blotting was used as loading and probing control. The dot plot on the right displays the quantitative analysis of total Msps level in control and tacc transheterozygotes.


A - C Live confocal images of ddaC neurons visualized by ppk - Gal4 driven mCD8 : : GFP expression at WP or 16 h APF . While expression of control RNAi in ddaC neurons ( A ) did not affect the dendrite morphogenesis nor the pruning processes , knockdown of tacc using RNAi # 1 ( B ) or tacc59 / tacc74 transheterozygous mutant ( C ) severely perturbed the dendrite arborization as well as pruning . Red arrowheads point to the ddaC somas .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O

A-C Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. While expression of control RNAi in ddaC neurons (A) did not affect the dendrite morphogenesis nor the pruning processes, knockdown of tacc using RNAi #1 (B) or tacc59/tacc74 transheterozygous mutant (C) severely perturbed the dendrite arborization as well as pruning. Red arrowheads point to the ddaC somas.


D Percentages of ddaC neurons showing severing defects in ( A - C ) at 16 h APF .

O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O

D Percentages of ddaC neurons showing severing defects in (A-C) at 16 h APF.


E Quantitative analysis of unpruned dendrite lengths in ( A - C ) at 16 h APF .

O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O

E Quantitative analysis of unpruned dendrite lengths in (A-C) at 16 h APF.


F - H Live confocal images of ddaC neurons visualized by ppk - Gal4 driven mCD8 : : GFP expression at WP or 16 h APF . Simultaneous knockdown of tacc and msps via co - expressing tacc RNAi and msps RNAi ( H ) in the ddaC neurons did not enhance the pruning defects , compared to those neurons co - expressing control RNAi and msps RNAi ( F ) . Red arrowheads point to the ddaC somas .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O

F-H Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown of tacc and msps via co-expressing tacc RNAi and msps RNAi (H) in the ddaC neurons did not enhance the pruning defects, compared to those neurons co-expressing control RNAi and msps RNAi (F). Red arrowheads point to the ddaC somas.


I Percentages of ddaC neurons showing severing defects in ( F - H ) at 16 h APF .

I Percentages of ddaC neurons showing severing defects in (F-H) at 16 h APF.


J Quantitative analysis of unpruned dendrite lengths in ( F - H ) at 16 h APF .

J Quantitative analysis of unpruned dendrite lengths in (F-H) at 16 h APF.


Confocal images of ddaC neurons expressing UAS - mCD8 : : GFP , UAS - Nod - lacZ or UAS - Kin - lacZ and immunostained for β - galactosidase at wL3 stages . ( A - C ) Wild - type ddaC neurons ( A ) displayed normal distribution of Nod - lacZ signals in dendrites . In contrast , in msps810 mutants ( B ) and tacc59 / tacc74 mutants ( C ) , ddaC neurons exhibited altered Nod - lacZ distribution patterns , with highly enriched stainings in the somas and decreased signals in the dendrites .

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O

Confocal images of ddaC neurons expressing UAS-mCD8::GFP, UAS-Nod-lacZ or UAS-Kin-lacZ and immunostained for β-galactosidase at wL3 stages. (A-C) Wild-type ddaC neurons (A) displayed normal distribution of Nod-lacZ signals in dendrites. In contrast, in msps810 mutants (B) and tacc59/tacc74 mutants (C), ddaC neurons exhibited altered Nod-lacZ distribution patterns, with highly enriched stainings in the somas and decreased signals in the dendrites.


Confocal images of ddaC neurons ( D - F ) Kin - lacZ signals were present in axons but not detectable in dendrites of the control ddaC neurons ( D ) , whereas part of the Kin - lacZ were mislocalized to dendrites in msps RNAi ( E ) and tacc RNAi ( F ) ddaC neurons . Asterisks indicate the location of ddaC somas . White arrows indicate the location of axons . Curly brackets mark the dendritic regions where fluorescence intensity of Nod - lacZ was measured . White arrowheads point to dendritically localized Kin - lacZ signals .

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O

Confocal images of ddaC neurons (D-F) Kin-lacZ signals were present in axons but not detectable in dendrites of the control ddaC neurons (D), whereas part of the Kin-lacZ were mislocalized to dendrites in msps RNAi (E) and tacc RNAi (F) ddaC neurons. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured. White arrowheads point to dendritically localized Kin-lacZ signals.


G Percentages of ddaC neurons showing defective Nod - lacZ distribution in mutants or RNAi - expressing neurons .

O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE O

G Percentages of ddaC neurons showing defective Nod-lacZ distribution in mutants or RNAi-expressing neurons.


H Quantitative analysis of normalized Nod - lacZ intensity for 20 μm of major dendrite located 40 μm away from the soma .

O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O

H Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 40 μm away from the soma.


I Percentages of ddaC neurons showing defective Kin - lacZ distribution .

O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O

I Percentages of ddaC neurons showing defective Kin-lacZ distribution.


A - F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL . Horizontal arrow indicates the direction towards the somas , and vertical arrow indicates the time . EB1 : : GFP expression was driven under Gal44 - 77 . In the control ddaC neurons ( A , D ) , EB1 comets move predominantly in retrograde direction . EB1 comets were largely diminished in msps810 / mspsP mutant neurons ( B ) . Interestingly , in msps810 / mspsP18 mutants ( C ) , tacc RNAi ( E ) and tacc59 / tacc74 mutant ( F ) , increased anterograde comets were detected in ddaC dendrites .

O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-CELL_TYPE B-SUBCELLULAR O

A-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. EB1::GFP expression was driven under Gal44-77. In the control ddaC neurons (A, D), EB1 comets move predominantly in retrograde direction. EB1 comets were largely diminished in msps810/mspsP mutant neurons (B). Interestingly, in msps810/mspsP18 mutants (C), tacc RNAi (E) and tacc59/tacc74 mutant (F), increased anterograde comets were detected in ddaC dendrites.


G - J Quantitative analysis of the percentages of anterograde EB1 : : GFP comets , the track length , the number ( per 30 µm ) and the velocity of the comets in each neuron imaged .

O O O O O O O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O B-CELL_TYPE O O

G-J Quantitative analysis of the percentages of anterograde EB1::GFP comets, the track length, the number (per 30 µm) and the velocity of the comets in each neuron imaged.


A - F Confocal images of ddaC neurons expressing UAS - mCD8 : : GFP ( Green channel ) , UAS - Nod - lacZ and immunostained for β - galactosidase at wL3 stages . Simultaneous knockdown of klp10A and msps ( B ) significantly restored Nod - lacZ localization in the dendrites , compared to msps and control RNAi ( A ) . ddaC neurons with colchicine treatment ( D ) or with Kat - 60 overexpression ( F ) exhibited perturbed Nod - lacZ distribution with highly enriched staining in the soma and decreased signals in the dendrites . Asterisks indicate the location of ddaC somas . White arrows indicate the location of axons . Curly brackets mark the dendritic regions where fluorescence intensity of Nod - lacZ was measured .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O

A-F Confocal images of ddaC neurons expressing UAS-mCD8::GFP (Green channel), UAS-Nod-lacZ and immunostained for β-galactosidase at wL3 stages. Simultaneous knockdown of klp10A and msps (B) significantly restored Nod-lacZ localization in the dendrites, compared to msps and control RNAi (A). ddaC neurons with colchicine treatment (D) or with Kat-60 overexpression (F) exhibited perturbed Nod-lacZ distribution with highly enriched staining in the soma and decreased signals in the dendrites. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured.


G - L Live confocal images of ddaC neurons visualized by ppk - Gal4 driven mCD8 : : GFP expression at WP or 16 h APF . Simultaneous knockdown klp10A and msps ( H ) almost fully rescued the dendrite morphological defects and the pruning defects compared with the msps , control RNAi neurons . Wild type larvae fed with colchicine ( J ) showed pruning defects in comparison with those feed with DMSO ( I ) . ddaC neurons with Kat - 60 overexpression ( L ) exhibited abnormal dendrite arborization and pruning defects . Red arrowheads point to the ddaC somas .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O

G-L Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown klp10A and msps (H) almost fully rescued the dendrite morphological defects and the pruning defects compared with the msps, control RNAi neurons. Wild type larvae fed with colchicine (J) showed pruning defects in comparison with those feed with DMSO (I). ddaC neurons with Kat-60 overexpression (L) exhibited abnormal dendrite arborization and pruning defects. Red arrowheads point to the ddaC somas.


M , N Quantitative analysis of normalized Nod - lacZ intensity for 20 μm of major dendrite located 55 μm or 40 μm away from the soma respectively .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O

M, N Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 55 μm or 40 μm away from the soma respectively.


O Percentages of ddaC neurons showing severing defects at 16 h APF .

O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O

O Percentages of ddaC neurons showing severing defects at 16 h APF.


P Quantitative analysis of unpruned dendrite lengths at 16 h APF .

O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O

P Quantitative analysis of unpruned dendrite lengths at 16 h APF.


- F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL . Horizontal arrow indicates the direction towards the somas , and vertical arrow indicates the time . ( B ) RNAi knockdown of klp10A in msps810 / mspsP18 mutants restored the retrograde movement pattern of EB1 comets . MT depolymerization or severing induced by Colchicine treatment ( D ) or overexpression of Kat - 60 ( F ) led to mixed MT orientation in ddaC neurons .

O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O

-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. (B) RNAi knockdown of klp10A in msps810/mspsP18 mutants restored the retrograde movement pattern of EB1 comets. MT depolymerization or severing induced by Colchicine treatment (D) or overexpression of Kat-60 (F) led to mixed MT orientation in ddaC neurons.


G - J Quantitative analysis of the percentages of anterograde EB1 comets , the track length , number ( per 30 µm ) and the velocity of EB1 comets in each neuron imaged .

O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE O O

G-J Quantitative analysis of the percentages of anterograde EB1 comets, the track length, number (per 30 µm) and the velocity of EB1 comets in each neuron imaged.


( A ) 2D STED micrographs of RPE1 cells co - stained with the indicated antibodies . Scale bar : 200 nm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

(A) 2D STED micrographs of RPE1 cells co-stained with the indicated antibodies. Scale bar: 200 nm.


( B ) Dot plots showing the average pixel intensities at individual parent centrioles expressed as the PIDD1 / ODF2 fluorescence ratio in the indicated cell lines and genotypes . Mean values ( red lines ) ± s . e . m . are reported . N ≥ 50 centrosomes were assessed for each condition , a . u . = arbitrary units . Unpaired Mann - Whitney test ( * * * * P < 0 . 0001 ) .

O O O O O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Dot plots showing the average pixel intensities at individual parent centrioles expressed as the PIDD1/ODF2 fluorescence ratio in the indicated cell lines and genotypes. Mean values (red lines) ± s.e.m. are reported. N ≥ 50 centrosomes were assessed for each condition, a.u. = arbitrary units. Unpaired Mann-Whitney test (****P < 0.0001).


( C ) Representative fluorescence micrographs from the indicated cell lines co - stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

(C) Representative fluorescence micrographs from the indicated cell lines co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( D ) Co - localization between ODF2 and PIDD1 ( n = 11 ) , FBF1 and PIDD1 ( n = 21 ) , and ANKRD26 and PIDD1 ( n = 17 ) assessed on images The value represents the fraction PIDD1 objects touching either ODF2 , FBF1 or ANKRD26 objects .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O

(D) Co-localization between ODF2 and PIDD1 (n = 11), FBF1 and PIDD1 (n = 21), and ANKRD26 and PIDD1 (n = 17) assessed on images The value represents the fraction PIDD1 objects touching either ODF2, FBF1 or ANKRD26 objects.


( E ) 2D STED micrographs of RPE1 cells co - stained with the indicated antibodies , scale bar : 200 nm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

(E) 2D STED micrographs of RPE1 cells co-stained with the indicated antibodies, scale bar: 200 nm.


( F ) Representative fluorescence micrographs of RPE1 cells of the indicated genotypes co - stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

(F) Representative fluorescence micrographs of RPE1 cells of the indicated genotypes co-stained with indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( G ) Dot plot showing the average pixel intensities at individual parent centrioles expressed as the PIDD1 / CEP128 fluorescence ratio in RPE1 of the indicated genotypes . Mean values ( red lines ) ± s . e . m . are reported . N ≥ 50 centrosomes were assessed from as many individual cells for each condition , a . u . = arbitrary units . Statistical significance was assessed by Kruskal - Wallis test , comparing each sample to the wild type ( * * * * P < 0 . 0001 ) .

O O O O O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Dot plot showing the average pixel intensities at individual parent centrioles expressed as the PIDD1/CEP128 fluorescence ratio in RPE1 of the indicated genotypes. Mean values (red lines) ± s.e.m. are reported. N ≥ 50 centrosomes were assessed from as many individual cells for each condition, a.u. = arbitrary units. Statistical significance was assessed by Kruskal-Wallis test, comparing each sample to the wild type (****P < 0.0001).


( B ) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . Cells were co - stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

(B) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Cells were co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( C ) Dot plot showing the average pixel intensities of the Myc signal at individual parent centrioles in RPE1 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . Mean values ( red lines ) ± s . e . m . are reported . N > 50 centrosomes were assessed for each condition in as many individual cells ; a . u . = arbitrary units . Kruskal - Wallis test ( * * * * P < 0 . 0001 ; n . s . = non - significant ) .

O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-SUBCELLULAR O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Dot plot showing the average pixel intensities of the Myc signal at individual parent centrioles in RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001; n.s. = non-significant).


( D ) Dot plot showing the average pixel intensities of the PIDD1 signal at individual parent centrioles in RPE1 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . Mean values ( red lines ) ± s . e . m . are reported . N > 50 centrosomes were assessed for each condition in as many individual cells ; a . u . = arbitrary units . Kruskal - Wallis test ( * * * * P < 0 . 0001 ; n . s . = non - significant ) .

(D) Dot plot showing the average pixel intensities of the PIDD1 signal at individual parent centrioles in RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001; n.s. = non-significant).


( E ) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . Cells were co - stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

(E) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Cells were co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( A ) RPE1 cells of the indicated genotypes were treated either with DMSO or with DHCB for 24h . A fraction of DHCB treated cells were released into fresh medium for other 24h ( release ) . Samples were subjected to immunoblotting ; n = 3 independent experiments .

O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

(A) RPE1 cells of the indicated genotypes were treated either with DMSO or with DHCB for 24h. A fraction of DHCB treated cells were released into fresh medium for other 24h (release). Samples were subjected to immunoblotting; n = 3 independent experiments.


( B ) Fluorescence micrographs of RPE1 cells of the indicated genotypes , either treated for 24h with DHCB or with vehicle alone ( DMSO ) . Blow - up without Hoechst 33342 is magnified 2 . 5X . Scale bar : 5 μm . ( C ) Immunofluorescence micrographs of RPE1 as in ( B ) were used to visually assess the percentage of cells presenting one or two nuclei . N = 3 , ≥ 50 cells from each independent experiment . Mean values ± s . e . m . are reported . The increase in the number of binucleated cells between the wild type sample and all the other genotypes was assessed ( ANOVA test ; * P < 0 . 05 ) . ( D ) Immunofluorescence micrographs of RPE1 as in ( B ) were used to visual score the number of centrosomes per cell by counting the number of γ - tubulin - positive centrioles . N = 3 , ≥ 50 cells from each independent experiment . Mean values ± s . e . m . are reported . The increase in the number of cells with > 2 centrosomes between the wild type sample and all the other genotypes was assessed ( ANOVA test ; * * P < 0 . 01 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Fluorescence micrographs of RPE1 cells of the indicated genotypes, either treated for 24h with DHCB or with vehicle alone (DMSO). Blow-up without Hoechst 33342 is magnified 2.5X. Scale bar: 5 μm. (C) Immunofluorescence micrographs of RPE1 as in (B) were used to visually assess the percentage of cells presenting one or two nuclei. N = 3, ≥ 50 cells from each independent experiment. Mean values ± s.e.m. are reported. The increase in the number of binucleated cells between the wild type sample and all the other genotypes was assessed (ANOVA test; *P < 0.05). (D) Immunofluorescence micrographs of RPE1 as in (B) were used to visual score the number of centrosomes per cell by counting the number of γ-tubulin-positive centrioles. N = 3, ≥ 50 cells from each independent experiment. Mean values ± s.e.m. are reported. The increase in the number of cells with > 2 centrosomes between the wild type sample and all the other genotypes was assessed (ANOVA test; **P < 0.01).


( E ) Quantitative assessment of the fraction of RPE1 cells undergoing cytokinesis failure upon DHCB treatment , inferred on the basis of the increase of ploidies ≥ 4C ( upper panel ) , and of the fraction of the abovementioned cells undergoing genome reduplication upon release after DHCB treatment ( lower panel ) . Individual values of biological replicates , their mean and standard deviations are reported .

O O O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(E) Quantitative assessment of the fraction of RPE1 cells undergoing cytokinesis failure upon DHCB treatment, inferred on the basis of the increase of ploidies ≥ 4C (upper panel), and of the fraction of the abovementioned cells undergoing genome reduplication upon release after DHCB treatment (lower panel). Individual values of biological replicates, their mean and standard deviations are reported.


( F ) RPE1 cells were either left untransduced ( mock ) or transduced with lentiviral vectors expressing the indicated Myc - ANKRD26 constructs . Subsequently , cells were treated either with DMSO or ZM447439 for 24h and subjected to immunoblotting ; n = 3 independent experiments .

O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O

(F) RPE1 cells were either left untransduced (mock) or transduced with lentiviral vectors expressing the indicated Myc-ANKRD26 constructs. Subsequently, cells were treated either with DMSO or ZM447439 for 24h and subjected to immunoblotting; n = 3 independent experiments.


( B ) A549 cells of the indicated genotypes were either left untransduced ( mock ) or transduced with lentiviral vectors expressing the indicated PIDD1 - V5 derivatives and subjected to immunoblotting . N = 2 independent experiments .

O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O

(B) A549 cells of the indicated genotypes were either left untransduced (mock) or transduced with lentiviral vectors expressing the indicated PIDD1-V5 derivatives and subjected to immunoblotting. N = 2 independent experiments.


( C ) Fluorescence micrographs of A549 cells of the indicated genotypes either left untransduced ( mock ) , or transduced with PIDD1 - V5 lentiviral vectors and co - stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

(C) Fluorescence micrographs of A549 cells of the indicated genotypes either left untransduced (mock), or transduced with PIDD1-V5 lentiviral vectors and co- stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( D ) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . N > 50 centrosomes were assessed for each condition in as many individual cells ; a . u . = arbitrary units . ( E ) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . N > 50 centrosomes were assessed for each condition in as many individual cells ; a . u . = arbitrary units .

O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-SUBCELLULAR O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-SUBCELLULAR O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O

(D) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. (E) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units.


( F ) Representative fluorescence micrographs of A549 cells of the indicated genotypes . Cells were either left untransduced ( mock ) or transduced with PIDD1 - V5 lentiviral vectors expressing the PIDD1 non - cleavable derivative ( PIDD1S446A - S588A ) or truncations thereof . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

(F) Representative fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors expressing the PIDD1 non-cleavable derivative (PIDD1S446A-S588A) or truncations thereof. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( A ) A549 cells of the indicated genotypes were either left untransduced or transduced with a lentiviral vector expressing PIDD1 - V5 in its wild type form . Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting . N = 2 independent experiments .

O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O

(A) A549 cells of the indicated genotypes were either left untransduced or transduced with a lentiviral vector expressing PIDD1-V5 in its wild type form. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments.


( B , C ) A549 cells of the indicated genotypes were either left untransduced ( mock ) , or transduced with lentiviral vectors expressing PIDD1 - V5 in its wild type form or carrying the indicated point mutations . Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting . N = 2 independent experiments .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O

(B,C) A549 cells of the indicated genotypes were either left untransduced (mock), or transduced with lentiviral vectors expressing PIDD1-V5 in its wild type form or carrying the indicated point mutations. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments.


( D ) Fluorescence micrographs of A549 cells of the indicated genotypes . Cells were either left untransduced ( mock ) or transduced with PIDD1 - V5 lentiviral vectors carrying the indicated point mutations , and stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

(D) Fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors carrying the indicated point mutations, and stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( A ) Representative fluorescence micrographs across the indicated cell cycle phases from RPE1 cells stably expressing CETN1 - GFP . Centrosomal antigens were stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

(A) Representative fluorescence micrographs across the indicated cell cycle phases from RPE1 cells stably expressing CETN1-GFP. Centrosomal antigens were stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


The centrioles subjected to fluorescence intensity measurement in ( B ) are labelled with numbers . ( B ) Quantification of the PIDD1 average pixel intensity at individual centrioles across the indicated cell cycle phases of RPE1 cells stained N > 50 centrioles were assessed for each phase , a . u . = arbitrary units .

O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O O O

The centrioles subjected to fluorescence intensity measurement in (B) are labelled with numbers. (B) Quantification of the PIDD1 average pixel intensity at individual centrioles across the indicated cell cycle phases of RPE1 cells stained N > 50 centrioles were assessed for each phase, a.u. = arbitrary units.


( C ) Fluorescence micrographs of RPE1 cells stably expressing CETN1 - GFP treated either with DMSO or with DHCB for 24h . Blow - ups 33342 are magnified 2 . 5X . Scale bar : 5 μm . ( D ) Dot plot showing PIDD1 average pixel intensities at individual parent centrioles calculated from images as in ( C ) . Mean values ( red lines ) ± s . e . m . are reported . N > 50 centrosomes were assessed for each condition , a . u . = arbitrary units .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

(C) Fluorescence micrographs of RPE1 cells stably expressing CETN1-GFP treated either with DMSO or with DHCB for 24h. Blow-ups 33342 are magnified 2.5X. Scale bar: 5 μm. (D) Dot plot showing PIDD1 average pixel intensities at individual parent centrioles calculated from images as in (C). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition, a.u. = arbitrary units.


( E ) Movie stills of a representative RPE1 cell stably expressing CETN1 - GFP treated with DHCB and subjected to time - lapse video microscopy in the presence of SiR - DNA . Time is expressed in minutes , relative to anaphase onset . The dashed line indicates the plasma membrane of the cell of interest , arrowheads indicate the centrosomes ' position .

O O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O

(E) Movie stills of a representative RPE1 cell stably expressing CETN1-GFP treated with DHCB and subjected to time-lapse video microscopy in the presence of SiR-DNA. Time is expressed in minutes, relative to anaphase onset. The dashed line indicates the plasma membrane of the cell of interest, arrowheads indicate the centrosomes' position.


( F ) Centrosomal distance over time in RPE1 cells stably expressing CETN1 - GFP and treated with DHCB . Time zero corresponds to the frame preceding anaphase onset . Coloured dots ( lower panel ) summarize the clustering time for each cell , mean ± standard deviation in black . Data calculated from four - dimensional imaging as in ( E ) . N = 10 cells .

O O O B-SUBCELLULAR O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

(F) Centrosomal distance over time in RPE1 cells stably expressing CETN1-GFP and treated with DHCB. Time zero corresponds to the frame preceding anaphase onset. Coloured dots (lower panel) summarize the clustering time for each cell, mean ± standard deviation in black. Data calculated from four-dimensional imaging as in (E). N = 10 cells.


( A ) Dot plot showing the distance between parent centrioles pairs in A549 cells following the indicated treatments ( ZM = ZM447439 ; REV = reversine ) . Nocodazole concentrations are 0 . 03 μM , 0 . 1 μM , 0 . 33 μM , 1 μM , 3 . 3 μM . Median ( red ) and 95 % confidence interval thereof ( black ) are shown . N > 50 cells were analysed . ( B ) Immunoblot of A549 cells subjected to the indicated treatments for 24h as in ( A ) . N = 3 independent experiments . ( C ) DNA content analysis of A549 cells subjected to the indicated treatments as in ( A ) either for 24h ( left panels ) or 48h ( right panels ) . N = 2 independent experiments .

O O O O O O O O O O B-SUBCELLULAR O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Dot plot showing the distance between parent centrioles pairs in A549 cells following the indicated treatments (ZM = ZM447439; REV = reversine). Nocodazole concentrations are 0.03 μM, 0.1 μM, 0.33 μM, 1 μM, 3.3 μM. Median (red) and 95% confidence interval thereof (black) are shown. N > 50 cells were analysed. (B) Immunoblot of A549 cells subjected to the indicated treatments for 24h as in (A). N = 3 independent experiments. (C) DNA content analysis of A549 cells subjected to the indicated treatments as in (A) either for 24h (left panels) or 48h (right panels). N = 2 independent experiments.


(E) Immunoblots of RPE1 cells synchronized


( F ) Representative fluorescence micrographs of RPE1 cells synchronized Centrosomal antigens were stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2X . Scale bar : 5 μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O

(F) Representative fluorescence micrographs of RPE1 cells synchronized Centrosomal antigens were stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2X. Scale bar: 5 μm.


( A ) A549 cells of the indicated genotypes were treated for 24h as indicated ( CPT = camptothecin , ZM = ZM447439 ) . Samples were subjected to immunoblotting ; n = 3 independent experiments . ( B ) A549 cells treated as in ( A ) were subjected to fluorescence microscopy and centrosome abundance was assessed by visually scoring γ - tubulin - positive centrioles per cell . N = 3 , ≥ 50 cells from each independent experiment .

O O O B-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O

(A) A549 cells of the indicated genotypes were treated for 24h as indicated (CPT = camptothecin, ZM = ZM447439). Samples were subjected to immunoblotting; n = 3 independent experiments. (B) A549 cells treated as in (A) were subjected to fluorescence microscopy and centrosome abundance was assessed by visually scoring γ-tubulin-positive centrioles per cell. N = 3, ≥ 50 cells from each independent experiment.


( C ) RT - qPCR analysis of PIDD1 mRNA expression in A549 cells upon treatment with increasing doses of Nutlin 3a ( i . e . 3 . 3 μM or 10 μM ) using two independent probes . The average fold of induction ± standard deviation is shown . N = 3 biological replicates with two technical replicates each . Comparisons were performed between treatments of every genotype and the corresponding treatment of wild type ( WT )

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) RT-qPCR analysis of PIDD1 mRNA expression in A549 cells upon treatment with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. The average fold of induction ± standard deviation is shown. N = 3 biological replicates with two technical replicates each. Comparisons were performed between treatments of every genotype and the corresponding treatment of wild type (WT) cells


with increasing doses of Nutlin 3a ( i . e . 3 . 3 μM or 10 μM ) using two independent probes . ( D ) Immunoblot analysis of samples treated N = 3 independent experiments .

O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. (D) Immunoblot analysis of samples treated N = 3 independent experiments.


( A ) Recombinant c10orf76 directly binds to PI4KB in vitro . His - pulldown assays of baculovirus / Sf9 produced 6xHis - c10orf76 ( 3 μM ) were carried out with untagged PI4KB ( 2 . 5 μM ) .

O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O B-ORGANISM O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O

(A) Recombinant c10orf76 directly binds to PI4KB in vitro. His-pulldown assays of baculovirus/Sf9 produced 6xHis-c10orf76 (3 μM) were carried out with untagged PI4KB (2.5 μM).


( B ) PI4KB and c10orf76 form a stable complex . The complex of c10orf76 - PI4KB eluted from a S200 superdex 10 / 300 GL increase gel filtration column ( GE ) at a volume consistent with a heterodimer ( 169 kDa ) , while c10orf76 alone eluted at a volume consistent with a monomer ( 79 kDa ) . Lines with MW values indicate elution of MW standards ( 158 kDa aldolase , 75 kDa conalbumin ) .

O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O

(B) PI4KB and c10orf76 form a stable complex. The complex of c10orf76-PI4KB eluted from a S200 superdex 10/300 GL increase gel filtration column (GE) at a volume consistent with a heterodimer (169 kDa), while c10orf76 alone eluted at a volume consistent with a monomer (79 kDa). Lines with MW values indicate elution of MW standards (158 kDa aldolase, 75 kDa conalbumin).


( C ) PI4KB is potently inhibited by c10orf76 in a dose - dependent manner in vitro . Kinase assays of PI4KB ( 20 nM ) in the presence of varying concentrations of c10orf76 ( 1 . 6 nM - 1 μM ) were carried out on pure PI lipid vesicles ( 0 . 5 mg / L ) in the presence of 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone . IC50 values were determined by one binding site , nonlinear regression ( curve fit ) using Graphpad . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

O O O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) PI4KB is potently inhibited by c10orf76 in a dose-dependent manner in vitro. Kinase assays of PI4KB (20 nM) in the presence of varying concentrations of c10orf76 (1.6 nM-1 μM) were carried out on pure PI lipid vesicles (0.5 mg/L) in the presence of 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. IC50 values were determined by one binding site, nonlinear regression (curve fit) using Graphpad. Error bars represent standard deviation of independent technical replicates (n=3).


( D ) PI4KB is potently inhibited by c10orf76 on pure PI vesicles and vesicles mimicking Golgi composition . Kinase assays of PI4KB and c10orf76 were carried out on lipid substrate composed of pure PI vesicles ( 0 . 5 mg / mL ) with 100 μM ATP , and Golgi mimic vesicles ( 0 . 5 mg / ml , 10 % PS , 20 % PI , 25 % PE , 45 % PC ) with 10 μM ATP . PI4KB was present at 20 and 300 nM in the PI and Golgi substrate assays respectively , with c10orf76 present at 500 nM in both experiments . The data is normalized to the kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

O O O B-GENEPROD O O O O B-GENEPROD O O B-SMALL_MOLECULE B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O

(D) PI4KB is potently inhibited by c10orf76 on pure PI vesicles and vesicles mimicking Golgi composition. Kinase assays of PI4KB and c10orf76 were carried out on lipid substrate composed of pure PI vesicles (0.5 mg/mL) with 100 μM ATP, and Golgi mimic vesicles (0.5 mg/ml, 10% PS, 20% PI, 25% PE, 45% PC) with 10 μM ATP. PI4KB was present at 20 and 300 nM in the PI and Golgi substrate assays respectively, with c10orf76 present at 500 nM in both experiments. The data is normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( E ) Changes in deuterium incorporation PI4KB in the presence of c10orf76 showed a profound ordering of the kinase domain N - lobe linker and smaller changes in the helical domain and C - lobe of the kinase domain . The sum of the difference mapped as the difference in number of deuterons incorporated for PI4KB ( 400 nM ) in the presence and absence of c10orf76 ( 400 nM ) over all time points ( 3s at 1 oC ; 3s , 30s , and 300s at 23 oC ) . Each dot represents a peptide graphed on the x - axis according to the central residue . The red boxes highlight key regions that showed significant changes ( > 7 % decrease in exchange , > 0 . 5 Da difference , and unpaired two - tailed student t - test p < 0 . 05 ) . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Changes in deuterium incorporation PI4KB in the presence of c10orf76 showed a profound ordering of the kinase domain N-lobe linker and smaller changes in the helical domain and C-lobe of the kinase domain. The sum of the difference mapped as the difference in number of deuterons incorporated for PI4KB (400 nM) in the presence and absence of c10orf76 (400 nM) over all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC). Each dot represents a peptide graphed on the x-axis according to the central residue. The red boxes highlight key regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).


( G ) Changes in the deuterium incorporation of c10orf76 in the presence of PI4KB . H / D exchange reactions displayed as the sum of the difference in HDX in the number of deuterons for c10orf76 ( 400 nM ) in the presence of PI4KB ( 400 nM ) at all time points ( 3s at 1 oC ; 3s , 30s , and 300s at 23 oC ) analyzed . Red boxes highlight regions that showed significant changes ( > 7 % decrease in exchange , > 0 . 5 Da difference , and unpaired two - tailed student t - test p < 0 . 05 ) . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Changes in the deuterium incorporation of c10orf76 in the presence of PI4KB. H/D exchange reactions displayed as the sum of the difference in HDX in the number of deuterons for c10orf76 (400 nM) in the presence of PI4KB (400 nM) at all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC) analyzed. Red boxes highlight regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).


( H ) The PI4KB N - lobe linker undergoes a disorder - to - order transition upon binding c10orf76 . Selected peptides ( including the sequence , domain information , and numbering ) of both PI4KB and c10orf76 displayed as the % deuteration incorporation over time . Error bars represent standard deviation of independent technical replicates ( n = 3 ) , and are typically smaller than the size of the point on the graph .

O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) The PI4KB N-lobe linker undergoes a disorder-to-order transition upon binding c10orf76. Selected peptides (including the sequence, domain information, and numbering) of both PI4KB and c10orf76 displayed as the % deuteration incorporation over time. Error bars represent standard deviation of independent technical replicates (n=3), and are typically smaller than the size of the point on the graph.


( B ) The N - lobe kinase linker of PI4KB can be efficiently phosphorylated by PKA . Recombinant PKA at different concentrations ( 0 , 7 , 34 , 168 , or 840 ng ) was incubated with recombinant ( E . coli ) wild - type PI4KB ( 20 μg ) for 1 hour with 200 μM ATP and the amount of phosphorylation was followed using mass spectrometry . Relative abundance of Ser496 phosphorylated PI4KB was calculated using the relative intensity ( total area ) of the phosphorylated vs non - phosphorylated peptide ( 486 - 506 ) .

O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O

(B) The N-lobe kinase linker of PI4KB can be efficiently phosphorylated by PKA. Recombinant PKA at different concentrations (0, 7, 34, 168, or 840 ng) was incubated with recombinant (E. coli) wild-type PI4KB (20 μg) for 1 hour with 200 μM ATP and the amount of phosphorylation was followed using mass spectrometry. Relative abundance of Ser496 phosphorylated PI4KB was calculated using the relative intensity (total area) of the phosphorylated vs non-phosphorylated peptide (486-506).


( C ) PI4KB phosphorylation by PKA alters the affinity for c10orf76 . The kinase activity of different variants of PI4KB ( 15 nM ) was measured in the presence of varying amounts of c10orf76 ( 23 nM - 1 . 5μM ) with 100 % PI lipid substrate ( 0 . 5 mg / L ) and 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O

(C) PI4KB phosphorylation by PKA alters the affinity for c10orf76. The kinase activity of different variants of PI4KB (15 nM) was measured in the presence of varying amounts of c10orf76 (23 nM-1.5μM) with 100% PI lipid substrate (0.5 mg/L) and 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone Error bars represent standard deviation of independent technical replicates (n=3).


( D ) PI4KB has the same kinase activity when Ser496 is phosphorylated or mutated to alanine . Kinase assay of PI4KB non - phosphorylated , phos - Ser496 or S496A ( 15 nM ) on pure PI lipid vesicles ( 0 . 5 mg / L ) with 100 μM ATP . The data was normalized to the kinase activity of WT PI4KB . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O

(D) PI4KB has the same kinase activity when Ser496 is phosphorylated or mutated to alanine. Kinase assay of PI4KB non-phosphorylated, phos-Ser496 or S496A (15 nM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).


( E ) Engineered RL494EA PI4KB mutant shows decreased binding to c10orf76 . His - pulldown assays of 6xHis - c10orf76 ( 3 μM ) with wild - type or RL494EA PI4KB ( 1 - 2 μM ) .

O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O

(E) Engineered RL494EA PI4KB mutant shows decreased binding to c10orf76. His-pulldown assays of 6xHis-c10orf76 (3 μM) with wild-type or RL494EA PI4KB (1-2 μM).


( F ) RL494EA PI4KB activity is not inhibited by c10orf76 . Kinase assays of either wild type or mutant RL494EA PI4KB ( 40 nM ) were carried out with varying concentrations of c10orf76 ( 3 . 9 nM - 2μM ) with 100 % PI lipid vesicles ( 0 . 5 mg / L ) and 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

O O O O B-GENEPROD O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O

(F) RL494EA PI4KB activity is not inhibited by c10orf76. Kinase assays of either wild type or mutant RL494EA PI4KB (40 nM) were carried out with varying concentrations of c10orf76 (3.9 nM-2μM) with 100% PI lipid vesicles (0.5 mg/L) and 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( G ) Wild - type PI4KB and RL494EA PI4KB mutant have the same lipid kinase activity . Kinase assays of either wild - type and mutant PI4KB ( 10 nM ) were carried out with 100 % PI lipid vesicles ( 0 . 5 mg / L ) , 100 μM ATP , and PI4KB ( 300 nM ) on Golgi - mimic vesicles ( 0 . 5 mg / mL ) with 10 μM ATP . The data was normalized to the kinase activity of WT PI4KB . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O

(G) Wild-type PI4KB and RL494EA PI4KB mutant have the same lipid kinase activity. Kinase assays of either wild-type and mutant PI4KB (10 nM) were carried out with 100% PI lipid vesicles (0.5 mg/L), 100 μM ATP, and PI4KB (300 nM) on Golgi-mimic vesicles (0.5 mg/mL) with 10 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).


( H ) FLH409AAA - c10orf76 mutant shows decreased affinity for PI4KB . His - pulldown assays of 6xHis - c10orf76 ( 1 μM ) with wild - type PI4KB ( 1 μM ) . Samples washed a total of 4 times .

O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

(H) FLH409AAA-c10orf76 mutant shows decreased affinity for PI4KB. His-pulldown assays of 6xHis-c10orf76 (1 μM) with wild-type PI4KB (1 μM). Samples washed a total of 4 times.


( I ) Kinase assay shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced . Kinase assay of PI4KB ( 40 nM ) and a concentration curve of c10orf76 ( 3 . 9 nM - 2μM ) on pure PI lipid vesicles ( 0 . 5 mg / L ) with 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O

(I) Kinase assay shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced. Kinase assay of PI4KB (40 nM) and a concentration curve of c10orf76 (3.9 nM-2μM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( A ) Transfections of HEK293 cells revealed that both wild - type GFP - PI4KB and RL494EA GFP - PI4KB localize to the Golgi .

O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O

(A) Transfections of HEK293 cells revealed that both wild-type GFP-PI4KB and RL494EA GFP-PI4KB localize to the Golgi.


( B ) WT c10orf76 also localized to the Golgi , however , the PI4KB binding deficient mutant of c10orf76 ( FLH409AAA ) predominantly localized to the cytosol .

O O O O B-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR O O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY O O B-SUBCELLULAR O

(B) WT c10orf76 also localized to the Golgi, however, the PI4KB binding deficient mutant of c10orf76 (FLH409AAA) predominantly localized to the cytosol.


( D ) Mitochondria recruitment experiment with wild - type PI4KB and c10orf76 . Left : AKAP1 - FRB - CFP is localized to the mitochondria before ( top ) and 5 minutes after rapamycin ( 100 nM ) treatment ( bottom ) . Middle : mRFP - FKBP12 - PI4KB at high expression level is located in the cytosol before rapamycin ( top ) and translocates to the mitochondria after rapamycin induction ( bottom ) . Right : eGFP - c10orf76 at a high expression level is located in the cytosol before rapamycin ( top ) and translocates to the mitochondria after rapamycin induction ( bottom ) .

O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O

(D) Mitochondria recruitment experiment with wild-type PI4KB and c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after rapamycin (100 nM) treatment (bottom). Middle: mRFP-FKBP12-PI4KB at high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 at a high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom).


( F ) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76 . Left : AKAP1 - FRB - CFP is localized to the mitochondria before ( top ) and 5 minutes after ( bottom ) rapamycin treatment . Middle : mRFP - FKBP12 - PI4KB ( RL494EA ) is located in the cytosol before rapamycin ( top ) and translocates to the mitochondria after rapamycin induction ( bottom ) . Right : eGFP - c10orf76 is located in the cytosol before ( top ) and after ( bottom ) rapamycin induction .

O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O O

(F) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after (bottom) rapamycin treatment. Middle: mRFP-FKBP12-PI4KB(RL494EA) is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 is located in the cytosol before (top) and after (bottom) rapamycin induction.


( A HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

O O B-CELL_LINE O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-GENEPROD O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O

(A HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


B ) HAP1 cells were fixed and stained with antibodies examining Golgi morphology markers ( B ) . ( C ) Western blot of GBF1 , PI4KB and B - Actin levels in both wild - type and c10orf76 KO HAP1 cells . Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

O O B-CELL_LINE O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O

B) HAP1 cells were fixed and stained with antibodies examining Golgi morphology markers (B). (C) Western blot of GBF1, PI4KB and B-Actin levels in both wild-type and c10orf76 KO HAP1 cells. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


( D ) Markers of Arf1 activation - the coatomer proteins COPIα / γ and βCOP act as a readout for GTP - bound Arf1 , while the native coatomer was detected with the CM1 antibody . Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

O O O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O

(D) Markers of Arf1 activation - the coatomer proteins COPIα/γ and βCOP act as a readout for GTP-bound Arf1, while the native coatomer was detected with the CM1 antibody. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


( A ) Viral infection assays determining viral titers by end - point titration at 0 hours and 8 hours in HAP1 wild - type or c10orf76 knockout cells . Left : Coxsackievirus A10 infection . Middle : Poliovirus - 1 infection . Right : Coxsackievirus B3 infection . Titre values for c10orf76 knockout cells at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours and wild - type titres at 8 hours using a one - way ANOVA . * * , P < 0 . 01 ; N . S . , P > 0 . 05 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O B-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Viral infection assays determining viral titers by end-point titration at 0 hours and 8 hours in HAP1 wild-type or c10orf76 knockout cells. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Titre values for c10orf76 knockout cells at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours and wild-type titres at 8 hours using a one-way ANOVA. **, P<0.01; N.S., P>0.05.


( B ) Viral infection assays determining virus titers by end point titration at 8 hours in HeLa wild - type and PI4KB knockout cells upon transfection of wild - type PI4KB , the complex - disrupting RL494EA PI4KB mutant or the kinase dead D674A PI4KB mutant . Left : Coxsackievirus A10 infection . Middle : Poliovirus - 1 infection . Right : Coxsackievirus B3 infection . Values were statistically evaluated compared to the GalT control using a one - way ANOVA . * * , P < 0 . 01 ; * , P < 0 . 05 ; N . S . , P > 0 . 05 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Viral infection assays determining virus titers by end point titration at 8 hours in HeLa wild-type and PI4KB knockout cells upon transfection of wild-type PI4KB, the complex-disrupting RL494EA PI4KB mutant or the kinase dead D674A PI4KB mutant. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Values were statistically evaluated compared to the GalT control using a one-way ANOVA. **, P<0.01; *, P<0.05; N.S., P>0.05.


Overall structure of 2019 - nCoV RBD bound with ACE2 . ACE2 is colored green . 2019 - nCoV RBD core is colored cyan and RBM is colored red . Disulfide bonds in the 2019 - nCoV RBD are shown as stick and indicated by yellow arrows . The N - terminal helix of ACE2 responsible for binding is labeled .

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O

Overall structure of 2019-nCoV RBD bound with ACE2. ACE2 is colored green. 2019-nCoV RBD core is colored cyan and RBM is colored red. Disulfide bonds in the 2019-nCoV RBD are shown as stick and indicated by yellow arrows. The N-terminal helix of ACE2 responsible for binding is labeled.


Alignment of the 2019 - nCoV RBD ( core in cyan and RBM in red ) and SARS - CoV RBD ( core in orange and RBM in blue ) structures .

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

Alignment of the 2019-nCoV RBD (core in cyan and RBM in red) and SARS-CoV RBD (core in orange and RBM in blue) structures.


Structural alignment of 2019 - nCoV RBD / ACE2 and SARS - CoV RBD / ACE2 complexes . 2019 - nCoV RBD is colored cyan and red , its interacting ACE2 is colored green . SARS - CoV RBD is colored orange and blue , its interacting ACE2 is colored salmon . The PDB code for SARS - CoV RBD / ACE2 complex : 2AJF .

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O

Structural alignment of 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 complexes. 2019-nCoV RBD is colored cyan and red, its interacting ACE2 is colored green. SARS-CoV RBD is colored orange and blue, its interacting ACE2 is colored salmon. The PDB code for SARS-CoV RBD/ACE2 complex: 2AJF.


Contacting residues shown as stick at the 2019 - nCoV RBD / ACE2 and SARS - CoV RBD / ACE2 interfaces . Positions in both RBDs involved in ACE2 binding are indicated by red labels .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O

Contacting residues shown as stick at the 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 interfaces. Positions in both RBDs involved in ACE2 binding are indicated by red labels.


( B - K ' ) The Drosophila posterior midguts containing Su ( H ) - Gal4 > UAS - CD8 : GFP ( Su ( H ) > GFP ) with sucrose ( Suc , control ) ( B - C ' ) , P . e for 36 h ( D - F ' ) , Ecc15 for 36 h ( G - I ' ) and DSS for 36 h ( J - K ' ) were immunostained for GFP ( green ) , PH3 ( red ) and DAPI ( blue ) . ( C - C ' , E - E ' , H - H ' and K - K ' ) Magnification of selected areas containing PH3 + GFP ─ cells from B , D , G and J . The EB cell close to the PH3 + cell ( white arrow ) in C - C ' is out of focus . ( F - F ' and I - I ' ) Magnification of selected areas containing PH3 + EBs ( GFP + ) from D and G . White arrowheads and red arrows indicate PH3 in GFP ─ and GFP + cells , respectively . ( L and M ) Quantification of PH3 GFP ─ ISCs ( L ) and PH3 + GFP + EBs ( M ) with given treatments . n = 21 guts for each treatment .

O O O O O O O B-ORGANISM O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O

(B-K') The Drosophila posterior midguts containing Su(H)-Gal4>UAS-CD8:GFP (Su(H)>GFP) with sucrose (Suc, control) (B-C'), P.e for 36 h (D-F'), Ecc15 for 36 h (G-I') and DSS for 36 h (J-K') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (C-C', E-E', H-H' and K-K') Magnification of selected areas containing PH3+GFP─ cells from B, D, G and J. The EB cell close to the PH3+ cell (white arrow) in C-C' is out of focus. (F-F' and I-I') Magnification of selected areas containing PH3+ EBs (GFP+) from D and G. White arrowheads and red arrows indicate PH3 in GFP─ and GFP+ cells, respectively. (L and M) Quantification of PH3 GFP─ ISCs (L) and PH3+GFP+ EBs (M) with given treatments. n = 21 guts for each treatment.


( D - F ' ' ) When the ISCs were marked by esgGal4 , su ( H ) - Gal80 > UAS - GFP , PH3 expression was found in GFP + ISCs with feeding of Suc ( control ) ( D - D ' ' ) , P . e ( E - E ' ' ) and Dss ( F - F ' ' ) . However , PH3 expression is present in EBs with GFP ─ Pros ─ and small nuclei upon P . e infection ( E - E ' ' , red arrows ) . The white arrows indicate GFP + cells with PH3 expression . ( G ) Quantification of PH3 + GFP ─ Pros ─ EBs with given treatments . n = 11 guts for each treatment . Data information : Three independent experiments were performed , and error bars are ± SEM . * * * , P < 0 . 001 ( Student ' s t - test ) ( G ) .

O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-F'') When the ISCs were marked by esgGal4, su(H)-Gal80>UAS-GFP, PH3 expression was found in GFP+ ISCs with feeding of Suc (control) (D-D''), P.e (E-E'') and Dss (F-F''). However, PH3 expression is present in EBs with GFP─Pros─ and small nuclei upon P.e infection (E-E'', red arrows). The white arrows indicate GFP+ cells with PH3 expression. (G) Quantification of PH3+GFP─Pros─ EBs with given treatments. n = 11 guts for each treatment. Data information: Three independent experiments were performed, and error bars are ± SEM. ***, P < 0.001 (Student's t-test) (G).


( D - N ' ) The Drosophila midguts expressing Su ( H ) ts > GFP with control ( D - E ' ) , UAS - RasV12 for 6 days ( F - H ' ) , EGFRA887T for 4 days ( I - K ' ) and UAS − λTop for 5 days ( L - N ' ) were immunostained for GFP ( green ) , PH3 ( red ) and DAPI ( blue ) . ( E - E ' , G - G ' , J - J ' and M - M ' ) Magnification of selected areas containing PH3 + GFP ─ cells from D , F , I and L . ( H - H ' , K - K ' and N - N ' ) Magnification of selected areas containing PH3 + GFP + cells from F , I and L . White arrowheads and red arrows indicate PH3 in GFP ─ ISCs and GFP + EBs , respectively . ( O , P ) Quantification of PH3 + GFP ─ ( P ) and PH3 + GFP + ( O ) in midguts with indicated genotypes . n = 13 guts for each genotype . ( Q ) Quantification of PH3 + EBs ( GFP + ) with the given genotypes in response to P . e infection . n = 11 guts for each genotype .

O O O O O O O B-ORGANISM B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-TISSUE O O O O

(D-N') The Drosophila midguts expressing Su(H)ts>GFP with control (D-E'), UAS-RasV12 for 6 days (F-H'), EGFRA887T for 4 days (I-K') and UAS−λTop for 5 days (L-N') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (E-E', G-G', J-J' and M-M') Magnification of selected areas containing PH3+GFP─ cells from D, F, I and L. (H-H', K-K' and N-N') Magnification of selected areas containing PH3+GFP+ cells from F, I and L. White arrowheads and red arrows indicate PH3 in GFP─ ISCs and GFP+ EBs, respectively. (O, P) Quantification of PH3+GFP─ (P) and PH3+GFP+ (O) in midguts with indicated genotypes. n = 13 guts for each genotype. (Q) Quantification of PH3+ EBs (GFP+) with the given genotypes in response to P. e infection. n = 11 guts for each genotype.


( B - C ' ' ' ) Flies expressing UAS - Flp ; Su ( H ) ts UAS - CD8 : GFP ; ActP > Stop > LacZ were fed with Suc ( B - B ' ' ' ) or P . e ( C - C ' ' ' ) for 36h and their midguts were dissected and immunostained with antibodies against GFP , β - gal and Dl . Blue arrowheads indicate Dl + ISCs ; white arrows indicate GFP + EBs ; and red arrowheads indicate EB progeny cells ( LacZ + GFP ─ ) . ( D ) Quantification of Dl + ISC - like cells from EBs ( GFP ─ LacZ + Dl + ) in the control , P . e infected midguts , n = 50 . ( E ) Quantification of Dl + ISC - like cells from EBs ( GFP ─ LacZ + Dl + ) in the control with Suc or P . e , or knockdown of EGFR with Suc or P . e infection , n = 50 .

O O O O O O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O B-SMALL_MOLECULE O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O

(B-C''') Flies expressing UAS-Flp; Su(H)ts UAS-CD8:GFP; ActP>Stop>LacZ were fed with Suc (B-B''') or P.e (C-C''') for 36h and their midguts were dissected and immunostained with antibodies against GFP, β-gal and Dl. Blue arrowheads indicate Dl+ ISCs; white arrows indicate GFP+ EBs; and red arrowheads indicate EB progeny cells (LacZ+GFP─). (D) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the control, P. e infected midguts, n=50. (E) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the control with Suc or P. e, or knockdown of EGFR with Suc or P. e infection, n=50.


( A - C ' ' ' ) The midguts from adult female flies expressing UAS - Flp ; Su ( H ) ts UAS - CD8 : GFP ; ActP > Stop > LacZ with ( B - B ' ' ' ) or without ( A - A ' ' ' ) UAS - RasV12 or with UAS - RasV12 and UAS - stg - RNAi ( C - C ' ' ' ) were dissected and immunostained with antibodies against GFP , β - gal and Dl . The bigger GFP ─ nucleus with LacZ expression ( only red arrowheads ) indicate differentiating or mature ECs . Blue arrowheads indicate Dl + ISCs ; white arrows indicate GFP + EBs ; and red arrowheads indicate EB progeny cells ( LacZ + GFP ─ ) . ( D ) Quantification of Dl + ISC - like cells from EBs ( GFP ─ LacZ + Dl + ) in the indicated genotypes , n = 50 .

O O O O O O O O O B-TISSUE O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

(A-C''') The midguts from adult female flies expressing UAS-Flp; Su(H)ts UAS-CD8:GFP; ActP>Stop>LacZ with (B-B''') or without (A-A''') UAS-RasV12 or with UAS-RasV12 and UAS-stg-RNAi (C-C''') were dissected and immunostained with antibodies against GFP, β-gal and Dl. The bigger GFP─ nucleus with LacZ expression (only red arrowheads) indicate differentiating or mature ECs. Blue arrowheads indicate Dl+ ISCs; white arrows indicate GFP+ EBs; and red arrowheads indicate EB progeny cells (LacZ+GFP─). (D) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the indicated genotypes, n=50.


( C - E ' ' ) The representative twin clones from midguts when EGFRA887T is overexpressed in EBs ( D - D ' ' , red and green arrows ) or adult flies were fed with P . e for 36h ( E - E ' ' , red and green arrows ) , but no twin clone is found in the control ( C - C ' ' ) . ( F ) Quantification of different types of twin clones in the control midguts , or in the midguts with EGFRA887T overexpression or P . e infection . n = 10 guts for each genotype .

O O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-TISSUE O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-TISSUE O O O O

(C-E'') The representative twin clones from midguts when EGFRA887T is overexpressed in EBs (D-D'', red and green arrows) or adult flies were fed with P.e for 36h (E-E'', red and green arrows), but no twin clone is found in the control (C-C''). (F) Quantification of different types of twin clones in the control midguts, or in the midguts with EGFRA887T overexpression or P.e infection. n = 10 guts for each genotype.


( A - C ' ' ) ISCs from EBs upon RasV12 expression or P . e infection could differentiate to generate EE or pre - EE cells ( B - C ' ' , LacZ + Pros + GFP ─ , blue arrowheads ) , but no EE or pre - EE cells from EBs are found in the control ( A - A ' ' , white arrowheads ) . ( D ) Quantification of EE cells from progenies of EBs with indicated genotypes or treatments . n = 9 guts for each genotype or treatment .

O O O O O O O O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-EXP_ASSAY O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O O O O O O

(A-C'') ISCs from EBs upon RasV12 expression or P. e infection could differentiate to generate EE or pre-EE cells (B-C'', LacZ+Pros+GFP─, blue arrowheads), but no EE or pre-EE cells from EBs are found in the control (A-A'', white arrowheads). (D) Quantification of EE cells from progenies of EBs with indicated genotypes or treatments. n = 9 guts for each genotype or treatment.


Flow cytometric analysis of γδ T cells in RORγtCRE - STAT3F / F ( Cre + ) and littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse and line the median . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 using Mann - Whitney test . Numbers of γδT17 cells in the LN ( A ) and skin ( B ) before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . Steady - state : n = 8 ; 4 experiments , IMQ : n = 11 - 12 ; 4 experiments .

B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O B-ORGANISM O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

Flow cytometric analysis of γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse and line the median. *p < 0.05, **p < 0.01, ***p < 0.001 using Mann-Whitney test. Numbers of γδT17 cells in the LN (A) and skin (B) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments.


Representative micrographs indicating ear skin sections stained for H & E before and after IMQ - induced psoriasis ( scale bar = 100μm ) . Quantification of epidermal thickening from H & E stained sections . Steady - state : n = 8 ; 4 experiments , IMQ : n = 11 - 12 ; 4 experiments . Each symbol is the average thickening from 5 micrographs measured in μm .

O O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

Representative micrographs indicating ear skin sections stained for H&E before and after IMQ-induced psoriasis (scale bar = 100μm). Quantification of epidermal thickening from H&E stained sections. Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments. Each symbol is the average thickening from 5 micrographs measured in μm.