D , H1299 cells transfected as indicated were treated with 12 μM erastin ( + ) or untreated ( - ) for 24 h . Representative phase - contrast images were recorded ( magnification , × 20 ) . E , Surviving cells from the assay shown in ( D ) were counted . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

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

D, H1299 cells transfected as indicated were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20). E, Surviving cells from the assay shown in (D) were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


G , Affected metal ion - binding genes were selected and subjected to cluster analysis .

G, Affected metal ion-binding genes were selected and subjected to cluster analysis.


H , Labile iron levels were assessed by flow cytometry with a standard method in H1299 cells .

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

H, Labile iron levels were assessed by flow cytometry with a standard method in H1299 cells.


I , Labile iron levels examined were quantified . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

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 O O O O O O O O O O O O O O O O O O

I, Labile iron levels examined were quantified. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


Western blot analysis was performed with H1299 or Hep3B cells transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) for 48 h .

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

Western blot analysis was performed with H1299 or Hep3B cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) for 48 h.


Western blot analysis was performed with H1299 cells transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) for 48 h .

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

Western blot analysis was performed with H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) for 48 h.


C , Immunofluorescence ( IF ) assay for H1299 cells transfected with a Flag - tagged p53 ( Flag - p53 ) or an empty plasmid ( Cont . ) for 48 h . Representative images are shown ( magnification , × 40 ) . Arrowhead indicates a Flag - p53 overexpressing cell .

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

C, Immunofluorescence (IF) assay for H1299 cells transfected with a Flag-tagged p53 (Flag-p53) or an empty plasmid (Cont.) for 48 h. Representative images are shown (magnification, ×40). Arrowhead indicates a Flag-p53 overexpressing cell.


D , Western blot analysis was performed with H1299 transfected with a Flag - tagged p53 or an empty vector ( Cont . ) for 48 h .

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

D, Western blot analysis was performed with H1299 transfected with a Flag-tagged p53 or an empty vector (Cont.) for 48 h.


E , 293T cells transfected with a control siRNA ( siCont . ) and three p53 - specific siRNAs ( sip53 ) were subjected to Western blot analysis .

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

E, 293T cells transfected with a control siRNA (siCont.) and three p53-specific siRNAs (sip53) were subjected to Western blot analysis.


F , SMMC cells were treated with 100 μM etoposide ( + ) or untreated ( - ) for 24 h , and Western blot analysis was performed .

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

F, SMMC cells were treated with 100 μM etoposide (+) or untreated (-) for 24 h, and Western blot analysis was performed.


, Western blot analysis of A549 cells and H1299 cells .

, Western blot analysis of A549 cells and H1299 cells.


H , H1299 cells transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) were analyzed by micrococcal nuclease ( MNase ) digestion assay .

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

H, H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) were analyzed by micrococcal nuclease (MNase) digestion assay.


I , Western blot analysis of H1299 cells transfected with a wild - type p53 ( p53 - WT ) or two " gain - of - function " mutants ( p53 - R273H and p53 - R175H ) for 48 h .

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

I, Western blot analysis of H1299 cells transfected with a wild-type p53 (p53-WT) or two "gain-of-function" mutants (p53-R273H and p53-R175H) for 48 h.


A , Coimmunoprecipitation ( Co - IP ) analysis was performed in A549 cells with antibodies against p53 or USP7 .

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

A, Coimmunoprecipitation (Co-IP) analysis was performed in A549 cells with antibodies against p53 or USP7.


B , H1299 cells were transfected with an empty vector ( Cont . ) , a Flag - tagged p53 , a USP7 - specific shRNA , or the last two plasmids together for 48 h . Western blot analysis was then performed .

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

B, H1299 cells were transfected with an empty vector (Cont.), a Flag-tagged p53, a USP7-specific shRNA, or the last two plasmids together for 48 h. Western blot analysis was then performed.


C , Nuclear and cytosolic extracts were differentially prepared from H1299 cells transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) . Western blot analysis was then performed .

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

C, Nuclear and cytosolic extracts were differentially prepared from H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.). Western blot analysis was then performed.


D , H1299 cells transfected with a Myc - tagged USP7 or a Myc - tagged USP7 together with a Flag - tagged p53 were subjected to IF analysis ( upper ) . A549 cells transfected with a p53 - specific siRNA or a control siRNA ( siCont . ) were examined by IF analysis , and the RNAi efficiency is shown ( lower ) . Representative images are shown ( magnification , × 40 ) .

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

D, H1299 cells transfected with a Myc-tagged USP7 or a Myc-tagged USP7 together with a Flag-tagged p53 were subjected to IF analysis (upper). A549 cells transfected with a p53-specific siRNA or a control siRNA (siCont.) were examined by IF analysis, and the RNAi efficiency is shown (lower). Representative images are shown (magnification, ×40).


F , IF analysis of A549 cells transfected with a Myc - tagged wild - type USP7 ( Myc - USP7 - WT ) or three Myc - tagged truncated mutants . Representative images are shown ( magnification , × 40 ) .

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

F, IF analysis of A549 cells transfected with a Myc-tagged wild-type USP7 (Myc-USP7-WT) or three Myc-tagged truncated mutants. Representative images are shown (magnification, ×40).


G , H1299 cells were transfected with a Flag - tagged p53 or an empty vector together with a USP7 - specific siRNA or a control siRNA as indicated for 48 h . Cells were then harvested and subjected to a MNase digestion assay .

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

G, H1299 cells were transfected with a Flag-tagged p53 or an empty vector together with a USP7-specific siRNA or a control siRNA as indicated for 48 h. Cells were then harvested and subjected to a MNase digestion assay.


A , Co - IP analysis was performed in 293T cells with antibodies against p53 or H2Bub1 .

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

A, Co-IP analysis was performed in 293T cells with antibodies against p53 or H2Bub1.


Peptide pull - down assay was performed with synthesized

Peptide pull-down assay was performed with synthesized peptides


D , Co - IP analysis for H1299 cells transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) treated with a USP7 - specific inhibitor P5091 .

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

D, Co-IP analysis for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) treated with a USP7-specific inhibitor P5091.


A , Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times .

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

A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times.


B , H1299 cells were transfected with Flag - tagged p53 for 24 h and then treated with 12 μM erastin or untreated for another 24 h . Co - IP analysis was performed with antibodies against Flag .

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

B, H1299 cells were transfected with Flag-tagged p53 for 24 h and then treated with 12 μM erastin or untreated for another 24 h. Co-IP analysis was performed with antibodies against Flag.


C , H1299 cells transfected with Flag - tagged p53 were treated with erastin or untreated for 24 h . Nuclear and cytosolic extracts were differentially prepared and subjected to Western blot analysis .

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

C, H1299 cells transfected with Flag-tagged p53 were treated with erastin or untreated for 24 h. Nuclear and cytosolic extracts were differentially prepared and subjected to Western blot analysis.


D , 293T cells transfected with a p53 - specific siRNA , a USP7 - specific siRNA , or a control siRNA ( siCont . ) were treated with 20 μM erastin or untreated for 24 h as indicated . Western blot analysis was then performed .

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

D, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA, or a control siRNA (siCont.) were treated with 20 μM erastin or untreated for 24 h as indicated. Western blot analysis was then performed.


, qRT - PCR ( above ) and Western blot ( below ) analyses for H1299 cells transfected with a Flag - tagged p53 or an empty plasmid together with a USP7 - specific siRNA or a control siRNA ( siCont . ) as indicated . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats .

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

, qRT-PCR (above) and Western blot (below) analyses for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as indicated. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.


F , ChIP analysis of H1299 cells transfected with a Flag - tagged p53 or an empty plasmid together with a USP7 - specific siRNA or a control siRNA ( siCont . ) as indicated . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats .

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

F, ChIP analysis of H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as indicated. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.


G , H1299 cells transfected with Flag - tagged p53 were treated with 12 μM erastin or untreated for 24 h . ChIP analysis with antibodies against Flag or USP7 was performed . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats .

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

G, H1299 cells transfected with Flag-tagged p53 were treated with 12 μM erastin or untreated for 24 h. ChIP analysis with antibodies against Flag or USP7 was performed. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.


H , 293T cells transfected with a p53 - specific siRNA , a USP7 - specific siRNA or a control were treated with 20 μM erastin or untreated for 24 h . ChIP analysis was performed with anti - H2Bub1 antibodies . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats .

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

H, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA or a control were treated with 20 μM erastin or untreated for 24 h. ChIP analysis was performed with anti-H2Bub1 antibodies. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.


A , H1299 cells transfected with a Flag - tagged p53 or an empty plasmid together with a USP7 - specific siRNA or a control siRNA ( siCont . ) as were treated with 12 μM erastin ( + ) or untreated ( - ) for 24 h as indicated . Representative phase - contrast images were recorded ( magnification , × 20 ) , and the surviving cells were counted . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 .

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

A, H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as were treated with 12 μM erastin (+) or untreated (-) for 24 h as indicated. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05.


B , H1299 cells treated were subjected to transmission electron microscopy . Representative images are shown . Arrow head indicates condensed and marginated chromatin .

O 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 O B-EXP_ASSAY O O O O

B, H1299 cells treated were subjected to transmission electron microscopy. Representative images are shown. Arrow head indicates condensed and marginated chromatin.


C , Lipid ROS levels were examined in cells treated Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 .

O 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

C, Lipid ROS levels were examined in cells treated Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05.


Examples of CT scan images of several metastases ( M032R1 , M032R2 , and M032R5 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O O O O O

Examples of CT scan images of several metastases (M032R1, M032R2, and M032R5).


Volumetric measurements based on the PET and CT scans could be generated from M032 , M032R1 , and M032R5 . Graph illustrates that the metastases R1 and R5 initially expanded before the start of the treatment , but regressed upon vemurafenib treatment . M032R5 showed progressive disease after 4 months , whereas M032R1 still displayed stable disease . M032 was excised and no recurrence was observed .

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

Volumetric measurements based on the PET and CT scans could be generated from M032, M032R1, and M032R5. Graph illustrates that the metastases R1 and R5 initially expanded before the start of the treatment, but regressed upon vemurafenib treatment. M032R5 showed progressive disease after 4 months, whereas M032R1 still displayed stable disease. M032 was excised and no recurrence was observed.


Hematoxylin - eosin ( H & amp ; amp ; E ) and p - ERK stainings on FFPE material of all metastases showed that all vemurafenib - resistant tumors had reactivation of the MAPK pathway . Scale bar represents 100

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

Hematoxylin-eosin (H&amp;E) and p-ERK stainings on FFPE material of all metastases showed that all vemurafenib-resistant tumors had reactivation of the MAPK pathway. Scale bar represents 100 μm


qPCR was performed on gDNA retrieved from each of the metastases , using primers for BRAF and CRAF and normalized on LINE levels . Bars represent the mean of three replicates , error bars indicate standard deviation . The results confirmed that BRAF was amplified in M032R1 , M032R2 , and M032R5 .

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

qPCR was performed on gDNA retrieved from each of the metastases, using primers for BRAF and CRAF and normalized on LINE levels. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in M032R1, M032R2, and M032R5.


Staining for BRAFV600E with a mutant epitope - specific antibody confirmed the upregulation of BRAFV600E in R1 , R2 , and R5 . Scale bar represents 100 μm .

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 O O O O

Staining for BRAFV600E with a mutant epitope-specific antibody confirmed the upregulation of BRAFV600E in R1, R2, and R5. Scale bar represents 100 μm.


Immunoblotting for MAPK pathway components confirmed reactivation of p - ERK and p - MEK in all resistant metastases .

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

Immunoblotting for MAPK pathway components confirmed reactivation of p-ERK and p-MEK in all resistant metastases.


Immunoblotting for M032 patient ' s samples using an antibody directed against either the mutant BRAFV600E epitope or an N - terminal region of BRAF revealed the expression of a shorter variant of BRAFV600E in M032R3 , of approximately 80 kDa . This 80 - kDa band could not be detected with the N - terminus - recognizing antibody .

B-EXP_ASSAY O 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 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

Immunoblotting for M032 patient's samples using an antibody directed against either the mutant BRAFV600E epitope or an N-terminal region of BRAF revealed the expression of a shorter variant of BRAFV600E in M032R3, of approximately 80 kDa. This 80-kDa band could not be detected with the N-terminus-recognizing antibody.


Validation of T55delinsRT was done by PCR using insertion - specific primers . As a control , primers amplifying exon 2 of MEK1WT were used .

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

Validation of T55delinsRT was done by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1WT were used.


Transfection of pQXCIP - GFP , MEK1 - WT , or MEK1 - T55delinsRT into HEK293T cells showed that this mutation induces p - ERK and p - RSK .

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

Transfection of pQXCIP-GFP, MEK1-WT, or MEK1-T55delinsRT into HEK293T cells showed that this mutation induces p-ERK and p-RSK.


Validation of expression of pQXCIP - GFP , MEK1WT , or MEK1T55delinsRT in A375 melanoma cells by immunoblotting .

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

Validation of expression of pQXCIP-GFP, MEK1WT, or MEK1T55delinsRT in A375 melanoma cells by immunoblotting.


Dose - response curves for A375 melanoma cells expressing GFP , MEK1WT , or MEK1T55delinsRT with indicated doses of BRAF inhibitor vemurafenib , ERK inhibitor SCH772984 , or MEK inhibitor trametinib . Error bars indicate standard deviation .

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

Dose-response curves for A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT with indicated doses of BRAF inhibitor vemurafenib, ERK inhibitor SCH772984, or MEK inhibitor trametinib. Error bars indicate standard deviation.


Colony formation assays with A375 melanoma cells , infected with GFP , MEK1WT , or MEK1T55delinsRT - encoding lentivirus , and treated with indicated doses and inhibitors .

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

Colony formation assays with A375 melanoma cells, infected with GFP, MEK1WT, or MEK1T55delinsRT-encoding lentivirus, and treated with indicated doses and inhibitors.


Treatment of A375 MEK1T55delinsRT melanoma cells with DMSO ( − ) , 250 nM dabrafenib ( D ) , 10 nM trametinib ( T ) , or a combination ( D + T ) .

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

Treatment of A375 MEK1T55delinsRT melanoma cells with DMSO (−), 250 nM dabrafenib (D), 10 nM trametinib (T), or a combination (D+T).


A375 melanoma cells expressing GFP , MEK1WT , or MEK1T55delinsRT were injected into immune - deficient mice ( n = 8 per group ) , and after the tumor size of ˜ 100 mm3 was reached , mice were treated with 30 mg / kg dabrafenib or vehicle . Graphs represent fold change in tumor volume normalized on the tumor volume on the day of the start of the treatment . Error bars indicate standard error of the mean .

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

A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT were injected into immune-deficient mice (n = 8 per group), and after the tumor size of ˜100 mm3 was reached, mice were treated with 30 mg/kg dabrafenib or vehicle. Graphs represent fold change in tumor volume normalized on the tumor volume on the day of the start of the treatment. Error bars indicate standard error of the mean.


Average tumor weight of all groups at the end of the experiment described in ( A ) . Error bars indicate standard deviation . Unpaired t - test was used to calculate statistical significance . P - values are indicated in the graph .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Average tumor weight of all groups at the end of the experiment described in (A). Error bars indicate standard deviation. Unpaired t-test was used to calculate statistical significance. P-values are indicated in the graph.


Immunoblotting for MAPK pathway components performed on tumors of all groups .

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

Immunoblotting for MAPK pathway components performed on tumors of all groups.


Immunohistochemistry for Sirius Red ( staining for collagen ) and H & amp ; amp ; E on tumors from all groups , to identify grade of demarcation and local invasion . Scale bar represents 500 ( upper row ) and 100 ( lower row ) μm .

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

Immunohistochemistry for Sirius Red (staining for collagen) and H&amp;E on tumors from all groups, to identify grade of demarcation and local invasion. Scale bar represents 500 (upper row) and 100 (lower row) μm.


gDNA was isolated from another fragment of the tumor resection , and the PCR with the insertion - specific primers was repeated . Primers amplifying exon 2 of MEK1WT were used as an input control . Source data are available online for this figure .

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

gDNA was isolated from another fragment of the tumor resection, and the PCR with the insertion-specific primers was repeated. Primers amplifying exon 2 of MEK1WT were used as an input control.Source data are available online for this figure.


Staining for hematoxylin - eosin ( H & amp ; amp ; E ) and p - ERK on the PDX samples M032 . X1 , M032R1 . X1 , M032R2 . X1 , M032R4 . X1 , and M032R5 . X1 . Stainings showed that p - ERK is higher in the PDX derived from the resistant metastases . Scale bar represents 100 μm .

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

Staining for hematoxylin-eosin (H&amp;E) and p-ERK on the PDX samples M032.X1, M032R1.X1, M032R2.X1, M032R4.X1, and M032R5.X1. Stainings showed that p-ERK is higher in the PDX derived from the resistant metastases. Scale bar represents 100 μm.


Immunoblotting for components of the MAPK pathway confirmed reactivation of p - ERK in the PDX derived from the vemurafenib - resistant metastases , although the p - ERK signal is heterogeneous in the pre - treatment PDX .

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

Immunoblotting for components of the MAPK pathway confirmed reactivation of p-ERK in the PDX derived from the vemurafenib-resistant metastases, although the p-ERK signal is heterogeneous in the pre-treatment PDX.


Validation of the BRAF amplification was performed by qPCR on gDNA derived from the PDX , using primers for BRAF and CRAF and normalized on LINE expression . Bars represent the mean of three replicates , error bars indicate standard deviation . The results confirmed that BRAF was amplified in PDX derived from M032R2 and M032R5 , but not from M032R1 .

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

Validation of the BRAF amplification was performed by qPCR on gDNA derived from the PDX, using primers for BRAF and CRAF and normalized on LINE expression. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in PDX derived from M032R2 and M032R5, but not from M032R1.


Presence of the MEK1T55delinsRT was analyzed by PCR using insertion - specific primers . As a control , primers amplifying exon 2 of MEK1WT were used . The insertion was found in M032R1 . X1 and M032R4 . X1 .

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

Presence of the MEK1T55delinsRT was analyzed by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1WT were used. The insertion was found in M032R1.X1 and M032R4.X1.


A - C Calcium flux measured by FACScan for splenic B cells isolated from B1 - 8 transgenic mice after stimulation with ( A ) NIP15 - BSA ( 30 pM ) or 1NIP - pep ( see Figure EV1 , 80 nM ) ; ( B ) Ac146 antibody ( 12 . 5 nM ) or Ac146Fab ( 25 nM ) ; ( C ) Ac38 antibody ( 12 . 5 nM ) or Ac38Fab ( 25 nM ) . The addition of the stimuli to the cells is indicated by arrows .

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

A-C Calcium flux measured by FACScan for splenic B cells isolated from B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (see Figure EV1, 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows.


D Representative microscopy images showing Fab - PLA results measuring the BCR proximity for the IgM - BCR ( upper ) and the IgD - BCR ( lower ) on untreated or treated B1 - 8 splenic B cells . PLA signals are shown as red dots and the nuclei are visualized by DAPI staining . Scale bar : 5 μm . E The Fab - PLA results are quantified by Blobfinder software and presented as box plots . The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample . Data from treated samples were compared to data from resting cells , p - values were calculated by Kruskal - Wallis one - way analysis of variance ( ANOVA ) .

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

D Representative microscopy images showing Fab-PLA results measuring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. PLA signals are shown as red dots and the nuclei are visualized by DAPI staining. Scale bar: 5 μm.E The Fab-PLA results are quantified by Blobfinder software and presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from treated samples were compared to data from resting cells, p-values were calculated by Kruskal-Wallis one-way analysis of variance (ANOVA).


A - C Calcium flux measured by FACScan for splenic B cells isolated from Lyn - deficient B1 - 8 transgenic mice after stimulation with ( A ) NIP15 - BSA ( 30 pM ) or 1NIP - pep ( 80 nM ) ; ( B ) Ac146 antibody ( 12 . 5 nM ) or Ac146Fab ( 25 nM ) ; ( C ) Ac38 antibody ( 12 . 5 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells .

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

A-C Calcium flux measured by FACScan for splenic B cells isolated from Lyn-deficient B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


D Representative microscopy images showing Fab - PLA results monitoring the BCR proximity for the IgM - BCR ( upper ) and the IgD - BCR ( lower ) on untreated or treated B1 - 8 splenic B cells . Scale bar : 5 μm . E Quantified Fab - PLA results are presented as box plots , where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample . p - values were calculated by Kruskal - Wallis one - way ANOVA .

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

D Representative microscopy images showing Fab-PLA results monitoring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. Scale bar: 5 μm.E Quantified Fab-PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. p-values were calculated by Kruskal-Wallis one-way ANOVA.


A - D Proximity between IgM - BCR on the surface of 3046SM ( A , C ) or 3046M ( B , D ) cells before and after a 1 - min stimulation with the indicated reagents , assayed by Fab - PLA . Results are presented as representative microscopy images ( A , B ) and box plots after quantification ( C , D ) . Scale bar : 5 μm . In the box plots , the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample and p - values were calculated by Kruskal - Wallis one - way ANOVA .

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

A-D Proximity between IgM-BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1-min stimulation with the indicated reagents, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.


E - H Calcium flux measured by FACScan for 3046SM ( E , F ) and 3046M ( G , H ) cells after stimulation with NIP15 - BSA ( 30 pM ) , 1NIP - pep ( 80 nM ) , Ac146Fab ( 25 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells .

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

E-H Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


A - D Proximity between IgD - BCRs on the surface of 3046SD ( A , C ) or 3046D ( B , D ) cells before and after a 1 - min stimulation , assayed by Fab - PLA . Results are presented as representative microscopy images ( A , B ) and box plots after quantification ( C , D ) . PLA signals are shown as red dots and nuclei were visualized by DAPI staining . Scale bar : 5 μm . In the box plots , the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample and p - values were calculated by Kruskal - Wallis one - way ANOVA .

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

A-D Proximity between IgD-BCRs on the surface of 3046SD (A, C) or 3046D (B, D) cells before and after a 1-min stimulation, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.


E - H Calcium flux measured by FACScan for 3046SD ( E , F ) and 3046D ( G , H ) cells after stimulation with NIP15 - BSA ( 30 pM ) , 1NIP - pep ( 80 nM ) , Ac146Fab ( 25 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells .

E-H Calcium flux measured by FACScan for 3046SD (E, F) and 3046D (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


Calcium flux measured by FACScan for 3046M ( A , D ) , 3046SD ( B , E ) and 3046D ( C , F ) cells stimulated with anti - HC ( 2 μl / ml ) ( A , B , C ) , or anti - λ ( 2 μl / ml ) ( D , E , F ) antibodies with or without 1 min prestimulation with 1NIP - pep ( 80 nM ) . The addition of antibodies is indicated by black arrows . The 1NIP - pep was added immediately before recording . Data are representative of three independent experiments .

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

Calcium flux measured by FACScan for 3046M (A, D), 3046SD (B, E) and 3046D (C, F) cells stimulated with anti-HC (2 μl/ml) (A, B, C), or anti-λ (2 μl/ml) (D, E, F) antibodies with or without 1 min prestimulation with 1NIP-pep (80 nM). The addition of antibodies is indicated by black arrows. The 1NIP-pep was added immediately before recording. Data are representative of three independent experiments.


A - E B1 - 8 B cells , 3046SM cells , 3046M cells , 3046SD cells , or 3046D cells were stimulated with 1NIP - pep ( 80 nM ) , or anti - CD19 antibody ( 62 nM ) , or costimulated with 1NIP - pep and anti - CD19 antibody . The calcium fluxes were measured by FACScan .

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

A-E B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD19 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD19 antibody. The calcium fluxes were measured by FACScan.


F - J B1 - 8 B cells , 3046SM cells , 3046M cells , 3046SD cells , or 3046D cells were stimulated with 1NIP - pep ( 80 nM ) , or anti - CD20 antibody ( 62 nM ) , or costimulated with 1NIP - pep and anti - CD20 antibody . The calcium fluxes were measured by FACScan .

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

F-J B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD20 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD20 antibody. The calcium fluxes were measured by FACScan.


API delivery is blocked at low temperatures . SEY6210 cells were pulse labeled for 5 min and chased for the times indicated . Identical experiments were performed at 10 ° , 14 ° , and 30 ° C . API was recovered by immunoprecipitation followed by SDS - PAGE and detected by a STORM phosphorimager ( Molecular Dynamics , Sunnyvale , CA ) . The positions of prAPI and mAPI are indicated .

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

API delivery is blocked at low temperatures. SEY6210 cells were pulse labeled for 5 min and chased for the times indicated. Identical experiments were performed at 10°, 14°, and 30°C. API was recovered by immunoprecipitation followed by SDS-PAGE and detected by a STORM phosphorimager (Molecular Dynamics, Sunnyvale, CA). The positions of prAPI and mAPI are indicated.


Precursor API is trapped in a prevacuolar compartment in vps18 cells . ( A ) Yeast strain JSR18Δ1 with plasmid pJSR9 ( vps18 ts ) was grown at the permissive temperature of 26 ° C . Before labeling , the cells were incubated at either 26 ° or 38 ° C for 10 min . Pulse labeling was for 5 min , followed by the indicated chase times .

O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM 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

Precursor API is trapped in a prevacuolar compartment in vps18 cells. (A) Yeast strain JSR18Δ1 with plasmid pJSR9 (vps18 ts) was grown at the permissive temperature of 26°C. Before labeling, the cells were incubated at either 26° or 38°C for 10 min. Pulse labeling was for 5 min, followed by the indicated chase times.


( B ) vps18 spheroplasts were shifted to 38 ° C for 5 min , pulse labeled for 5 min , and then chased for either 0 or 60 min . At each time point the spheroplasts were collected by centrifugation and resuspended in PS0 buffer . The Total ( T ) fraction either received no treatment , was treated with 50 μg / ml proteinase K , or received proteinase K in the presence of 0 . 2 % Triton X - 100 . The supernatant ( S0 ) and pellet ( P0 ) fractions were collected after centrifugation at 10 , 000 g for 5 min .

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

(B) vps18 spheroplasts were shifted to 38°C for 5 min, pulse labeled for 5 min, and then chased for either 0 or 60 min. At each time point the spheroplasts were collected by centrifugation and resuspended in PS0 buffer. The Total (T) fraction either received no treatment, was treated with 50 μg/ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100. The supernatant (S0) and pellet (P0) fractions were collected after centrifugation at 10,000 g for 5 min.


A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment . ( A ) Spheroplasts from an ape1Δ strain containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer . The Total ( T ) fraction ( after differential lysis ) either received no treatment , was treated with 50 μg / ml proteinase K , or received proteinase K in the presence of 0 . 2 % Triton X - 100 .

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

A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment. (A) Spheroplasts from an ape1Δ strain containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer. The Total (T) fraction (after differential lysis) either received no treatment, was treated with 50 μg/ ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100.


( B ) The supernatant ( S200 ) and pellet ( P200 ) fractions were collected after centrifugation at 5 , 000 g for 5 min . The P200 fraction was resuspended in 10 % Ficoll , and vacuoles were isolated by flotation through 4 % Ficoll . V , vacuole ; I , 4 / 10 % Ficoll interface ; P , gradient pellet . Proteins were detected by Western blotting . The positions of prAPI , mAPI , and mPrA are indicated .

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

(B) The supernatant (S200) and pellet (P200) fractions were collected after centrifugation at 5,000 g for 5 min. The P200 fraction was resuspended in 10% Ficoll, and vacuoles were isolated by flotation through 4% Ficoll. V, vacuole; I, 4/10% Ficoll interface; P, gradient pellet. Proteins were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.


( C ) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min , harvested by centrifugation , and subjected to a nonradioactive chase in either nitrogen - containing ( SMD ) or nitrogen starvation ( SD - N ) medium . Samples were collected at the times indicated and immunoprecipitated with API antiserum . The resulting SDS - PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager .

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

(C) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min, harvested by centrifugation, and subjected to a nonradioactive chase in either nitrogen-containing (SMD) or nitrogen starvation (SD-N) medium. Samples were collected at the times indicated and immunoprecipitated with API antiserum. The resulting SDS-PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager.


Precursor API is trapped within a vesicle in cvt17 mutants . ( A ) Precursor API is in a protease - protected compartment in cvt17 mutants . THY32 ( cvt17 ) spheroplasts were subjected to differential lysis and protease treatment in PS200 buffer as in Fig

O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O

Precursor API is trapped within a vesicle in cvt17 mutants. (A) Precursor API is in a protease-protected compartment in cvt17 mutants. THY32 (cvt17) spheroplasts were subjected to differential lysis and protease treatment in PS200 buffer as in Fig 3


B ) Precursor API is not free in the vacuolar lumen in cvt17 mutants . Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant ( S0 ) and pellet ( P0 ) fractions by centrifugation at 10 , 000 g . The S0 and P0 fractions were treated with proteinase K in the presence and absence of Triton X - 100 as indicated . All fractions were precipitated with 10 % TCA and resolved by SDS PAGE ; proteins of interest were detected by Western blotting . The positions of prAPI , mAPI , and mPrA are indicated .

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

B) Precursor API is not free in the vacuolar lumen in cvt17 mutants. Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant (S0) and pellet (P0) fractions by centrifugation at 10,000 g. The S0 and P0 fractions were treated with proteinase K in the presence and absence of Triton X-100 as indicated. All fractions were precipitated with 10% TCA and resolved by SDS PAGE; proteins of interest were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.


Precursor API is contained within subvacuolar vesicles in cvt17 mutants . ( A ) Isolated vacuoles ( V ) from cvt17 cells were lysed in PS0 buffer , loaded on top of an Optiprep step gradient , and centrifuged at 170 , 000 g for 60 min as described in Materials and Methods . Fractions VV , 0 / 1 . 06 interface ; LD , 1 . 06 region ; SV , 1 . 06 / 1 . 12 interface ; HD , 1 . 12 region .

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

Precursor API is contained within subvacuolar vesicles in cvt17 mutants. (A) Isolated vacuoles (V) from cvt17 cells were lysed in PS0 buffer, loaded on top of an Optiprep step gradient, and centrifuged at 170,000 g for 60 min as described in Materials and Methods. Fractions VV, 0/1.06 interface; LD, 1.06 region; SV, 1.06/1.12 interface; HD, 1.12 region.


( B ) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X - 100 as indicated . Because of the presence of protease inhibitors in the vesicle isolation procedure , an intermediate - sized API degradation product results from proteinase K treatment . The V fraction represents one - tenth of the load of the Optiprep gradient . Proteins were detected by Western blotting . The positions of prAPI and mALP are indicated .

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

(B) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X-100 as indicated. Because of the presence of protease inhibitors in the vesicle isolation procedure, an intermediate-sized API degradation product results from proteinase K treatment. The V fraction represents one-tenth of the load of the Optiprep gradient. Proteins were detected by Western blotting. The positions of prAPI and mALP are indicated.


Immuno - EM of cvt17 cells . Yeast strains SEY6210 ( parental wild type ) and THY32 ( cvt17 ) were grown in YPD and prepared for microscopy as described in Materials and Methods . ( A ) Wild - type cells probed with antibody against mature API . ( B ) A cvt17 section showing a cytosolic vesicle containing prAPI probed with antibody against the API proregion . ( C ) A cvt17 section showing subvacuolar vesicles containing API probed with antibody against mature API . N , nucleus ; V , vacuole ; ( arrows ) API containing vesicles . Bars , 0 . 5 μm .

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

Immuno-EM of cvt17 cells. Yeast strains SEY6210 (parental wild type) and THY32 (cvt17) were grown in YPD and prepared for microscopy as described in Materials and Methods. (A) Wild-type cells probed with antibody against mature API. (B) A cvt17 section showing a cytosolic vesicle containing prAPI probed with antibody against the API proregion. (C) A cvt17 section showing subvacuolar vesicles containing API probed with antibody against mature API. N, nucleus; V, vacuole; (arrows) API containing vesicles. Bars, 0.5 μm.


Characterization of API - containing vesicles . ( A ) Fractionations were performed as in Fig . 5 . The total ( T ) , vacuole ( V ) , vacuolar vesicle ( VV ) , and subvacuolar vesicle ( SV ) fractions are shown . Proteins were detected by immunoblotting as indicated in the figure .

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

Characterization of API-containing vesicles. (A) Fractionations were performed as in Fig. 5. The total (T), vacuole (V), vacuolar vesicle (VV), and subvacuolar vesicle (SV) fractions are shown. Proteins were detected by immunoblotting as indicated in the figure.


( B ) SYPRO orange staining detected by fluorescent scanning mode on a Molecular Dynamics STORM phosphorimager . The relative mobility of the molecular mass standards are indicated on the left . Polypeptides enriched in the subvacuolar vesicles are indicated on the right .

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

(B) SYPRO orange staining detected by fluorescent scanning mode on a Molecular Dynamics STORM phosphorimager. The relative mobility of the molecular mass standards are indicated on the left. Polypeptides enriched in the subvacuolar vesicles are indicated on the right.


C Maximal cytochrome c release for all DX / REL pairs with ≥ 3 biological replicates ) ; box - whisker plots summarize data ( box 25 % - 75 % ; belt = median ; dot = mean ; whiskers = minimum and maximum values ) . Statistical analyses were performed using an unpaired two - sided Student ' s t - test , with significance p < 0 . 05 ( trend p < 0 . 10 ) . Cyto c , cytochrome

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

C Maximal cytochrome c release for all DX/REL pairs with ≥3 biological replicates); box-whisker plots summarize data (box 25%-75%; belt=median; dot=mean; whiskers=minimum and maximum values). Statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend p<0.10). Cyto c, cytochrome c


D Bak oligomerization in response to escalating tBid concentration for DX / REL pairs . Relative mitochondrial protein loading per lane is assessed by densitometry , showing ration of loading control in REL lane with patient - matched DX lane at same tBid exposure ( no REL lane is underloaded compared to DX lane ) .

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

D Bak oligomerization in response to escalating tBid concentration for DX/REL pairs. Relative mitochondrial protein loading per lane is assessed by densitometry, showing ration of loading control in REL lane with patient-matched DX lane at same tBid exposure (no REL lane is underloaded compared to DX lane).


A In vitro viability curves after 72hr exposure to mafosfamide or doxorubicin . Results are shown for three DX / REL neuroblastoma cell line pairs , two with attenuated cytochrome c release ( CHLA15 / CHLA20 and SKNBE1 / SKNBE2C ) and one without ( CHLA122 / CHLA136 ) . Data information : data points are mean and SD from triplicate wells , experiments are representative of at least three biological replicates dotted - line represents 50 % viability .

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

A In vitro viability curves after 72hr exposure to mafosfamide or doxorubicin. Results are shown for three DX/REL neuroblastoma cell line pairs, two with attenuated cytochrome c release (CHLA15/CHLA20 and SKNBE1/SKNBE2C) and one without (CHLA122/CHLA136). Data information: data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates dotted-line represents 50% viability.


C , In vitro viability for CHLA15 ( DX ) and CHLA20 ( REL ) following ionizing radiation at 7 days Data information : data points are mean and SD from triplicate wells , experiments are representative of at least three biological replicates , dotted - line represents 50 % viability .

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

C, In vitro viability for CHLA15 (DX) and CHLA20 (REL) following ionizing radiation at 7 days Data information: data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates, dotted-line represents 50% viability.


F Cytochrome c release from mitochondria after exposure to tBid or BimBH3 peptide for parental NB1643 and SY5Y cells , in comparison to cells cultured in escalating concentrations of crizotinib until resistant ( NB1643 - ALKR and SY5Y - ALKR ) . Data information : data points are mean of duplicate wells ( SD < 0 . 05 at all points ) in a representative experiment from at least two biological replicates .

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

F Cytochrome c release from mitochondria after exposure to tBid or BimBH3 peptide for parental NB1643 and SY5Y cells, in comparison to cells cultured in escalating concentrations of crizotinib until resistant (NB1643-ALKR and SY5Y-ALKR). Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.


A , B , C Transmission electron microscopy image analysis was used to quantify mitochondrial size , circularity and roundness in DX / REL neuroblastoma pairs ( mean + / - SD shown ) . Data information : , n = 137 - 241 mitochondria per cell line statistical analyses were performed using a two - tailed Mann - Whitney U test , with significance p < 0 . 05 ( trend p < 0 . 10 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O O O O O B-DISEASE 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

A, B, C Transmission electron microscopy image analysis was used to quantify mitochondrial size, circularity and roundness in DX/REL neuroblastoma pairs (mean +/- SD shown). Data information: , n=137-241 mitochondria per cell line statistical analyses were performed using a two-tailed Mann-Whitney U test, with significance p<0.05 (trend p<0.10).


H In vitro viability of CHLA15 - ctrl , CHLA15 - shMFN2 , CHLA15 - shPACS2 , and CHLA20 cells following 72hr exposure to ABT - 737 ; dotted - line represents 50 % viability . Data information : data points are mean and SD from triplicate wells , experiments are representative of at least two biological replicates .

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

H In vitro viability of CHLA15-ctrl, CHLA15-shMFN2, CHLA15-shPACS2, and CHLA20 cells following 72hr exposure to ABT-737; dotted-line represents 50% viability. Data information: data points are mean and SD from triplicate wells, experiments are representative of at least two biological replicates.


I Mitochondrial cytochrome c release in response to tBid and BimBH3 peptide in CHLA15 - Ctrl , CHLA15 - shMFN2 and CHLA15 - shPACS2 cells . Data information : , data points are mean of duplicate wells ( SD < 0 . 05 at all points ) in a representative experiment from at least two biological

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

I Mitochondrial cytochrome c release in response to tBid and BimBH3 peptide in CHLA15-Ctrl, CHLA15-shMFN2 and CHLA15-shPACS2 cells. Data information: , data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates


A , B Cytosolic ( black - tracing ) and mitochondrial ( colored - tracing ) Ca2 + concentration measured using fluorescent reporters in the CHLA15 ( n = 29 cells ) / CHLA20 ( n = 65 cells ) and SKNBE1 ( n = 37 cells ) / SKNBE2C ( n = 52 cells ) pairs . ER Ca2 + release was induced by 100μM carbachol , an IP3R - agonist ; time added indicated by arrow ; tracings are mean + / - SD error bars . Coupling time ( time between achieving 50 % of maximal cytosolic and mitochondrial concentrations ) is an index of ER - mitochondrial proximity and transfer efficiency ; plotted below . Data information : data is derived from at least 3 separate cell transfections ( biological replicates ) . statistical analyses were performed using an unpaired two - sided Student ' s t - test , with significance p < 0 . 05 .

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

A, B Cytosolic (black-tracing) and mitochondrial (colored-tracing) Ca2+ concentration measured using fluorescent reporters in the CHLA15 (n=29 cells)/CHLA20 (n=65 cells) and SKNBE1 (n=37 cells)/SKNBE2C (n=52 cells) pairs. ER Ca2+ release was induced by 100μM carbachol, an IP3R-agonist; time added indicated by arrow; tracings are mean +/- SD error bars. Coupling time (time between achieving 50% of maximal cytosolic and mitochondrial concentrations) is an index of ER-mitochondrial proximity and transfer efficiency; plotted below. Data information: data is derived from at least 3 separate cell transfections (biological replicates). statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05.


F Cytochrome c release in response to tBid and BimBH3 assessed . Data information : data points are mean of duplicate wells ( SD < 0 . 05 at all points ) in a representative experiment from at least two biological replicates .

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

F Cytochrome c release in response to tBid and BimBH3 assessed. Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.


A , B , C Concentration of ceramide species as measured by LC / MS from DX and REL pair whole cell pellets . Data information : show mean of 3 - 4 biological replicates plotted ; 3 technical replicates

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

A, B, C Concentration of ceramide species as measured by LC/MS from DX and REL pair whole cell pellets. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each


D , E Cumulative ceramides and sphingomyelins ( of C32 - C36 chain length ) for DX / REL pairs . Data information : show mean of 3 - 4 biological replicates plotted ; 3 technical replicates each error bars are + / - SD .

O O O O B-SMALL_MOLECULE 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 O

D, E Cumulative ceramides and sphingomyelins (of C32-C36 chain length) for DX/REL pairs. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each error bars are +/- SD.


H Summary data for CHLA15 / CHLA20 and SKNBE1 / SKNBE2C showing relative cytochrome c release when pre - incubated with GW4869 to inhibit ceramide generation , compared with untreated cells . Data information : All data points from two ( CHLA15 ) or three ( SKNBE1 ) biological replicates are depicted in H . Statistical analyses were performed using an unpaired two - sided Student ' s t - test , with significance p < 0 . 05 ( trend , p < 0 . 10 ) .

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

H Summary data for CHLA15/CHLA20 and SKNBE1/SKNBE2C showing relative cytochrome c release when pre-incubated with GW4869 to inhibit ceramide generation, compared with untreated cells. Data information: All data points from two (CHLA15) or three (SKNBE1) biological replicates are depicted in H. Statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend, p<0.10).


A - D Mesenteric blood ( PECAM1 , red ) and lymphatic vessels ( LYVE1 , green ; PROX1 , gray ) . Asterisks indicate lymphatic valves , arrow indicates a lymphatic vessel stub , and arrowhead indicates an isolated lymphatic vessel fragmentE - H Blood vessels ( PECAM1 , green ) and lymphatic vessels ( VEGFR - 3 , red ) in the small intestinal wall .

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

A-D Mesenteric blood (PECAM1, red) and lymphatic vessels (LYVE1, green; PROX1, gray). Asterisks indicate lymphatic valves, arrow indicates a lymphatic vessel stub, and arrowhead indicates an isolated lymphatic vessel fragmentE-H Blood vessels (PECAM1, green) and lymphatic vessels (VEGFR-3, red) in the small intestinal wall.


I , J Detection of chylous ascites ( arrows ) in the VCiΔR26 mice at P6 .

O O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O

I, J Detection of chylous ascites (arrows) in the VCiΔR26 mice at P6.


K , L LYVE1 staining of intestinal lymphatic vessels at P6 .

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

K, L LYVE1 staining of intestinal lymphatic vessels at P6.


M - T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice . Genotypes and deletion lengths are indicated in ( U ) . U Quantification of LYVE1 areas in ( M - T ) . Length of the i ∆ Vegfc gene deletion is indicated in months ( mo ) , and Vegfd indicates the VEGF - D genotype . Data are represented as mean ± SEM . Significant differences were determined using one - way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in ( M ) . * P = 0 . 003 , * * P = 0 . 001 , * * * P = 0 . 0008 , ♯ P = 0 . 0002 , § P = 0 . 0001 .

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

M-T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice. Genotypes and deletion lengths are indicated in (U).U Quantification of LYVE1 areas in (M-T). Length of the i∆Vegfc gene deletion is indicated in months (mo), and Vegfd indicates the VEGF-D genotype. Data are represented as mean ± SEM. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in (M). *P = 0.003, **P = 0.001, ***P = 0.0008, ♯P = 0.0002, §P = 0.0001.


Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in ( B - F ) ; ( I ) for the entire villus and ( II ) for the villus base . β - Gal staining pattern of the villus in wild - type ( Ctrl ) , Vegfc / LacZ ( VC ) , Vegfr3 / LacZ ( VR - 3 ) , and Vegfr2 / LacZ ( VR - 2 ) mice . Higher magnification images representing β - Gal staining of the villus base in Vegfc / LacZmice . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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

Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in (B-F); (I) for the entire villus and (II) for the villus base. β-Gal staining pattern of the villus in wild-type (Ctrl), Vegfc/LacZ (VC), Vegfr3/LacZ (VR-3), and Vegfr2/LacZ (VR-2) mice.Higher magnification images representing β-Gal staining of the villus base in Vegfc/LacZmice.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Vegfc / LacZβ - Gal staining reaction with smooth muscle actin ( SMA ) peroxidase staining . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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

Vegfc/LacZβ-Gal staining reaction with smooth muscle actin (SMA) peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Vegfc / LacZβ - Gal staining and LYVE1 peroxidase staining . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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

Vegfc/LacZβ-Gal staining and LYVE1 peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Surface image of Vegfc / LacZβ - Gal - stained intestine ( left ) and cross section counterstaining with PECAM1 ( right ) . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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

Surface image of Vegfc/LacZβ-Gal-stained intestine (left) and cross section counterstaining with PECAM1 (right).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Immunofluorescence staining of lacteal lymphatic vessel ( LYVE1 ) , blood capillaries ( PECAM1 ) , and smooth muscle cells ( SMA ) . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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

Immunofluorescence staining of lacteal lymphatic vessel (LYVE1), blood capillaries (PECAM1), and smooth muscle cells (SMA).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Body weight change during seven weeks of HFD , expressed as average fold change in comparison with the starting weight . n = 16 , WT ; n = 6 , Vegfd − / − ; n = 16 , VCiΔR26 ; n = 5 , Vegfd − / − ; VCiΔR26 . Body weight comparisons at seven weeks of HFD . Significant differences were determined using one - way ANOVA and Bonferroni post hoc analysis compared to WT . * P = 0 . 004 ; * * P = 0 . 003 . n = 16 , WT ; n = 6 , Vegfd − / − ; n = 16 , VCiΔR26 ; n = 5 , Vegfd − / − ; VCiΔR26 .

O O O O O B-ORGANISM 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 O O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-GENEPROD 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 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 B-ORGANISM O O O O O B-GENEPROD O O O O B-ORGANISM O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Body weight change during seven weeks of HFD, expressed as average fold change in comparison with the starting weight. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.Body weight comparisons at seven weeks of HFD. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT. *P = 0.004; **P = 0.003. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Glucose tolerance test ( GTT ) after six weeks of HFD . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 014 ; * * P = 0 . 041 . n = 5 , WT ; n = 6 , VCiΔR26 .

O O O O O B-ORGANISM O B-SMALL_MOLECULE 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 O O O O O 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-ORGANISM O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Glucose tolerance test (GTT) after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.014; **P = 0.041. n = 5, WT; n = 6, VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Total fatweight , fat percentage from body composition measurements after six weeks of HFD , and weights of visceral fat ( VF ) and subcutaneous fat ( SF ) at the time of necropsy . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 006 ; * * P = 0 . 001 ; # P = 0 . 008 ; § P = 0 . 006 . n = 4 in each group .

O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE I-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 O O O O O O O O O O O O O O O O O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Total fatweight, fat percentage from body composition measurements after six weeks of HFD, and weights of visceral fat (VF) and subcutaneous fat (SF) at the time of necropsy. Significant differences were determined using unpaired two-tailed t-test. *P = 0.006; **P = 0.001; #P = 0.008; §P = 0.006. n = 4 in each group.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Food consumption during the fifth week of HFD . n = 9 , WT ; n = 10 , VCiΔR26 .

O O O O O B-ORGANISM O B-SMALL_MOLECULE 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 B-ORGANISM O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Food consumption during the fifth week of HFD. n = 9, WT; n = 10, VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Whole - mount immunofluorescence staining of blood ( PECAM1 , red ) and lymphatic vessels ( LYVE1 , green ) in intestinal villi and intestinal wall . Quantification of the lacteal and villus length ( solid and striped color bars , respectively ) and the intestinal wallLYVE1 + area percentage from images represented in ( F ) . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 0002 ; * * P = 0 . 00007 . n = 5 , WT ; n = 6 , VCiΔR26 .

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

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Whole-mount immunofluorescence staining of blood (PECAM1, red) and lymphatic vessels (LYVE1, green) in intestinal villi and intestinal wall.Quantification of the lacteal and villus length (solid and striped color bars, respectively) and the intestinal wallLYVE1+ area percentage from images represented in (F). Significant differences were determined using unpaired two-tailed t-test. *P = 0.0002; **P = 0.00007. n = 5, WT; n = 6, VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Free fatty acid ( FFA ) and cholesterol measurements from the feces after six weeks of HFD . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 001 ; * * P = 0 . 007 . n = 5 , WT ; n = 6 , VCiΔR26 .

O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 O O O B-ORGANISM O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Free fatty acid (FFA) and cholesterol measurements from the feces after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.001; **P = 0.007. n = 5, WT; n = 6, VCiΔR26.


D , RT - qPCR time courses . Promoters of the indicated oscillating genes were used to drive expression of a destabilized , nuclear GFP protein from a single copy integrated transgene ; gfp mRNA and the endogenous transcript driven by the same promoter were quantified from the same RNA samples . Relative expression was calculated as - dCT = - ( target CT values - actin CT values ) and then mean normalized for each trace individually . Peak phases ( ϕPeak ) and amplitudes ( A ) for the endogenous transcripts are from ( Meeuse et al . , 2020 ) . qPCR was performed in technical replicate , shown are averages .

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

D, RT-qPCR time courses. Promoters of the indicated oscillating genes were used to drive expression of a destabilized, nuclear GFP protein from a single copy integrated transgene; gfp mRNA and the endogenous transcript driven by the same promoter were quantified from the same RNA samples. Relative expression was calculated as -dCT = - (target CT values - actin CT values) and then mean normalized for each trace individually. Peak phases (ϕPeak) and amplitudes (A) for the endogenous transcripts are from (Meeuse et al., 2020). qPCR was performed in technical replicate, shown are averages.


B , C Heatmap showing trend - corrected luminescence as in ( A ) , for indicated RNAi conditions causing altered numbers ( B ) or durations ( C ) of molts , respectively .

O O O O O O 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

B, C Heatmap showing trend-corrected luminescence as in (A), for indicated RNAi conditions causing altered numbers (B) or durations (C) of molts, respectively.


C , Image sequence of an L1 synchronized grh - 1 : : aid animal ( HW2418 ) grown at 20 ° C on a 250 μM auxin - containing plates . A lethargic animal was transferred to an agar - pad containing microscopy slide and images were collected every 1 sec , using DIC , 100x magnification . Selected images of Movie EV1 are shown . Time stamp ( min : sec ) is indicated . Arrows indicate phenotypic features : loosening of the cuticle ( 0 : 00 ) ; back - and - forth movements ( 0 : 05 ) ; inflation of the cuticle ( 2 : 46 ) ; vesicles underneath loosened cuticle ( 6 : 11 ) ; rupturing of the cuticle ( 6 : 20 , 6 : 40 ) .

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

C, Image sequence of an L1 synchronized grh-1::aid animal (HW2418) grown at 20°C on a 250 μM auxin-containing plates. A lethargic animal was transferred to an agar-pad containing microscopy slide and images were collected every 1 sec, using DIC, 100x magnification. Selected images of Movie EV1 are shown. Time stamp (min:sec) is indicated. Arrows indicate phenotypic features: loosening of the cuticle (0:00); back-and-forth movements (0:05); inflation of the cuticle (2:46); vesicles underneath loosened cuticle (6:11); rupturing of the cuticle (6:20, 6:40).


A , Quantification of the percentage of grh - 1 : : aid animals constitutively expressing luciferase ( HW2434 ) that enter each of four molts molt upon hatching into increasing concentrations of auxin as indicated .

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

A, Quantification of the percentage of grh-1::aid animals constitutively expressing luciferase (HW2434) that enter each of four molts molt upon hatching into increasing concentrations of auxin as indicated.


B , Boxplot showing the duration of M1 , M2 and M3 of animals treated with indicated concentrations of auxin . Animals that failed to develop beyond M3 in ( A ) were excluded . The horizontal line represents the median , hinges extend to first and third quartiles , and the whiskers extend up to the most extreme value within 1 . 5 * IQR ( interquartile range ) of the median . Significant differences relative to 0 nM auxin are indicated . P - values were determined by a non - parametric Wilcoxon rank sum test that does not assume normal distribution .

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 O O O O O O O O O O O O O O O O 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 O O O O

B, Boxplot showing the duration of M1, M2 and M3 of animals treated with indicated concentrations of auxin. Animals that failed to develop beyond M3 in (A) were excluded. The horizontal line represents the median, hinges extend to first and third quartiles, and the whiskers extend up to the most extreme value within 1.5*IQR (interquartile range) of the median. Significant differences relative to 0 nM auxin are indicated. P-values were determined by a non-parametric Wilcoxon rank sum test that does not assume normal distribution.


A - D , Heatmaps showing trend - corrected luminescence ( Lum , arbitrary units ) of grh - 1 : : aid animals constitutively expressing luciferase ( HW2434 ) . T = 0h corresponds to time of plating embryos , which subsequently hatch at different times . Arrow indicates time point when 250 μM auxin was added , i . e . prior to the first molt ( A ; M1 ) , M2 ( B ) , M3 ( C ) or M4 ( D ) larval stage . Note that for technical convenience in ( A ) , auxin was provided at time of plating . Animals are sorted by entry into M1 ( A ) , M2 ( B ) , M3 ( C ) , M4 ( D ) , respectively .

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

A-D, Heatmaps showing trend-corrected luminescence (Lum, arbitrary units) of grh-1::aid animals constitutively expressing luciferase (HW2434). T = 0h corresponds to time of plating embryos, which subsequently hatch at different times. Arrow indicates time point when 250 μM auxin was added, i.e. prior to the first molt (A; M1), M2 (B), M3 (C) or M4 (D) larval stage. Note that for technical convenience in (A), auxin was provided at time of plating. Animals are sorted by entry into M1 (A), M2 (B), M3 (C), M4 (D), respectively.


Time - lapse imaging of grh - 1 : : gfp : : 3xflag animals producing endogenously GFP - tagged GRH - 1 protein . Average + / - 95 % confidence interval ( cyan shading ) are shown ; gray boxes indicate average time of molts .

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

Time-lapse imaging of grh-1::gfp::3xflag animals producing endogenously GFP-tagged GRH-1 protein. Average +/- 95% confidence interval (cyan shading) are shown; gray boxes indicate average time of molts.


D , Western blot revealing rapid GRH - 1 depletion in the grh - 1 : : aid strain ( HW2434 ) upon addition of 250 µM auxin . A synchronized culture of animals was grown in liquid at 20 ° C . After 21 h ( denoted t = 0 h in the figure ) , the culture was split in two and either auxin or vehicle were added as indicated . Cultures were sampled hourly and protein lysates probed by Western blotting using anti - FLAG and anti - actin antibodies as indicated .

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

D, Western blot revealing rapid GRH-1 depletion in the grh-1::aid strain (HW2434) upon addition of 250 µM auxin. A synchronized culture of animals was grown in liquid at 20°C. After 21 h (denoted t=0 h in the figure), the culture was split in two and either auxin or vehicle were added as indicated. Cultures were sampled hourly and protein lysates probed by Western blotting using anti-FLAG and anti-actin antibodies as indicated.


A , Scatter plot of GRH - 1 : : GFP : : 3xFLAG ChIP - seq peak enrichments across two biological replicates , using an anti - GFP antibody to immunoprecipitate the endogenously tagged protein from L2 stage larval lysates . Peaks assigned to genes encoding rhythmically expressed transcription factors are plotted in red , all others in blue . Peaks assigned to candidate core clock genes identified in this study are labeled .

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

A, Scatter plot of GRH-1::GFP::3xFLAG ChIP-seq peak enrichments across two biological replicates, using an anti-GFP antibody to immunoprecipitate the endogenously tagged protein from L2 stage larval lysates. Peaks assigned to genes encoding rhythmically expressed transcription factors are plotted in red, all others in blue. Peaks assigned to candidate core clock genes identified in this study are labeled.


Dose - dependent increase of C . albicans ( SC5314 ) aggregation after 4 - h exposure ( a ) and filamentation after 24 - h exposure ( b ) to different concentrations of IL - 17A in RPMI - 1640 liquid medium . Cells aliquots were imaged by microscopy and magnified in the insets . Also note the increased turbidity of cell suspension at 24 h . Scale bars , 100 μm .

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

Dose-dependent increase of C. albicans (SC5314) aggregation after 4-h exposure (a) and filamentation after 24-h exposure (b) to different concentrations of IL-17A in RPMI-1640 liquid medium. Cells aliquots were imaged by microscopy and magnified in the insets. Also note the increased turbidity of cell suspension at 24 h. Scale bars, 100 μm.


( c ) Percentages of germination of C . albicans untreated ( vehicle - treated , open circle ) or treated with IL - 17A at 1 ( black triangle ) or 10 ng ml − 1 ( black square ) for 4 or 24 h . Data are mean ± s . d . P 0 . 05 , IL - 17A - treated versus vehicle - treated cells , at 4 h ; P 0 . 01 ( 1 ng ) and P 0 . 001 ( 10 ng ) at 24 h ( one - way analysis of variance Bonferroni post - test ) ( n = 3 ) .

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

(c) Percentages of germination of C. albicans untreated (vehicle-treated, open circle) or treated with IL-17A at 1 (black triangle) or 10 ng ml−1 (black square) for 4 or 24 h. Data are mean±s.d. P 0.05, IL-17A-treated versus vehicle-treated cells, at 4 h; P 0.01 (1 ng) and P 0.001 (10 ng) at 24 h (one-way analysis of variance Bonferroni post-test) (n=3).


( d , e ) IL - 17A promotes biofilm formation . SEM images of C . albicans SC5314 untreated ( d ) or exposed to 10 ng ml − 1 IL - 17A ( e ) for 4 h and subsequently cultured onto plastic discs for 24 h . Scale bars , 10 μm .

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

(d,e) IL-17A promotes biofilm formation. SEM images of C. albicans SC5314 untreated (d) or exposed to 10 ng ml−1 IL-17A (e) for 4 h and subsequently cultured onto plastic discs for 24 h. Scale bars, 10 μm.


( f ) IL - 17A increases resistance to nitrogen starvation . Cells were first grown in YPD medium overnight and then switched to nitrogen - free SLAD medium in the presence of 10 ng ml − 1 IL - 17A . Viability , expressed as a percentage of colony - forming units at zero time , was measured over a 15 - day period . Cells were exposed to 0 . 1 ( black circle ) , 1 ( black triangle ) or 10 ng ml − 1 ( black square ) IL - 17A or drug vehicle ( open circle ) at the beginning of the cultures or , in the case of 1 ng ml − 1 ( open triangle ) , also added every two other days . Data are mean ± s . d . P - values ranged from 0 . 05 to 0 . 001 for all samples treated with 10 ng IL - 17A ; P0 . 05 , on days 3 and 5 for samples treated 1 ng IL - 17A every two other days ( one - way analysis of variance Bonferroni post - test ) ( n = 3 ) .

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

(f) IL-17A increases resistance to nitrogen starvation. Cells were first grown in YPD medium overnight and then switched to nitrogen-free SLAD medium in the presence of 10 ng ml−1 IL-17A. Viability, expressed as a percentage of colony-forming units at zero time, was measured over a 15-day period. Cells were exposed to 0.1 (black circle), 1 (black triangle) or 10 ng ml−1 (black square) IL-17A or drug vehicle (open circle) at the beginning of the cultures or, in the case of 1 ng ml−1 (open triangle), also added every two other days. Data are mean±s.d. P-values ranged from 0.05 to 0.001 for all samples treated with 10 ng IL-17A; P0.05, on days 3 and 5 for samples treated 1 ng IL-17A every two other days (one-way analysis of variance Bonferroni post-test) (n=3).