( H ) Kymographs showing dynamics of Rab11 - GFP in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . ( I ) Quantification of Rab11 - GFP dynamics in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n numbers are axon sections analysed for each condition . ( J ) Quantification of total Rab11 - GFP in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n = 43 for control , 53 for co - transfected with p110δ . n numbers are axon sections analysed for each condition .

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

(H) Kymographs showing dynamics of Rab11-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. (I) Quantification of Rab11-GFP dynamics in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n numbers are axon sections analysed for each condition. (J) Quantification of total Rab11-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n=43 for control, 53 for co-transfected with p110δ. n numbers are axon sections analysed for each condition.


C ) Retinal sections from AAV - injected mice , immunolabelled for phospho - S6 . Blue colour is DAPI to indicate nuclei . Inset images show individual colours at the same scale as the full image .

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

C) Retinal sections from AAV-injected mice, immunolabelled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image.


( D ) Percentage of phospho - S6 positive cells in the RGC layer 2 weeks after delivery of AAV - Cre - GFP or AAV - GFP .

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

(D) Percentage of phospho-S6 positive cells in the RGC layer 2 weeks after delivery of AAV-Cre-GFP or AAV-GFP.


( E ) Retinal whole mounts of AAV - injected mice immunolabelled for the RGC marker Brn3A to indicate cell survival .

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

(E) Retinal whole mounts of AAV-injected mice immunolabelled for the RGC marker Brn3A to indicate cell survival.


( F ) Analysis of RGC survival 28 days after optic nerve crush ( counts of Brn3A positive RGC neurons ) .

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

(F) Analysis of RGC survival 28 days after optic nerve crush (counts of Brn3A positive RGC neurons).


G ) Representative images of CTB - labelled RGC axons 4 weeks after optic nerve crush in wild type ( c57bl / 6 ) , Rosa26 - p110αH1047R , and Rosa26 - p110δ transgenic mice injected with AAV2 . Cre . GFP . Dashed line indicates crush site .

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

G) Representative images of CTB-labelled RGC axons 4 weeks after optic nerve crush in wild type (c57bl/6), Rosa26-p110αH1047R, and Rosa26-p110δ transgenic mice injected with AAV2.Cre.GFP. Dashed line indicates crush site.


( H ) Regenerating axons at different distances distal to lesion sites .

(H) Regenerating axons at different distances distal to lesion sites.


( A ) Retinal sections from mice injected with AAV2 viruses as indicated , immunolabeled for phospho - S6 . Blue colour is DAPI to indicate nuclei . Inset images show individual colours at the same scale as the full image . ( B ) Percentage of phospho S6 positive cells in the retinal ganglion layer of mice injected with AAV2 . shScramble . GFP or AAV2 . shPTEN . GFP . ( C ) Number of pS6 - postive RGCs of mice injected with AAV2 . GFP or AAV2 . p110δ .

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

(A) Retinal sections from mice injected with AAV2 viruses as indicated, immunolabeled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image. (B) Percentage of phospho S6 positive cells in the retinal ganglion layer of mice injected with AAV2.shScramble.GFP or AAV2.shPTEN.GFP. (C) Number of pS6-postive RGCs of mice injected with AAV2.GFP or AAV2.p110δ.


( D ) Representative images of retinal whole mounts from AAV - injected mice stained for the RGC marker Brn3A to indicate RGC survival . ( E ) RGC survival 28 days after optic nerve crush .

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

(D) Representative images of retinal whole mounts from AAV-injected mice stained for the RGC marker Brn3A to indicate RGC survival. (E) RGC survival 28 days after optic nerve crush.


( F ) CTB - labelled RGC axons 4 weeks after optic nerve crush . Boxes i . to iv . show regenerating axons . Dashed line indicates crush site . White arrows mark the end of axons . Graph shows quantification of regenerating axons at different distances distal to the lesion sites .

O O O B-SMALL_MOLECULE O O B-CELL_TYPE B-SUBCELLULAR O O O B-TISSUE I-TISSUE 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 B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O

(F) CTB-labelled RGC axons 4 weeks after optic nerve crush. Boxes i. to iv. show regenerating axons. Dashed line indicates crush site. White arrows mark the end of axons. Graph shows quantification of regenerating axons at different distances distal to the lesion sites.


( C ) NAD + / NADH ratio was measured in mBA cell lines and 17 . 5 days embryo liver . Data represented as mean ± SEMs , and obtained from three different cell line and embryo liver . Significance determined by Student ' s t test , two - tailed , * P < 0 . 05 , * * P < 0 . 01 .

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

(C) NAD+/NADH ratio was measured in mBA cell lines and 17.5 days embryo liver. Data represented as mean ±SEMs, and obtained from three different cell line and embryo liver. Significance determined by Student's t test, two-tailed, *P < 0.05, **P < 0.01.


( D ) Immunostaining of 17 . 5 days embryo tissues ( mid - brain , heart , liver , and skin ) with anti - PAR antibody . Insets show higher magnification . Scale bar indicates 100 μm ( insert ) .

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

(D) Immunostaining of 17.5 days embryo tissues (mid-brain, heart, liver, and skin) with anti-PAR antibody. Insets show higher magnification. Scale bar indicates 100 μm (insert).


( E ) Wild - type ( WT ) and Ews - / - embryos mid - brain at E17 . 5 days were subjected to TUNEL assay . Scale bar indicates 100 μm ( lnsert ) .

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

(E) Wild-type (WT) and Ews-/- embryos mid-brain at E17.5 days were subjected to TUNEL assay. Scale bar indicates 100 μm (lnsert).


( A ) Western blot analysis of Parp1 and DNA damage markers in Wild - type ( WT ) and Ews - / - mBA cells . Cells were treated with MMS ( 0 . 02 % ) in a time dependent manner . Proteins were fractionated into two groups , chromatin - bound ( Chro ) proteins and soluble ( Solu ) proteins . ( B ) After treatment with MMS ( 0 . 02 % , 1hour ) , the media was replaced to release the DNA damage . The proteins were fractionated and the specific protein kinetics at chromatin was analyzed by western blot .

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

(A) Western blot analysis of Parp1 and DNA damage markers in Wild-type (WT) and Ews-/- mBA cells. Cells were treated with MMS (0.02%) in a time dependent manner. Proteins were fractionated into two groups, chromatin-bound (Chro) proteins and soluble (Solu) proteins. (B) After treatment with MMS (0.02%, 1hour), the media was replaced to release the DNA damage. The proteins were fractionated and the specific protein kinetics at chromatin was analyzed by western blot.


( C ) GFP - PARP1 U2OS cells were transfected with siControl and siEWS . Local DNA damage was induced by micro - irradiation using a 405nm laser . Data represented as mean ± SEMs and more than six cells were analyzed from six independent experiments . The statistical significance determined by One - way ANOVA , * * * P < 0 . 001 . Scale bar indicates 5 μm .

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

(C) GFP-PARP1 U2OS cells were transfected with siControl and siEWS. Local DNA damage was induced by micro-irradiation using a 405nm laser. Data represented as mean ±SEMs and more than six cells were analyzed from six independent experiments. The statistical significance determined by One-way ANOVA, ***P< 0.001. Scale bar indicates 5 μm.


( D ) Fluorescence correlation spectroscopy ( FCS ) was measured after 15 minutes later following micro - irradiation ( Upper ) or under normal condition in GFP - PARP1 U2OS cells with either siControl or siEWS . Data represented as mean ± SEMs , more than 50 cells were analyzed , and significance determined by One - way ANOVA , * * * P < 0 . 001 . Scale bar indicate 5 μm .

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

(D) Fluorescence correlation spectroscopy (FCS) was measured after 15 minutes later following micro-irradiation (Upper) or under normal condition in GFP-PARP1 U2OS cells with either siControl or siEWS. Data represented as mean ±SEMs, more than 50 cells were analyzed, and significance determined by One-way ANOVA, ***P< 0.001. Scale bar indicate 5 μm.


( E ) AsiSI endonuclease - integrated U2OS cells were transfected with siControl and siEWS . Cells were incubated with doxycycline for 4 hours to induce DSBs . With or without changing the Dox - added media to fresh media for 2 hours ( for release samples ) , the amount of chromatin - associated PARP1 was measured using ChIP assay . ( N . T : Non - treat , T : AsiSI treat , Release : Damage released samples ) . Data represented as mean ± SEMs , and technical repeats ( n = 3 ) , significance determined by two - way ANOVA , * * * P < 0 . 001 .

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

(E) AsiSI endonuclease-integrated U2OS cells were transfected with siControl and siEWS. Cells were incubated with doxycycline for 4 hours to induce DSBs. With or without changing the Dox-added media to fresh media for 2 hours (for release samples), the amount of chromatin-associated PARP1 was measured using ChIP assay. (N.T: Non-treat, T: AsiSI treat, Release: Damage released samples). Data represented as mean ±SEMs, and technical repeats (n=3), significance determined by two-way ANOVA, ***P< 0.001.


( A ) Total levels of PAR in whole cell lysate were measured using western blot analysis . Wild - type ( WT ) and Ews - / - mBA cells were treated with H2O2 , ( 1 mM , 20 minutes ) with or without Olaparib ( 5 μM , 7 hours ) .

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

(A) Total levels of PAR in whole cell lysate were measured using western blot analysis. Wild-type (WT) and Ews-/- mBA cells were treated with H2O2, (1 mM, 20 minutes) with or without Olaparib (5 μM, 7 hours).


( B ) The kinetics of PAR accumulation was analyzed by western blotting . WT and Ews - / - cells were treated with H2O2 ( 1 mM , 20 minutes ) , followed by incubation in fresh media for the indicated times to allow release of DNA

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

(B) The kinetics of PAR accumulation was analyzed by western blotting. WT and Ews-/- cells were treated with H2O2 (1 mM, 20 minutes), followed by incubation in fresh media for the indicated times to allow release of DNA damage


( C ) Immunohistochemistry detection of PAR by ADP - ribose antibody in WT and Ews - / - cells after H2O2 treatment ( 1 mM , 20 minutes ) and recovery from DNA damage . Right graph displays mean of intensities measured from 500 cells . Data represented as mean ± SEMs , significance determined by One - way ANOVA , * * * P < 0 . 001 . Scale bar indicate 20 μm .

O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O 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 O O O O O O O O O O O O O O O O O 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) Immunohistochemistry detection of PAR by ADP-ribose antibody in WT and Ews-/- cells after H2O2 treatment (1 mM, 20 minutes) and recovery from DNA damage. Right graph displays mean of intensities measured from 500 cells. Data represented as mean ±SEMs, significance determined by One-way ANOVA, ***P < 0.001. Scale bar indicate 20 μm.


( D , E ) Whole cell level of PAR was measured in WT and Ews - / - mBA cells upon treatment with ( D ) MMS ( 0 . 02 % , 60 minutes ) ( E ) with or without recovery from DNA damage .

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

(D, E) Whole cell level of PAR was measured in WT and Ews-/- mBA cells upon treatment with (D) MMS (0.02%, 60 minutes) (E) with or without recovery from DNA damage.


( F ) NAD + / NADH ratio was measured in mBA cell lines following treatment of H2O2 ( 1 mM , 10 minutes ) . Data represented as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Student ' s t test , two - tailed , * * P < 0 . 01 , * * * P < 0 . 001 .

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

(F) NAD+/NADH ratio was measured in mBA cell lines following treatment of H2O2 (1 mM, 10 minutes). Data represented as mean ±SEMs, and technical repeats (n=3). Significance determined by Student's t test, two-tailed, **P < 0.01, ***P< 0.001.


( A ) PARP1 and EWS interaction was visualized in cells using the CUPID system . After transfection of mRCD - EWS and GC3 - PARP1 ( EGFP - PARP1 ) plasmid , the cells were treated with either H2O2 ( 1 mM , 10 minutes ) or PMA . Scale bar indicates 5 μm .

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

(A) PARP1 and EWS interaction was visualized in cells using the CUPID system. After transfection of mRCD-EWS and GC3-PARP1 (EGFP-PARP1) plasmid, the cells were treated with either H2O2 (1 mM, 10 minutes) or PMA. Scale bar indicates 5 μm.


( B ) Endogenous interaction between EWS and PARP1 was measured using immunoprecipitation western blot analysis . Cells were treated with H2O2 ( 1 mM , 10 minutes ) with or without Olaparib ( 5 μM , 7 hours ) .

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

(B) Endogenous interaction between EWS and PARP1 was measured using immunoprecipitation western blot analysis. Cells were treated with H2O2 (1 mM, 10 minutes) with or without Olaparib (5 μM, 7 hours).


( C ) MMS - treated cells , with or without Olaparib , were immunoprecipitated using an anti - EWS antibody and immunoblotted by anti - PAR antibody ( upper ) . Pre - Olaparib group was treated with Olaparib for 3 hours before MMS treatment ( 0 . 02 % 30 minutes ) and after - Olaparib group treated Olaparib after 10 minutes following treatment of MMS . Lower blot indicates input of anti - EWS antibody .

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

(C) MMS-treated cells, with or without Olaparib, were immunoprecipitated using an anti-EWS antibody and immunoblotted by anti-PAR antibody (upper). Pre-Olaparib group was treated with Olaparib for 3 hours before MMS treatment (0.02% 30 minutes) and after-Olaparib group treated Olaparib after 10 minutes following treatment of MMS. Lower blot indicates input of anti-EWS antibody.


( D ) In vitro PAR binding assay . THRAP3 binding to PAR was used as a positive control and BSA binding to PAR was used as a negative control .

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

(D) In vitro PAR binding assay. THRAP3 binding to PAR was used as a positive control and BSA binding to PAR was used as a negative control.


( E ) Schematic map of nine EWS mutants ( left ) and IP - western blot analysis ( right ) . GFP - PARP1 and siPARG were transfected into HEK - 293 cells with each EWS mutants plasmid . After H2O2 treatment , the cells were conducted to IP with anti - Flag antibody .

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

(E) Schematic map of nine EWS mutants (left) and IP-western blot analysis (right). GFP-PARP1 and siPARG were transfected into HEK-293 cells with each EWS mutants plasmid. After H2O2 treatment, the cells were conducted to IP with anti-Flag antibody.


( F ) Western blot analysis of PARP1 accumulation on chromatin . EWS - WT and M4 mutants were transfected into EWS depleted HEK - 293 cells . After treatment of MMS ( 0 . 02 % , 1 hour ) , the proteins were fractionated to either chromatin - bound or soluble fraction . ( G ) Quantification of relative amount of PARP1 and γH2AX on chromatin , divided by chromatin H3 , from three independent western experiments are presented . Data represented as mean ± SEMs , were measured from 3 independent experiments . Significance determined by t - test . * P < 0 . 05 , * * P < 0 . 01 . n . s indicate non -

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

(F) Western blot analysis of PARP1 accumulation on chromatin. EWS-WT and M4 mutants were transfected into EWS depleted HEK-293 cells. After treatment of MMS (0.02%, 1 hour), the proteins were fractionated to either chromatin-bound or soluble fraction. (G) Quantification of relative amount of PARP1 and γH2AX on chromatin, divided by chromatin H3, from three independent western experiments are presented. Data represented as mean ±SEMs, were measured from 3 independent experiments. Significance determined by t-test. * P < 0.05, **P < 0.01. n.s indicate non-significance


( H ) WT and Ews - / - cells were transfected with EWS - WT and - M4 mutant and cell viability was measured 24 hours after MMS treatment ( indicated concentration ) . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * P < 0 . 01 . n . s indicate non - significance ,

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

(H) WT and Ews-/- cells were transfected with EWS-WT and -M4 mutant and cell viability was measured 24 hours after MMS treatment (indicated concentration). Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, **P < 0.01. n.s indicate non-significance,


( A ) Relative cell viability of cells transfected with siCon ( WT ) , siEWS ( EWS KD ) , PARP1 ( PARP - 1 KD ) , and double siRNA ( DKD ) after MMS treatment . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 . Analysis ( * * * ) indicates differences between DKD with EWS KD .

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

(A) Relative cell viability of cells transfected with siCon (WT), siEWS (EWS KD), PARP1 (PARP-1 KD), and double siRNA (DKD) after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. Analysis (***) indicates differences between DKD with EWS KD.


( B ) Additional Parp1 knockout in Ews knockout cells ( DKO ) were subjected to viability test after MMS treatment . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 . * * * indicated differences between DKO with EWS - KO .

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

(B) Additional Parp1 knockout in Ews knockout cells (DKO) were subjected to viability test after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. *** indicated differences between DKO with EWS-KO.


C ) After transfection of siCon , siEWS , PARP1 , and double siRNA , the effect of co - depletion of PARP1 and EWS on the γH2AX and phosphorylated CHK1 upon MMS treatment was analyzed by western blot .

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C) After transfection of siCon, siEWS, PARP1, and double siRNA, the effect of co-depletion of PARP1 and EWS on the γH2AX and phosphorylated CHK1 upon MMS treatment was analyzed by western blot.


D ) After treatment of MMS , the effect of additional Parp1 knockout in Ews knockout cells ( Double KO ) to the γH2AX and phosphorylated CHK1 were analyzed using western blot .

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D) After treatment of MMS, the effect of additional Parp1 knockout in Ews knockout cells (Double KO) to the γH2AX and phosphorylated CHK1 were analyzed using western blot.


E ) Whole cell expression of PAR in mBA ( WT : Ews - WT , KO : Ews - KO , DKO : double KO ) .

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E) Whole cell expression of PAR in mBA (WT: Ews-WT, KO: Ews-KO, DKO: double KO).


F ) Relative NAD + / NADH ratios were measured in WT , Ews - / - and DKO cells . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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F) Relative NAD+/NADH ratios were measured in WT, Ews-/- and DKO cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.


G ) PARGi ( 5 μM , 24 hours ) treated WT and Ews - / - cells were subjected to cellular viability assay . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA .

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G) PARGi (5 μM, 24 hours) treated WT and Ews-/- cells were subjected to cellular viability assay. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA.


H ) Relative cellular viability was measured after NMN treatment , with or without MMS pretreatment , in WT and Ews - / - cells . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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H) Relative cellular viability was measured after NMN treatment, with or without MMS pretreatment, in WT and Ews-/- cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.


I ) Chromatin - bound PARP1 was quantified by western blot in WT and Ews - / - cells treated with Olaparib ( 5 μM , 24 hours ) treatment .

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I) Chromatin-bound PARP1 was quantified by western blot in WT and Ews-/- cells treated with Olaparib (5 μM, 24 hours) treatment.


J ) Relative viability was measured in WT and Ews - / - cells upon treatment of Olaparib for 24 hours . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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J) Relative viability was measured in WT and Ews-/- cells upon treatment of Olaparib for 24 hours. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.


( A ) Western blot analysis of PARP1 in chromatin . Two Ewing Sarcoma cells ( CHP100 and A4573 ) were treated with MMS ( 0 . 02 % , 1 hour ) with or without release ( 1 hour ) from MMS ( N . T : non treat , T : treat , R : release sample ) .

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(A) Western blot analysis of PARP1 in chromatin. Two Ewing Sarcoma cells (CHP100 and A4573) were treated with MMS (0.02%, 1 hour) with or without release (1 hour) from MMS (N.T: non treat, T: treat, R: release sample).


( B ) U2OS and two Ewing Sarcoma cells were treated MMS ( 0 . 02 % , 30 minutes ) and subjected to immunofluorescence by PARP1 antibody after chromatin bound fraction . Scale bar indicates 20 μm ( left ) . Red line indicates mean and more than 300 cells were analyzed . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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(B) U2OS and two Ewing Sarcoma cells were treated MMS (0.02%, 30 minutes) and subjected to immunofluorescence by PARP1 antibody after chromatin bound fraction. Scale bar indicates 20 μm (left). Red line indicates mean and more than 300 cells were analyzed. Significance determined by Two-way ANOVA, ***P < 0.001.


( C ) Human Ewing Sarcoma histology samples were analyzed by immuno - histochemistry using PAR antibody . Scale bar represents 100 mm .

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(C) Human Ewing Sarcoma histology samples were analyzed by immuno-histochemistry using PAR antibody. Scale bar represents 100 mm.


( A - C ) Atg7 + / + and Atg7 - / - MEFs stably expressing GFP ‐ Gal3 ( A , C ) or GFP ‐ Ub ( B ) were treated with 1000 μM LLOMe or 250 μg / ml silica for 3 h . Cells were subjected to immunocytochemistry using the following antibodies : anti ‐ LC3 and anti ‐ Lamp1 ( A ) , anti ‐ p62 and anti ‐ Lamp1 ( B ) , or anti ‐ FK2 and anti ‐ p62 ( C ) . Bars : 10 μm .

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(A-C) Atg7+/+ and Atg7-/- MEFs stably expressing GFP‐Gal3 (A, C) or GFP‐Ub (B) were treated with 1000 μM LLOMe or 250 μg/ml silica for 3 h. Cells were subjected to immunocytochemistry using the following antibodies: anti‐LC3 and anti‐Lamp1 (A), anti‐p62 and anti‐Lamp1 (B), or anti‐FK2 and anti‐p62 (C). Bars: 10 μm.


( A , B ) NIH3T3 cells stably expressing GFP ‐ Gal3 and either empty vector ( control ) or mStrawberry ‐ Atg4BC74A ( Atg4B mutant ) were treated with 1000 μM LLOMe for 1 h . After LLOMe washout , cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI ( blue ) ( A ) . The number of GFP ‐ Gal3 or Lamp1 puncta per cell was quantified using G ‐ Count ( see also Supplementary Figure S4A and B ) . Then , the percent of GFP ‐ Gal3 ‐ positive Lamp1 puncta was determined ( B ) . The data represent means ± s . d . At least 70 cells were counted ( n = 3 ) . Bars : 20 μm . Source data for this figure is available on the online supplementary information page .

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(A, B) NIH3T3 cells stably expressing GFP‐Gal3 and either empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI (blue) (A). The number of GFP‐Gal3 or Lamp1 puncta per cell was quantified using G‐Count (see also Supplementary Figure S4A and B). Then, the percent of GFP‐Gal3‐positive Lamp1 puncta was determined (B). The data represent means±s.d. At least 70 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.


( C - E ) HeLa cells transfected with tfGal3 and either One ‐ STrEP ‐ FLAG ‐ tagged Atg4BC74A ( Atg4B mutant ) or empty vector ( control ) were observed by confocal microscopy after treatment as shown in Figure 2A and B . The number of GFP ± RFP + or GFP + RFP + puncta per cell was quantified using G ‐ Count ( D ) . Then , the percent of GFP + RFP + tfGal3 puncta was calculated ( E ) . The data represent means ± s . d . At least 30 cells were counted ( n = 3 ) . Bar : 10 μm .

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(C-E) HeLa cells transfected with tfGal3 and either One‐STrEP‐FLAG‐tagged Atg4BC74A (Atg4B mutant) or empty vector (control) were observed by confocal microscopy after treatment as shown in Figure 2A and B. The number of GFP±RFP+ or GFP+RFP+ puncta per cell was quantified using G‐Count (D). Then, the percent of GFP+RFP+tfGal3 puncta was calculated (E). The data represent means±s.d. At least 30 cells were counted (n=3). Bar: 10 μm.


( F - H ) NIH3T3 cells stably expressing empty vector ( control ) or mStrawberry ‐ Atg4BC74A ( Atg4B mutant ) were treated with 1000 μM LLOMe for 1 h . After LLOMe washout , cells were cultured in the presence or absence of both 10 μg / ml E64d and Pepstatin A , fixed at the indicated time points , and subjected to immunocytochemistry for Gal3 ( green ) and DAPI ( blue ) ( F ) . The number of endogenous Gal3 puncta per control ( G ) or Atg4B ‐ mutant ( H ) cells was quantified by G ‐ Count . The data represent means ± s . d . At least 50 cells were counted ( n = 3 ) . Bars : 20 μm . Source data for this figure is available on the online supplementary information page .

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(F-H) NIH3T3 cells stably expressing empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were cultured in the presence or absence of both 10 μg/ml E64d and Pepstatin A, fixed at the indicated time points, and subjected to immunocytochemistry for Gal3 (green) and DAPI (blue) (F). The number of endogenous Gal3 puncta per control (G) or Atg4B‐mutant (H) cells was quantified by G‐Count. The data represent means±s.d. At least 50 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.


( A - F ) HeLa cells stably expressing GFP ‐ LC3 were transfected with mStrawberry ‐ Gal3 , and treated with 1000 μM LLOMe for 1 h . Then , cells were fixed and observed by confocal microscopy ( A ) . The same specimens were further examined by transmission electron microscopy ( B - F ) . Green : LC3 ; magenta : Gal3 ; blue : DAPI .

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(A-F) HeLa cells stably expressing GFP‐LC3 were transfected with mStrawberry‐Gal3, and treated with 1000 μM LLOMe for 1 h. Then, cells were fixed and observed by confocal microscopy (A). The same specimens were further examined by transmission electron microscopy (B-F). Green: LC3; magenta: Gal3; blue: DAPI.


( G - I ) NIH3T3 cells stably expressing CFP ‐ Gal3 and YFP ‐ LC3 , and either empty vector ( control ) ( G , H ) or mStrawberry ‐ Atg4BC74A ( I ) were treated with 1000 μM LLOMe for 2 h , fixed , and observed by confocal microscopy . The electron micrographs were taken in the same sample field as the transmission electron microscope . Green : LC3 ; blue : Gal3 and DAPI ; black arrow : single membrane ; white arrow : autophagosome ; white arrowhead : ER membrane .

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(G-I) NIH3T3 cells stably expressing CFP‐Gal3 and YFP‐LC3, and either empty vector (control) (G, H) or mStrawberry‐Atg4BC74A (I) were treated with 1000 μM LLOMe for 2 h, fixed, and observed by confocal microscopy. The electron micrographs were taken in the same sample field as the transmission electron microscope. Green: LC3; blue: Gal3 and DAPI; black arrow: single membrane; white arrow: autophagosome; white arrowhead: ER membrane.


( A ) Plasmacreatinine and ureanitrogen in Atg5F / F and Atg5F / F ; KAPmice treated with vehicle or UA + OA . The values displayed represent means ± s . e . Statistically significant differences ( * P0 . 05 ) are indicated . F / F : Atg5F / Fmice ; F / F ; KAP : Atg5F / F ; KAPmice .

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(A) Plasmacreatinine and ureanitrogen in Atg5F/F and Atg5F/F;KAPmice treated with vehicle or UA+OA. The values displayed represent means±s.e. Statistically significant differences (*P0.05) are indicated. F/F: Atg5F/Fmice; F/F;KAP: Atg5F/F;KAPmice.


( B , C ) PAS ‐ stained renal cortical region of Atg5F / F and Atg5F / F ; KAP mice treated with vehicle or UA + OA ( n = 4 - 7 ) . Bars : 40 μm ( B ) . Kidney injury score of Atg5F / F and Atg5F / F ; KAP mice treated with vehicle or UA + OA ( C ) .

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(B, C) PAS‐stained renal cortical region of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (n=4-7). Bars: 40 μm (B). Kidney injury score of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (C).


( D ) Kidney cortexes from Atg5F / F and Atg5F / F ; KAP mice treated with vehicle or UA + OA were subjected to immunohistocheminal analysis of Lamp1 and LC3 . Green : LC3 ; magenta : Lamp1 ; blue : DAPI . F / F : Atg5F / F mice ; F / F ; KAP : Atg5F / F ; KAP mice . Bar : 5 μm .

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(D) Kidney cortexes from Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA were subjected to immunohistocheminal analysis of Lamp1 and LC3. Green: LC3; magenta: Lamp1; blue: DAPI. F/F: Atg5F/F mice; F/F;KAP: Atg5F/F;KAP mice. Bar: 5 μm.


( E ) Electron micrographs of kidney proximal tubules in Atg5 F / F and Atg5F / F ; KAP mice treated with UA + OA . Yellow arrowhead : autophagosome ; asterisk : lysosome ; Mt : mitochondrion . Bar : 1 μm .

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(E) Electron micrographs of kidney proximal tubules in Atg5 F/F and Atg5F/F;KAP mice treated with UA+OA. Yellow arrowhead: autophagosome; asterisk: lysosome; Mt: mitochondrion. Bar: 1 μm.


Wild - type , apg12 - 1 mutant and Δapg12 cells were cultured in nitrogen - starvation medium containing 1 mM PMSF . After incubation for 6 h , cells were observed under a phase - contrast microscope . Arrows indicate autophagic bodies .

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Wild-type, apg12-1 mutant and Δapg12 cells were cultured in nitrogen-starvation medium containing 1 mM PMSF. After incubation for 6 h, cells were observed under a phase-contrast microscope. Arrows indicate autophagic bodies.


c , Wild - type ( squares ) and Δapg12 ( circles ) were cultured in nitrogen - starvation medium and their viability was determined by phloxine B staining .

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c, Wild-type (squares) and Δapg12 (circles) were cultured in nitrogen-starvation medium and their viability was determined by phloxine B staining.


d , Quantification of autophagic activity of wild - type and Δapg12 cells by alkaline phosphatase ( ALP ) assay before ( black bars ) and after ( white bars ) nitrogen starvation for 4 h . Error bars indicate s . d . of three independent experiments .

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d, Quantification of autophagic activity of wild-type and Δapg12 cells by alkaline phosphatase (ALP) assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate s.d. of three independent experiments.


a , b , Lysates from Δapg12 cells carrying only vector or 3 × HA - APG12 ( a ) , and Δapg5 , apg7 - 1 , apg10 - 1 and Δapg1 cells carrying 3 × HA - Apg12 plasmid ( b ) were immunoblotted using anti - HA antibody . The positions of 3 × HA - Apg12 and the larger product ( asterisks ) are indicated .

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a, b, Lysates from Δapg12 cells carrying only vector or 3 × HA-APG12 (a), and Δapg5, apg7-1, apg10-1 and Δapg1 cells carrying 3 × HA-Apg12 plasmid (b) were immunoblotted using anti-HA antibody. The positions of 3 × HA-Apg12 and the larger product (asterisks) are indicated.


c , Immunoblot analysis of wild - type and Δapg12 cells harbouring HA - APG5 plasmid .

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c, Immunoblot analysis of wild-type and Δapg12 cells harbouring HA-APG5 plasmid.


d , Δapg5 Δapg12 cells were co - transformed with Myc - APG12 and HA - APG5 . Their lysates were immunoprecipitated with anti - Myc or anti - HA antibodies and detected by immunoblotting using anti - Myc antibody . The position of the crossreacting IgG heavy chain is

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d, Δapg5 Δapg12 cells were co-transformed with Myc-APG12 and HA-APG5. Their lysates were immunoprecipitated with anti-Myc or anti-HA antibodies and detected by immunoblotting using anti-Myc antibody. The position of the crossreacting IgG heavy chain is indicated


b , Δapg12 cells were transformed with the mutant plasmids and their lysates were immunoblotted with anti - HA antibody .

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b, Δapg12 cells were transformed with the mutant plasmids and their lysates were immunoblotted with anti-HA antibody.


c , Autophagic activity was measured as described for Fig . 1d .

c, Autophagic activity was measured as described for Fig. 1d.


d , Transport of pro - API to the vacuole was examined by immunoblotting with anti - API antiserum . The positions of pro - API and mature API are indicated .

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d, Transport of pro-API to the vacuole was examined by immunoblotting with anti-API antiserum. The positions of pro-API and mature API are indicated.


Δapg5 cells were transformed with vector alone , wild - type APG5 or APG5K149R , and then immunoblotted with anti - HA ( b )

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Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted with anti-HA (b)


Autophagic activity was determined by alkaline phosphatase assay ( c ) .

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Autophagic activity was determined by alkaline phosphatase assay (c).


Δapg5 cells were transformed with vector alone , wild - type APG5 or APG5K149R , and then immunoblotted anti - API ( d ) .

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Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted anti-API (d).


c , In vitro conjugation of Apg5 and Apg12 . A cell lysate of Δapg12 carrying HA - APG5 ( lane 1 ) was incubated with an equal amount of lysate from Δapg5 carrying HA - APG12 ( 2 µ plasmid ) ( lane 2 ) at 30 ° C with ( lane 4 - 6 ) or without ( lane 7 ) 5 mM ATP . Samples were mixed with SDS - sample buffer at the times indicated .

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c, In vitro conjugation of Apg5 and Apg12. A cell lysate of Δapg12 carrying HA-APG5 (lane 1) was incubated with an equal amount of lysate from Δapg5 carrying HA-APG12 (2 µ plasmid) (lane 2) at 30 °C with (lane 4-6) or without (lane 7) 5 mM ATP. Samples were mixed with SDS-sample buffer at the times indicated.


Spheroplasts were generated from cells expressing either HA - Apg12 or HA - Apg5 . Their lysates were mixed and layered on top of a 10 - step ( 18 - 54 % w / w ) sucrose gradient , and centrifuged at 174 , 000g for 2 . 5 h ( ref . 29 ) . Fifteen fractions were collected and the positions of free Apg5 , free Apg12 and Apg5 / Apg12 conjugate were examined by western blotting . The peak fractions of alcohol dehydrogenase ( ADH ) ( cytosol ) , ALP ( vacuole ) , Kex2 ( Golgi ) and Sec12 ( endoplasmic reticulum ) are indicated by arrows .

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Spheroplasts were generated from cells expressing either HA-Apg12 or HA-Apg5. Their lysates were mixed and layered on top of a 10-step (18-54 % w/w) sucrose gradient, and centrifuged at 174,000g for 2.5 h (ref. 29). Fifteen fractions were collected and the positions of free Apg5, free Apg12 and Apg5/Apg12 conjugate were examined by western blotting. The peak fractions of alcohol dehydrogenase (ADH)(cytosol), ALP (vacuole), Kex2 (Golgi) and Sec12 (endoplasmic reticulum) are indicated by arrows.


b ) Caco - 2 cells were infected with SARS - CoV - 2 as per Figure 1A . Cells were washed , fixed and stained with DAPI ( blue ) to visualise cell nuclei and incubated with anti - N protein ( red ) to visualise SARS - CoV - 2 infected cells . Scale bar denotes 10 µM .

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b) Caco-2 cells were infected with SARS-CoV-2 as per Figure 1A. Cells were washed, fixed and stained with DAPI (blue) to visualise cell nuclei and incubated with anti-N protein (red) to visualise SARS-CoV-2 infected cells. Scale bar denotes 10 µM.


c ) Infection rates were calculated using automated imaging software ( see materials and methods ) calculated from six fields of view per time point per biological replicate . Data point indicates the sample means and the error bars the standard error of the mean ( SEM ) ( n = 3 ) . d ) SARS - CoV - 2 detected proteins plotted as a function of time where each data point denotes a different replicate ( n = 3 ) .

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c) Infection rates were calculated using automated imaging software (see materials and methods) calculated from six fields of view per time point per biological replicate. Data point indicates the sample means and the error bars the standard error of the mean (SEM) (n=3). d) SARS-CoV-2 detected proteins plotted as a function of time where each data point denotes a different replicate (n=3).


a ) Total number of significantly up - or down - regulated proteins ( | z - score | > 1 . 96 , q - value < 0 . 05 ) per time point of SARS - CoV - 2 infected Caco - 2 cells as described in 1A . Proteins that were significantly changed in abundance ( left ) and stability ( right ) are sub - grouped according to either down - regulation / destabilization ( red ) or up - regulation / stabilisation ( turquoise ) .

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a) Total number of significantly up- or down-regulated proteins (|z-score| >1.96, q-value <0.05) per time point of SARS-CoV-2 infected Caco-2 cells as described in 1A. Proteins that were significantly changed in abundance (left) and stability (right) are sub-grouped according to either down-regulation/destabilization (red) or up-regulation/stabilisation (turquoise).


c ) SAFB phosphopeptide abundance changes during SARS - CoV - 2 infection . Vero cell phosphopeptide data from ( Bouhaddou et al , 2020 ) . Data previously acquired from Vero cells infected with SARS - CoV - 2 ( Bouhaddou et al , 2020 ) . S604 phosphosite that temporally coincides with SAFB thermal destabilisation ( Potel et al , 2020 ) . Shown above the line plot are the thermostability and abundance profiles of SAFB in SARS - CoV - 2 infected Caco - 2 cells as described in 1A ( n = 3 ) . Asterisk denotes significant regulation ( see methods ) .

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c) SAFB phosphopeptide abundance changes during SARS-CoV-2 infection. Vero cell phosphopeptide data from(Bouhaddou et al, 2020). Data previously acquired from Vero cells infected with SARS-CoV-2 (Bouhaddou et al, 2020). S604 phosphosite that temporally coincides with SAFB thermal destabilisation (Potel et al, 2020). Shown above the line plot are the thermostability and abundance profiles of SAFB in SARS-CoV-2 infected Caco-2 cells as described in 1A (n=3). Asterisk denotes significant regulation (see methods).


D ) Thermal stability profile for all unmodified SAFB peptides ( green ) and the phosphorylated S604 ( pS604 ) ( purple ) in HeLa cells . Data from ( Potel et al , 2020 ) .

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D) Thermal stability profile for all unmodified SAFB peptides (green) and the phosphorylated S604 (pS604) (purple) in HeLa cells. Data from (Potel et al, 2020).


b ) The corresponding HSP90 inhibitor , Tanespimycin , effectively suppressed SARS - CoV - 2 proliferation ( n = 2 ) . Calu - 3 cells were pre - incubated with the compound , followed by the addition of SARS - CoV - 2 and subsequent co - incubation in the presence of the compound for 20 - 24 hours . Viral load was quantified in parallel using antibody - based detection of double stranded RNA ( orange ) . Compound toxicity in Calu - 3 cells was assessed ( purple ) treated with the compound alone for 20 - 24 hours .

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b) The corresponding HSP90 inhibitor, Tanespimycin, effectively suppressed SARS-CoV-2 proliferation (n=2). Calu-3 cells were pre-incubated with the compound, followed by the addition of SARS-CoV-2 and subsequent co-incubation in the presence of the compound for 20-24 hours. Viral load was quantified in parallel using antibody-based detection of double stranded RNA (orange). Compound toxicity in Calu-3 cells was assessed (purple) treated with the compound alone for 20-24 hours.


d ) The Cyp1a1 inhibitor suppressed SARS - CoV - 2 proliferation ( n = 2 ) . Dose response curves were performed as described in 4A . See Appendix Figure S7 for additional tested drugs . Data displayed as described in 5B .

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d) The Cyp1a1 inhibitor suppressed SARS-CoV-2 proliferation (n=2). Dose response curves were performed as described in 4A. See Appendix Figure S7 for additional tested drugs. Data displayed as described in 5B.


B . Coomassie - stained SDS - PAGE ( from left to right ) of Hchim , V1ΔH , VoND , and C .

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B. Coomassie-stained SDS-PAGE (from left to right) of Hchim, V1ΔH, VoND, and C.


C . Representative ATPase activity assay ( ATP regenerating system ) . At the indicated time , 200 nM ConA is added to the assay .

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C. Representative ATPase activity assay (ATP regenerating system). At the indicated time, 200 nM ConA is added to the assay.


C . Growth phenotypes of wild type and c ″ - mutant yeast strains probed on ( 1 ) rich media buffered to pH 5 ( YPD pH 5 ) ; ( 2 ) synthetic dropout ( SD ) plus all amino acids ( SD + all a . a . ) ; ( 3 ) SD minus uracil ( SD - ura ) ; ( 4 , 5 ) YPD buffered to pH 7 plus 4 mM Zn2 + or 60 mM Ca2 + , respectively ; ( 6 ) YPD buffered to pH 7 ; ( 7 ) YPD plus 60 mM Ca2 + .

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C. Growth phenotypes of wild type and c″-mutant yeast strains probed on (1) rich media buffered to pH 5 (YPD pH 5); (2) synthetic dropout (SD) plus all amino acids (SD + all a.a.); (3) SD minus uracil (SD - ura); (4,5) YPD buffered to pH 7 plus 4 mM Zn2+ or 60 mM Ca2+, respectively; (6) YPD buffered to pH 7; (7) YPD plus 60 mM Ca2+.


B . Mass spectrometry analysis of V1Hchim . Protein IDs are plotted as a function of overall peptide spectrum matches ( PSM ) and the posterior error probability ( PEP ) scores .

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B. Mass spectrometry analysis of V1Hchim. Protein IDs are plotted as a function of overall peptide spectrum matches (PSM) and the posterior error probability (PEP) scores.


A . Size exclusion chromatography elution profile ( 1 . 6 cm × 50 cm Superdex S75 ) and SDS - PAGE of full - length N - terminally 7 × His tagged yeast Oxr1 . B . Inhibition of V1ΔH and V1Hchim ' s MgATPase activity by Oxr1 in absence and presence of subunit C . The data from one representative of two experiments using two different preparations are shown . Individual data points ( n = 2 ) and mean from one representative of two biological replicates are shown with error bars representing S . E . M . . C . Oxr1 binds wild type V1 . Samples of V1 , Oxr1 , and mixtures of V1 with a 2 - fold molar ratio of Oxr1 in absence or presence of a 3 - fold molar ratio of subunit C were subjected to size exclusion chromatography on a 1 cm × 30 cm Superose 6 Increase column . SDS - PAGE is shown for all four samples and immunoblot analysis of column fractions using a penta - His antibody is shown for samples containing Oxr1 and Oxr1 plus C . Note that incubation of V1 with Oxr1 in presence of excess subunit C leads to partial release of subunit H ( highlighted by the rectangular box in the V1 + Oxr1 + C gel ) . Also , some degradation of Oxr1 ' s unstructured N - terminus is observed as indicated by the asterisk ( only the full - length protein is detected by the αHis - tag antibody ) . The data from one representative of two experiments using two different preparations are shown .

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A. Size exclusion chromatography elution profile (1.6 cm × 50 cm Superdex S75) and SDS-PAGE of full-length N-terminally 7×His tagged yeast Oxr1. B. Inhibition of V1ΔH and V1Hchim's MgATPase activity by Oxr1 in absence and presence of subunit C. The data from one representative of two experiments using two different preparations are shown. Individual data points (n=2) and mean from one representative of two biological replicates are shown with error bars representing S.E.M. . C. Oxr1 binds wild type V1. Samples of V1, Oxr1, and mixtures of V1 with a 2-fold molar ratio of Oxr1 in absence or presence of a 3-fold molar ratio of subunit C were subjected to size exclusion chromatography on a 1 cm × 30 cm Superose 6 Increase column. SDS-PAGE is shown for all four samples and immunoblot analysis of column fractions using a penta-His antibody is shown for samples containing Oxr1 and Oxr1 plus C. Note that incubation of V1 with Oxr1 in presence of excess subunit C leads to partial release of subunit H (highlighted by the rectangular box in the V1 + Oxr1 + C gel). Also, some degradation of Oxr1's unstructured N-terminus is observed as indicated by the asterisk (only the full-length protein is detected by the αHis-tag antibody). The data from one representative of two experiments using two different preparations are shown.


D . Dissociation kinetics of Hwt and Hchim from V1ΔH in presence of Oxr1 and subunit C as probed by BLI . Recombinant wild type ( left ) and chimeric subunit H ( right ) with an N - terminal fusion to maltose binding protein ( MBP ) was immobilized on anti - mouse Fc ( AMC ) BLI sensors via an αMBP monoclonal antibody . The sensors were then dipped into wells with V1ΔH followed by wells with buffer in absence or presence of Oxr1 and C to monitor the kinetics of V1ΔH dissociation . Only the dissociation phase is shown . The data from one representative of three experiments using two different preparations are shown .

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D. Dissociation kinetics of Hwt and Hchim from V1ΔH in presence of Oxr1 and subunit C as probed by BLI. Recombinant wild type (left) and chimeric subunit H (right) with an N-terminal fusion to maltose binding protein (MBP) was immobilized on anti-mouse Fc (AMC) BLI sensors via an αMBP monoclonal antibody. The sensors were then dipped into wells with V1ΔH followed by wells with buffer in absence or presence of Oxr1 and C to monitor the kinetics of V1ΔH dissociation. Only the dissociation phase is shown. The data from one representative of three experiments using two different preparations are shown.


E . Oxr1 mediated inhibition of assembly of holo V - ATPase from V1Hchim , subunit C and VoND . The data from one representative of two experiments using two different preparations are shown . Individual data points ( n = 2 ) and mean from one representative of two biological replicates are shown with error bars representing S . E . M . .

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E. Oxr1 mediated inhibition of assembly of holo V-ATPase from V1Hchim, subunit C and VoND. The data from one representative of two experiments using two different preparations are shown. Individual data points (n=2) and mean from one representative of two biological replicates are shown with error bars representing S.E.M. .


F . Oxr1 prevents binding of V1Hchim to VoND as probed by BLI . Streptavidin coated BLI sensors were loaded with Vo in lipid nanodiscs containing biotinylated MSP , and sensors were then dipped into wells containing recombinant C and V1Hchim in absence and presence of Oxr1 . Only the V1Hchim association and dissociation phase from one representative of two experiments using two different preparations is shown .

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F. Oxr1 prevents binding of V1Hchim to VoND as probed by BLI. Streptavidin coated BLI sensors were loaded with Vo in lipid nanodiscs containing biotinylated MSP, and sensors were then dipped into wells containing recombinant C and V1Hchim in absence and presence of Oxr1. Only the V1Hchim association and dissociation phase from one representative of two experiments using two different preparations is shown.


G . Oxr1 mediated dissociation of holo V - ATPase as analyzed by negative stain electron microscopy . V1HchimVoND was incubated without and with a 3 - fold molar ratio of Oxr1 for 16 h . Samples were then spotted on carbon coated copper grids , stained with 1 % uranyl acetate and observed by transmission electron microscopy . Number of assembled V1Vo and disassembled V1 and Vo particles were counted on a total of 20 micrographs each . The horizontal dotted lines indicate the mean . A few holoenzyme and free V1 and VoND particles are highlighted by white circles and boxes , respectively . Bar = 50 nm . The data from one representative of two experiments using two different preparations are shown .

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G. Oxr1 mediated dissociation of holo V-ATPase as analyzed by negative stain electron microscopy. V1HchimVoND was incubated without and with a 3-fold molar ratio of Oxr1 for 16 h. Samples were then spotted on carbon coated copper grids, stained with 1% uranyl acetate and observed by transmission electron microscopy. Number of assembled V1Vo and disassembled V1 and Vo particles were counted on a total of 20 micrographs each. The horizontal dotted lines indicate the mean. A few holoenzyme and free V1 and VoND particles are highlighted by white circles and boxes, respectively. Bar = 50 nm. The data from one representative of two experiments using two different preparations are shown.


SIRT1 activity is required for resveratrol - induced autophagy but not for spermidine - mediated autophagy induction in mammalian cultured cells . ( A - C ) Human colon carcinoma HCT 116 cells were left untransfected ( Co , control ) or transfected with an irrelevant siRNA ( UNR , unrelated ) or siRNAs specific for ATG5 , ATG7 , or SIRT1 and then retransfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and left untreated or treated for 4 h with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) . The same experiment was performed in the presence of bafilomycin A1 ( BafA1 ) , which inhibits the fusion between lysosomes and autophagosomes , to evaluate the autophagic flux . ( A ) Representative images . ( B ) Quantitative data .

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SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A-C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data.


SIRT1 activity is required for resveratrol - induced autophagy but not for spermidine - mediated autophagy induction in mammalian cultured cells . ( A - C ) Human colon carcinoma HCT 116 cells were left untransfected ( Co , control ) or transfected with an irrelevant siRNA ( UNR , unrelated ) or siRNAs specific for ATG5 , ATG7 , or SIRT1 and then retransfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and left untreated or treated for 4 h with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) . The same experiment was performed in the presence of bafilomycin A1 ( BafA1 ) , which inhibits the fusion between lysosomes and autophagosomes , to evaluate the autophagic flux . ( A ) Representative images . ( B ) Quantitative data . ( C ) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1 - specific siRNA showing LC3 lipidation after treatment with 100 - µM spermidine in the presence or absence of bafilomycin A1 .

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SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A-C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data. (C) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1-specific siRNA showing LC3 lipidation after treatment with 100-µM spermidine in the presence or absence of bafilomycin A1.


( D - F ) HCT 116 cells were left transfected with a GFP - LC3 plasmid , cultured in complete medium for 24 h , and treated with either vehicle ( Co ) , 100 - µM resveratrol , or 100 - µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h . ( D ) Representative images . ( E ) Quantitative data . ( B and E ) Bars depict the percentages of cells showing accumulation of GFP - LC3 in puncta ( GFP - LC3vac ; means ± SEM ; n = 3 ; * , P < 0 . 05 ) . ( F ) Representative immunoblots showing LC3 lipidation in HCT 116 cells treated with 100 - µM spermidine in the presence or absence of EX527 . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(D-F) HCT 116 cells were left transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h, and treated with either vehicle (Co), 100-µM resveratrol, or 100-µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h. (D) Representative images. (E) Quantitative data. (B and E) Bars depict the percentages of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac; means ± SEM; n = 3; *, P < 0.05). (F) Representative immunoblots showing LC3 lipidation in HCT 116 cells treated with 100-µM spermidine in the presence or absence of EX527. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


The lifespan - extending and autophagy - inducing effects of spermidine in yeast are not mediated by Sir2 . ( A - E ) EGFP - Atg8p was ectopically expressed in wild - type ( WT ) or Δsir2 S . cerevisiae undergoing chronological aging on small synthetic 2 % glucose media with or without ( Co , control ) supplementation of 4 - mM spermidine ( Spd ) . ( A ) Representative images . EGFP - Atg8p localization ( bottom ) was visualized by fluorescence microscopy . Yeast cells undergoing autophagy ( in which EGFP - Atg8p exhibits a prominent vacuolar localization ) are indicated by arrows . Yeast morphology was monitored by differential interference contrast ( DIC ; top ) .

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The lifespan-extending and autophagy-inducing effects of spermidine in yeast are not mediated by Sir2. (A-E) EGFP-Atg8p was ectopically expressed in wild-type (WT) or Δsir2 S. cerevisiae undergoing chronological aging on small synthetic 2% glucose media with or without (Co, control) supplementation of 4-mM spermidine (Spd). (A) Representative images. EGFP-Atg8p localization (bottom) was visualized by fluorescence microscopy. Yeast cells undergoing autophagy (in which EGFP-Atg8p exhibits a prominent vacuolar localization) are indicated by arrows. Yeast morphology was monitored by differential interference contrast (DIC; top).


( B ) Representative immunoblots against EGFP . Free EGFP indicates the vacuolar degradation of EGFP - Atg8p fusion , thereby representing the autophagic flux . Notice that both WT and Δsir2 yeast cells show similar free EGFP levels after spermidine - mediated autophagy induction .

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(B) Representative immunoblots against EGFP. Free EGFP indicates the vacuolar degradation of EGFP-Atg8p fusion, thereby representing the autophagic flux. Notice that both WT and Δsir2 yeast cells show similar free EGFP levels after spermidine-mediated autophagy induction.


( C ) Relative alkaline phosphatase ( ALP ) activity indicative of autophagy . n = 3 .

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(C) Relative alkaline phosphatase (ALP) activity indicative of autophagy. n = 3.


( E ) Quantification of reactive oxygen species . Bars indicate the percentages of cells exhibiting the reactive oxygen species - mediated conversion of dihydroethidine ( DHE ) into ethidium ( Eth ; n = 4 ) . Data represent means ± SEM ; * , P < 0 . 001 as compared with untreated cells of the same genotype . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase . RFU , relative fluorescence unit .

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(E) Quantification of reactive oxygen species. Bars indicate the percentages of cells exhibiting the reactive oxygen species-mediated conversion of dihydroethidine (DHE) into ethidium (Eth; n = 4). Data represent means ± SEM; *, P < 0.001 as compared with untreated cells of the same genotype. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. RFU, relative fluorescence unit.


The life - extending and autophagy - inducing effects of spermidine in C . elegans are not mediated by Sir2 . ( A ) Fluorescence microscopy of C . elegans transgenic embryos expressing a full - length plgg - 1DsRed : : LGG - 1 fusion protein indicative of autophagic activity . Two representative pictures of wild - type ( WT ) and sir - 2 . 1 embryos untreated ( Co , control ) or treated with 0 . 2 - mM spermidine ( Spd ) supplementation of food are shown . ( B ) Quantification of autophagic activity through the measurement of DsRed : : LGG - 1 pixel intensity from images of WT animals shown in A . Data represent means ± SEM ( n = 3 ) with ≥ 25 images processed for each trial .

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The life-extending and autophagy-inducing effects of spermidine in C. elegans are not mediated by Sir2. (A) Fluorescence microscopy of C. elegans transgenic embryos expressing a full-length plgg-1DsRed::LGG-1 fusion protein indicative of autophagic activity. Two representative pictures of wild-type (WT) and sir-2.1 embryos untreated (Co, control) or treated with 0.2-mM spermidine (Spd) supplementation of food are shown. (B) Quantification of autophagic activity through the measurement of DsRed::LGG-1 pixel intensity from images of WT animals shown in A. Data represent means ± SEM (n = 3) with ≥25 images processed for each trial.


C ) Survival of WT C . elegans during aging with and without ( control ) supplementation of food ( UV - killed E . coli ) with 0 . 2 - mM spermidine ( n = 110 ; P < 0 . 005 ) . ( D ) Survival of sir - 2 . 1 C . elegans ( ok434 phenotype ) during aging with and without ( control ) supplementation of food ( UV - killed E . coli ) with 0 . 2 - mM spermidine ( n = 110 ; P < 0 . 01 ) . P - values were calculated using the log - rank test as described in Materials and methods .

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C) Survival of WT C. elegans during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.005). (D) Survival of sir-2.1 C. elegans (ok434 phenotype) during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.01). P-values were calculated using the log-rank test as described in Materials and methods.


Convergent alterations in the phosphoproteome status after resveratrol and / or spermidine treatment . ( A - C ) Human colon carcinoma HCT 116 cells were treated for 2 h with vehicle ( Co , control ) , 100 - µM resveratrol ( Resv ) , and 100 - µM spermidine ( Spd ) , alone or in combination ( Resv + Spd ) . ( A ) Representative phosphoprotein arrays are shown .

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Convergent alterations in the phosphoproteome status after resveratrol and/or spermidine treatment. (A-C) Human colon carcinoma HCT 116 cells were treated for 2 h with vehicle (Co, control), 100-µM resveratrol (Resv), and 100-µM spermidine (Spd), alone or in combination (Resv + Spd). (A) Representative phosphoprotein arrays are shown.


( B ) Clustering analysis for the effects on protein kinase phosphorylation .

(B) Clustering analysis for the effects on protein kinase phosphorylation.


( C ) Representative immunoblots of selected kinases whose phosphorylation status was unaffected ( PRKAA1 , RPS6KB1 , and acetyl - CoA carboxylase a [ ACACA ] ) or affected by resveratrol or / and spermidine treatment ( PTKB , AKT1 , MAPK8 , and CDKN1B ) , validating phosphoprotein array data .

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(C) Representative immunoblots of selected kinases whose phosphorylation status was unaffected (PRKAA1, RPS6KB1, and acetyl-CoA carboxylase a [ACACA]) or affected by resveratrol or/and spermidine treatment (PTKB, AKT1, MAPK8, and CDKN1B), validating phosphoprotein array data.


( D ) Human colorectal carcinoma HCT 116 cells were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and then treated with either vehicle or the indicated dose of resveratrol or spermidine , alone or in combination , for 2 h . Quantitative data . Bars depict the percentages ( means ± SD ; n = 3 ; * , P < 0 . 05 ) of cells showing the accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(D) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle or the indicated dose of resveratrol or spermidine, alone or in combination, for 2 h. Quantitative data. Bars depict the percentages (means ± SD; n = 3; *, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


Convergent acetylproteome modification after resveratrol or spermidine treatment . ( A - C ) Colon carcinoma HCT 116 cells were cultured for 2 wk in three different SILAC media containing different arginine and lysine isotopes . Cells were treated with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) for 2 h , fractioned into cytoplasmic , nuclear , and mitochondrial extracts , processed for acetyl lysine peptide enrichment , and analyzed by MS . ( A ) Hierarchical clustering of drug - specific organellar distributions of all acetylated sites quantified in at least one fraction . Fold changes are calculated relative to untreated cells . Only sites regulated > 1 . 5 - fold were included in statistical analyses . ( B ) Graphical representation of peptides whose acetylation status was affected in a convergent or divergent way . n = 560 .

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Convergent acetylproteome modification after resveratrol or spermidine treatment. (A-C) Colon carcinoma HCT 116 cells were cultured for 2 wk in three different SILAC media containing different arginine and lysine isotopes. Cells were treated with 100-µM resveratrol (Resv) or spermidine (Spd) for 2 h, fractioned into cytoplasmic, nuclear, and mitochondrial extracts, processed for acetyl lysine peptide enrichment, and analyzed by MS. (A) Hierarchical clustering of drug-specific organellar distributions of all acetylated sites quantified in at least one fraction. Fold changes are calculated relative to untreated cells. Only sites regulated >1.5-fold were included in statistical analyses. (B) Graphical representation of peptides whose acetylation status was affected in a convergent or divergent way. n = 560.


Significant motifs among sites undergoing acetylation . ( A ) Hierarchical clustering of the organellar distributions of sites whose acetylation was increased by > 1 . 5 - fold in response to resveratrol or spermidine , at least in one organellar fraction . ( B ) Consensus acetylation motifs identified upon resveratrol ( left ) or spermidine ( right ) treatment are depicted using the MotifX algorithm ( Schwartz and Gygi , 2005 ) . Among sites that were hyperacetylated in response to both agents , the K ( F / Y ) motif is significantly enriched when tested against the whole proteome ( P < 0 . 00001 ) . ( C ) When testing against the largest acetylation site dataset from Choudhary et al . ( 2009 ) ( acetylation background dataset [ ABD ] ) , the SxK motif is significant ( P < 0 . 0001 ) for sites whose acetylation increased upon spermidine treatment . No general consensus motifs were found for sites whose acetylation increased in response to both agents .

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

Significant motifs among sites undergoing acetylation. (A) Hierarchical clustering of the organellar distributions of sites whose acetylation was increased by >1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Consensus acetylation motifs identified upon resveratrol (left) or spermidine (right) treatment are depicted using the MotifX algorithm (Schwartz and Gygi, 2005). Among sites that were hyperacetylated in response to both agents, the K(F/Y) motif is significantly enriched when tested against the whole proteome (P < 0.00001). (C) When testing against the largest acetylation site dataset from Choudhary et al. (2009) (acetylation background dataset [ABD]), the SxK motif is significant (P < 0.0001) for sites whose acetylation increased upon spermidine treatment. No general consensus motifs were found for sites whose acetylation increased in response to both agents.


Significant motifs among sites undergoing deacetylation . ( A ) Hierarchical clustering of the organellar distributions of sites whose deacetylation was increased by > 1 . 5 - fold in response to resveratrol or spermidine , at least in one organellar fraction . ( B ) Among sites that were hypoacetylated in response to both agents , the KP motif is significantly enriched when tested against ABD ( P < 0 . 001 ) . ( C and D ) When tested against the ABD ( C ) or the whole proteome ( D ) , the KP motif is significant for sites undergoing hypoacetylation upon spermidine treatment ( P < 0 . 0001 ) . No general consensus motifs were found for sites whose acetylation decreased in response to both agents .

O O O O O O O O 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 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 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

Significant motifs among sites undergoing deacetylation. (A) Hierarchical clustering of the organellar distributions of sites whose deacetylation was increased by >1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Among sites that were hypoacetylated in response to both agents, the KP motif is significantly enriched when tested against ABD (P < 0.001). (C and D) When tested against the ABD (C) or the whole proteome (D), the KP motif is significant for sites undergoing hypoacetylation upon spermidine treatment (P < 0.0001). No general consensus motifs were found for sites whose acetylation decreased in response to both agents.


Autophagy can be efficiently regulated by cytoplasmic ( de ) acetylation reactions . ( A and B ) HCT 116 cells were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and enucleated to obtain cytoplasts . The cytoplasts were treated with either vehicle ( Co , control ) , 100 - µM resveratrol ( Resv ) , or 100 - µM spermidine ( Spd ) for 4 h . 1 - µM rapamycin ( Rapa ) was used as a positive control . ( A ) Representative images of cytoplasts indicative of autophagic activity . Arrows indicate GFP - LC3 - transfected cytoplasts , whereas insets show nonenucleated transfected cells . ( B ) Quantitative data . Bars show the percentages of cells or cytoplasts showing the accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) .

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Autophagy can be efficiently regulated by cytoplasmic (de)acetylation reactions. (A and B) HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and enucleated to obtain cytoplasts. The cytoplasts were treated with either vehicle (Co, control), 100-µM resveratrol (Resv), or 100-µM spermidine (Spd) for 4 h. 1-µM rapamycin (Rapa) was used as a positive control. (A) Representative images of cytoplasts indicative of autophagic activity. Arrows indicate GFP-LC3-transfected cytoplasts, whereas insets show nonenucleated transfected cells. (B) Quantitative data. Bars show the percentages of cells or cytoplasts showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac).


( C - E ) HCT 116 cells were cotransfected with a plasmid for the expression of RFP - LC3 together with an empty vector ( pcDNA3 ) or plasmids encoding wild - type ( WT ) SIRT1 or a SIRT1 variant with a mutation in the nuclear localization signal fused to GFP ( WT GFP - SIRT1 and mtNLS GFP - SIRT1 , respectively ) for 12 or 24 h , fixed , and analyzed by fluorescence microscopy . ( C ) Representative images indicative of 24 - h autophagic activity .

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(C-E) HCT 116 cells were cotransfected with a plasmid for the expression of RFP-LC3 together with an empty vector (pcDNA3) or plasmids encoding wild-type (WT) SIRT1 or a SIRT1 variant with a mutation in the nuclear localization signal fused to GFP (WT GFP-SIRT1 and mtNLS GFP-SIRT1, respectively) for 12 or 24 h, fixed, and analyzed by fluorescence microscopy. (C) Representative images indicative of 24-h autophagic activity.


( D ) Quantitative data . Bars depict the percentages of cells exhibiting the accumulation of RFP - LC3 in puncta ( RFP - LC3vac ) . ( B and D ) Means ± SEM ; n = 3 ; * , P < 0 . 05 .

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(D) Quantitative data. Bars depict the percentages of cells exhibiting the accumulation of RFP-LC3 in puncta (RFP-LC3vac). (B and D) Means ± SEM; n = 3; *, P < 0.05.


( E ) Representative immunoblots for 24 - h endogenous LC3 lipidation . The asterisk stands for a nonspecific band . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(E) Representative immunoblots for 24-h endogenous LC3 lipidation. The asterisk stands for a nonspecific band. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


Low doses of resveratrol and spermidine synergistically induce autophagy in vitro and in vivo . ( A ) Human colorectal carcinoma HCT 116 cells were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and then treated with either vehicle ( Co , control ) or the indicated dose of resveratrol ( Resv ) or spermidine ( Spd ) , alone or in combination , for 2 h . ( top ) Quantitative data . Bars depict the percentages ( means ± SD ; n = 3 ; * * , P < 0 . 05 ) of cells showing the accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) . ( bottom ) Representative immunoblots showing endogenous LC3 lipidation .

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Low doses of resveratrol and spermidine synergistically induce autophagy in vitro and in vivo. (A) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle (Co, control) or the indicated dose of resveratrol (Resv) or spermidine (Spd), alone or in combination, for 2 h. (top) Quantitative data. Bars depict the percentages (means ± SD; n = 3; **, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). (bottom) Representative immunoblots showing endogenous LC3 lipidation.


B ) Representative immunoblots showing endogenous LC3 lipidation in the presence of bafilomycin A1 ( BafA1 ) . ( C - E ) Transgenic C57BL / 6 mice expressing a GFP - LC3 fusion protein were injected with resveratrol and spermidine at the indicated concentrations . 3 h later , mice were killed , and tissues were processed for immunofluorescence microscopy determinations of GFP - LC3vac .

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B) Representative immunoblots showing endogenous LC3 lipidation in the presence of bafilomycin A1 (BafA1). (C-E) Transgenic C57BL/6 mice expressing a GFP-LC3 fusion protein were injected with resveratrol and spermidine at the indicated concentrations. 3 h later, mice were killed, and tissues were processed for immunofluorescence microscopy determinations of GFP-LC3vac.


( D ) Quantitative results . Bars represent the percentages ( means ± SEM ; n = 3 ; * , P < 0 . 05 ; and * * , P < 0 . 01 as compared with the same tissue from untreated animals ) of cells exhibiting GFP - LC3vac .

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(D) Quantitative results. Bars represent the percentages (means ± SEM; n = 3; *, P < 0.05; and **, P < 0.01 as compared with the same tissue from untreated animals) of cells exhibiting GFP-LC3vac.


( E ) Representative immunoblots showing endogenous LC3 lipidation and p62 protein content in WT C57BL / 6 mouse tissues . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(E) Representative immunoblots showing endogenous LC3 lipidation and p62 protein content in WT C57BL/6 mouse tissues. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


A . Schematic representation of the experimental protocol . Abbreviations : LCM , laser capture microdissection . B , C . RT - PCR performed on RNA extracted from axons collected by LCM ( Ax ) or from eyes . ( B ) β - Actin mRNA is present both in eyes and axons , while MAP2 and H4 are present only in the eye sample , suggesting the absence of contamination from cell bodies or other cells in LCM axonal samples ( Bellon et al , 2017 ) . Abbreviations : Ax , axonal sample ; Eye , stage 37 / 38 eye ; LCM , laser capture microdissection ; - , PCR no template control ; NT , RT no template control .

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

A. Schematic representation of the experimental protocol. Abbreviations: LCM, laser capture microdissection. B, C. RT-PCR performed on RNA extracted from axons collected by LCM (Ax) or from eyes. (B) β-Actin mRNA is present both in eyes and axons, while MAP2 and H4 are present only in the eye sample, suggesting the absence of contamination from cell bodies or other cells in LCM axonal samples (Bellon et al, 2017). Abbreviations: Ax, axonal sample; Eye, stage 37/38 eye; LCM, laser capture microdissection; -, PCR no template control; NT, RT no template control.