( f ) Immunoblot for indicated in immunoprecipitates ( IP ) for Cx26 or Cx32 ( left ) or Atg16 ( right ) from liver homogenates . Inp : input , FT : flow through . BIP is shown as negative control for immunoprecipitation . values are mean + s . e . m significant for * P 0 . 01 ( of variance + Bonferroni was used in b ) . Scale bars : 5 μm in ; 1 μm 0 . 1 μm in top panels insets of e . Uncropped of blots are shown in Supplementary Fig . 9 .

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot for indicated proteins in immunoprecipitates (IP) for Cx26 or Cx32 (left) or Atg16 (right) from liver homogenates. Inp: input, FT: flow through. BIP is shown as the negative control for immunoprecipitation. All values are mean + s.e.m and significant for *P 0.01 (analysis of variance + Bonferroni test was used in b). Scale bars: 5 μm in fluorescence images; 1 μm and 0.1 μm in top panels and insets of e. Uncropped images of blots are shown in Supplementary Fig. 9.


A . Localization of TRAPP components after sodium arsenite ( SA ) treatment in HeLa treated with 300 µM SA for 30 min . microscopy of fixed using antibodies against TRAPPC2 eIF3 ( to label SGs ) at steady state after SA treatment ( top two rows ) . Other panels show localization of GFP - TRAPPC3 endogenous TRAPPC1 to eIF3 - labeled SGs of endogenous TRAPPC9 TRAPPC8 to G3BP - labeled SGs . DAPI ( blue ) . Scale bar , 10 µm .

O O O O O O 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-MEASURED_VAR I-MEASURED_VAR 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-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O

A. Localization of TRAPP complex components after sodium arsenite (SA) treatment in HeLa cells treated with 300 µM SA for 30 min. Fluorescence microscopy of fixed cells using antibodies against TRAPPC2 and eIF3 (to label SGs) at steady state and after SA treatment (top two rows). Other panels show localization of GFP-TRAPPC3 and endogenous TRAPPC1 to eIF3-labeled SGs and of endogenous TRAPPC9 and TRAPPC8 to G3BP-labeled SGs. DAPI (blue). Scale bar, 10 µm.


B . Representative of a time course of TRAPPC2 redistribution to SGs . were treated graph ( B ' ) shows of TRAPPC2 localization at SGs over time as between TRAPPC2 ( mean ) in SG TRAPPC2 . Mean ± s . e . m . n = 50 per experiment , N = 3 . Scale bar , 10 µm .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O 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. Representative images of a time course analysis of TRAPPC2 redistribution to SGs. Cells were treated The graph (B') shows quantification of TRAPPC2 localization at SGs over time as the ratio between TRAPPC2 (mean fluorescence intensity) in SG puncta and cytosolic TRAPPC2. Mean ± s.e.m. n = 50 cells per experiment, N = 3. Scale bar, 10 µm.


C . of TRAPPC2 ( green ) , TRAPPC8 ( blue ) TRAPPC9 ( orange ) localization at SGs upon TRAPPC2 , TRAPPC4 TRAPPC8 TRAPPC9 depletion . Mean ± s . e . m . n > 100 . * p < 0 . 05 ; ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison .

O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O 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. Analysis of TRAPPC2 (green), TRAPPC8 (blue) and TRAPPC9 (orange) localization at SGs upon TRAPPC2, TRAPPC4 TRAPPC8 and TRAPPC9 depletion. Mean ± s.e.m. n>100. *p<0.05; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.


E . of TRAPPC2 localization at SGs upon CLIC1 , CRYAB ENO1 depletion . Mean ± s . e . m . n > 100 . * * * * p < 0 . 0001 ; ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison .

O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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. Analysis of TRAPPC2 localization at SGs upon CLIC1, CRYAB and ENO1 depletion. Mean ± s.e.m. n>100. ****p<0.0001; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.


A . HeLa , untreated or treated with SA , were fixed visualized by microscopy using anti - Sec24C Ab , anti - eIF3 Ab , DAPI ( blue ) . Data information : Scale bars , 10 µm .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

A. HeLa cells, untreated or treated with SA, were fixed and visualized by fluorescence microscopy using anti-Sec24C Ab, anti-eIF3 Ab, and DAPI (blue). Data information: Scale bars, 10 µm.


B . of Sec24C redistribution to SGs over time after SA treatment ( between Sec24C ( mean ) in SG Sec24C ) . Mean ± s . e . m . n = 50 per experiment , N = 3 .

O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

B. Quantification of Sec24C redistribution to SGs over time after SA treatment (the ratio between Sec24C (mean fluorescence intensity) in SG puncta and cytosolic Sec24C). Mean ± s.e.m. n = 50 cells per experiment, N = 3.


C . of residual Sec24C in ERES after SA treatment . data are expressed as of steady state values ( CTRL ) . Mean ± s . e . m . n = 100 per experiment , N = 3 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O 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. Quantification of residual Sec24C in ERES after SA treatment. The data are expressed as percentage of steady state values (CTRL). Mean ± s.e.m. n = 100 cells per experiment, N = 3.


D . Representative of TRAPPC2 localization in Sec23AB - KD Sec24ABCD - KD treated with SA . were fixed visualized by microscopy using anti - TRAPPC2 Ab , anti - G3BP Ab ( to label SGs ) DAPI ( blue ) . Data information : Scale bars , 10 µm .

O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

D. Representative images of TRAPPC2 localization in Sec23AB-KD and Sec24ABCD-KD cells treated with SA. Cells were fixed and visualized by fluorescence microscopy using anti-TRAPPC2 Ab, anti-G3BP Ab (to label SGs) and DAPI (blue). Data information: Scale bars, 10 µm.


E . of TRAPPC2 redistribution to SGs after KD of indicated Sec23 Sec24 combinations , calculated as between TRAPPC2 ( mean ) in SG TRAPPC2 expressed as % of CTRL . Mean ± s . e . m . , n = 40 - 60 per experiment , N = 3 . ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O 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. Quantification of TRAPPC2 redistribution to SGs after KD of the indicated Sec23 and Sec24 combinations, calculated as the ratio between TRAPPC2 (mean intensity) in SG puncta and cytosolic TRAPPC2 and expressed as % of CTRL. Mean ± s.e.m., n = 40-60 cells per experiment, N = 3. ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.


F . Representative of Sec24C localization at SGs ( stained for G3BP ) in TRAPPC3 - KD TRAPPC2 - KD treated with SA . Depletion of entire TRAPP ( via TRAPPC3 depletion ) or of only TRAPPC2 reduces Sec24C recruitment . Graph , of Sec24C at SGs , calculated as in ( E ) . Mean ± s . e . m . , n = 40 - 60 per experiment , N = 3 . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison . Data information : Scale bars , 10 µm .

O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC3-KD and TRAPPC2-KD cells treated with SA. Depletion of the entire TRAPP complex (via TRAPPC3 depletion) or of only TRAPPC2 reduces Sec24C recruitment. Graph, quantification of Sec24C at SGs, calculated as in (E). Mean ± s.e.m., n = 40-60 cells per experiment, N = 3. ****p< 0.0001, One-way ANOVA with Dunnett's multiple comparison test. Data information: Scale bars, 10 µm.


G . Representative of Sec24C localization at SGs ( stained for G3BP ) in TRAPPC2 - KO treated with SA . Data information : Scale bars , 10 µm .

O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

G. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC2-KO cells treated with SA. Data information: Scale bars, 10 µm.


A . TRAPP COPII associate more stably with SGs than with ERES . association of Sec24C was evaluated in non - permeabilized or permeabilized with or without SA treatment , as indicated . G3BP was used as an SG marker . Left panel insets , G3BP in non - permeabilized . Dashed lines show outline of permeabilized SA - untreated . Blue , DAPI . Data information ; Scale bars , 10 µm .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O 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. TRAPP and COPII associate more stably with SGs than with ERES. The membrane association of Sec24C was evaluated in non-permeabilized or permeabilized cells with or without SA treatment, as indicated. G3BP was used as an SG marker. Left panel insets, G3BP staining in non-permeabilized cells. Dashed white lines show the outline of permeabilized SA-untreated cells. Blue, DAPI. Data information; Scale bars, 10 µm.


B . Stabilizing TRAPP COPII at ERES prevents their relocalization to SGs . HeLa overexpressing GFP - Sar1H79G were treated with SA immunostained for Sec24C , TRAPPC2 , eIF3 , as indicated . Blue , DAPI . Graphs show of Sec24C or TRAPPC2 at SGs in GFP - Sar1H79G - expressing . Data are between Sec24C or TRAPPC2 mean in SG Sec24C or TRAPPC2 mean at ERES , expressed as a of non - transfected ( NT ) . Mean ± s . e . m . n = 60 - 70 , three independent experiments . * * * * p < 0 . 0001 , Student ' s unpaired two - t - . Data information ; Scale bars , 10 µm .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Stabilizing TRAPP and COPII at the ERES prevents their relocalization to SGs. HeLa cells overexpressing GFP-Sar1H79G were treated with SA and immunostained for Sec24C, TRAPPC2, and eIF3, as indicated. Blue, DAPI. Graphs show quantification of Sec24C or TRAPPC2 at SGs in GFP-Sar1H79G-expressing cells. Data are the ratio between Sec24C or TRAPPC2 mean intensity in SG puncta and Sec24C or TRAPPC2 mean intensity at ERES, expressed as a percentage of the non-transfected (NT) cells. Mean ± s.e.m. n = 60-70, three independent experiments. ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.


C . Effect of Sec31A depletion on TRAPPC2 recruitment to SGs . were mock - treated or KD for Sec31 , treated with SA , then immunostained for TRAPPC2 eIF3 as indicated . Graphs , of TRAPPC2 at ERES SGs after SA treatment . Mean ± s . e . m . , one representative experiment ; n = 60 - 80 . * * p < 0 . 001 , * * * * p < 0 . 0001 , Student ' s unpaired two - t - . Data information ; Scale bars , 10 µm .

O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Effect of Sec31A depletion on TRAPPC2 recruitment to SGs. Cells were mock-treated or KD for Sec31, treated with SA, and then immunostained for TRAPPC2 and eIF3 as indicated. Graphs, quantification of TRAPPC2 at ERES and SGs after SA treatment. Mean ± s.e.m., one representative experiment; n=60-80. ** p<0.001, ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.


D . Effect of Sec31A depletion on Sec24C recruitment to SGs . were mock - treated or KD for Sec31 then immunostained for Sec24C cTAGE5 ( to stain ERES , top panels ) for eIF3 ( bottom panels ) . Insets show eIF3 . Graphs , of Sec24C at ERES in SGs after SA treatment . n = 80 . * * * * p < 0 . 0001 , Student ' s unpaired two - t - . Data information ; Scale bars , 10 µm .

O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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. Effect of Sec31A depletion on Sec24C recruitment to SGs. Cells were mock-treated or KD for Sec31 and then immunostained for Sec24C and cTAGE5 (to stain ERES, top panels) and for eIF3 (bottom panels). Insets show eIF3. Graphs, quantification of Sec24C at ERES and in SGs after SA treatment. n=80. ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.


A . were treated with 273 kinase inhibitors ( 10 µM ) from Sellekchem library with SA , Sec24C localization in G3BP ( % of total G3BP ) was analyzed by automated microscopy under High content screening is reported as a of control ( treated with SA alone ) . Gray dashed , mean of control ; gray box , control standard deviation ( ± 18 . 2 % ) ; red , threshold of positive hits ; red box : positive hits . B . Enrichment of positive hits ; for each class of inhibitor , enrichment ( in ) of compounds falling into positive hits enrichment ( in ) in total number of compounds was calculated expressed as a .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cells were treated with 273 kinase inhibitors (10 µM) from the Sellekchem library and with SA, and Sec24C localization in G3BP puncta (% of total G3BP area) was analyzed by automated microscopy under High content screening and is reported as a percentage of the control (cells treated with SA alone). Gray dashed line, mean of control; gray box, control standard deviation (± 18.2%); red line, threshold of positive hits; red box: positive hits. B. Enrichment Analysis of the positive hits; for each class of inhibitor, the enrichment (in percentage) of compounds falling into positive hits and the enrichment (in percentage) in the total number of compounds was calculated and expressed as a ratio.


C . Evaluation of CDK kinase activity upon SA treatment . Western blot of phosphoSer - CDK substrates in control , SA ( 300µM , 30 min ) , Dinaciclib ( 10 µM , 180 min ) SA + Dinaciclib ( 150 min Dinaciclib 30 min SA ) . CDKs are hyperactivated upon oxidative stress this activation is partially prevented upon CDK inhibition . Left , Western blot , right : Ponceau was used as loading control . Image , one representative experiment out of 3 independent replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Evaluation of CDK kinase activity upon SA treatment. Western blot analysis of phosphoSer-CDK substrates in control, SA (300µM, 30 min), Dinaciclib (10 µM, 180 min) and SA+Dinaciclib (150 min Dinaciclib and 30 min SA). CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition. Left, Western blot, right: Ponceau was used as loading control. Image, one representative experiment out of 3 independent replicates.


D . Drug specificity from high ( + + + + ) to low ( + ) of different CDKs as described by Selleckchem , Sec24C recruitment to SGs as a of control .

O 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-MEASURED_VAR O O O O O O O O

D. Drug specificity from high (++++) to low (+) of the different CDKs as described by Selleckchem, and Sec24C recruitment to SGs as a percentage of control.


E . of Sec24C recruitment to SGs in control CDK1 + 2 - KD HeLa . Different of CDK1 + 2 KD were achieved by transfecting with different amounts of siRNA for different times ( 2 nM 24 h ; 5 nM 24 h ; 5 nM 48 h ; 20 nM 48 h ) to give 50 , 30 , 20 10 % , respectively , of CDK1 + 2 in control . CDK1 + 2 measured by qRT - PCR Sec24C recruitment to SGs were evaluated from parallel cultures . Sec24C recruitment to SGs was performed by automated microscopy as described in ( A ) is expressed as a of mock ( CTRL , set at 100 % ) ± s . d . N = 8 .

O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O 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. Analysis of Sec24C recruitment to SGs in control and CDK1+2-KD HeLa cells. Different levels of CDK1+2 KD were achieved by transfecting cells with different amounts of siRNA for different times (2 nM 24 h; 5 nM 24 h; 5 nM 48 h; 20 nM 48 h) to give 50, 30, 20 and 10%, respectively, of the CDK1+2 levels in the control. CDK1+2 levels measured by qRT-PCR and Sec24C recruitment to SGs were evaluated from parallel cultures. Sec24C recruitment to SGs was performed by automated microscopy as described in (A) and is expressed as a percentage of mock (CTRL, set at 100%) ± s.d. N=8.


F . Representative of HeLa treated with three best hits ( Flavopiridol , SNS - 032 , Dinaciclib ; 1µM 150 min ) then exposed to SA ( 300 µM , 30 min ) , followed by immunostaining for Sec24C G3BP . Scale bar , 10 µm .

O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O

F. Representative images of HeLa cells treated with the three best hits (Flavopiridol, SNS-032, Dinaciclib; 1µM 150 min) and then exposed to SA (300 µM, 30 min), followed by immunostaining for Sec24C and G3BP. Scale bar, 10 µm.


G . HeLa were treated with Flavopiridol , SNS - 032 , or Dinaciclib for 150 min at indicated concentrations , subsequently treated with SA ( 300 µM , 30 min ) , then immunostained for Sec24C G3BP imaged by OPERA . BS - 181 , a specific CDK7 inhibitor , was used as negative control . Sec24C localization to SGs is expressed as a of control ( treated with SA alone ) . Dashed red , mean of control ; yellow box , standard deviation of control ( ± 9 . 8 % ) , N = 12 .

O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O 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. HeLa cells were treated with Flavopiridol, SNS-032, or Dinaciclib for 150 min at the indicated concentrations, subsequently treated with SA (300 µM, 30 min), and then immunostained for Sec24C and G3BP and imaged by OPERA. BS-181, a specific CDK7 inhibitor, was used as negative control. Sec24C localization to SGs is expressed as a percentage of the control (cells treated with SA alone). Dashed red line, mean of control; yellow box, standard deviation of control (± 9.8%), N=12.


H . were treated as in ( G ) immunostained for TRAPPC2 eIF3 . Scale bar , 10 µm . graph shows of TRAPPC2 localization in SG ( mean ) . Data are mean ± s . e . m . expressed as a of TRAPPC2 signal in SGs after Flavopiridol , SNS - 032 or Dinaciclib treatment compared to control ( treated with SA alone ) . N = 3 , three independent experiments , n = 60 - 80 per experiment . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cells were treated as in (G) and immunostained for TRAPPC2 and eIF3. Scale bar, 10 µm. The graph shows the quantification of TRAPPC2 localization in SG puncta (mean intensity). Data are mean ± s.e.m. expressed as a percentage of TRAPPC2 signal in SGs after Flavopiridol, SNS-032 or Dinaciclib treatment compared to the control (cells treated with SA alone). N=3, three independent experiments, n=60-80 cells per experiment. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


A . Immunofluorescence of Sec24C TRAPPC2 in vehicle - treated SNS - 032 - treated ( 1 µM , 3 h ) . Graphs show of Sec24C TRAPPC2 at ERES ( mean ) normalized in SNS - 032 - treated relative to vehicle - treated ( set as 100 % ) . Mean ± s . e . m . of three independent experiments . * * p < 0 . 05 , * * * * p < 0 . 0001 , Student ' s unpaired two - t - . Data information : Scale bars , 10 µm .

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunofluorescence images of Sec24C and TRAPPC2 in vehicle-treated and SNS-032-treated (1 µM, 3 h) cells. Graphs show the quantification of Sec24C and TRAPPC2 at ERES (mean intensity) normalized in SNS-032-treated cells relative to vehicle-treated cells (set as 100%). Mean ± s.e.m. of three independent experiments. **p<0.05, ****p<0.0001, Student's unpaired two-tailed t-test. Data information: Scale bars, 10 µm.


B . HeLa were treated with Dinaciclib ( 1 µM , 3 h ) permeabilized or not with digitonin A GM130 antibody was added to buffer of living to monitor permeabilization efficiency . Upper panels , non - permeabilized ( NP ) control ; middle panels , permeabilized control ; lower panels , permeabilized Dinaciclib - treated . Data information : Scale bars , 10 µm .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O 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. HeLa cells were treated with Dinaciclib (1 µM, 3 h) and permeabilized or not with digitonin A GM130 antibody was added to the buffer of living cells to monitor permeabilization efficiency. Upper panels, non-permeabilized (NP) control cells; middle panels, permeabilized control cells; lower panels, permeabilized Dinaciclib-treated cells. Data information: Scale bars, 10 µm.


C . of Sec24C association after CDK inhibitor treatment . Digitonin - permeabilized treated with vehicle ( CTRL ) or indicated CDK inhibitor ( 1 µM , 3 h ) were immunostained for Sec24C . mean of Sec24C in perinuclear normalized for Sec24C signal in drug - treated is expressed as fold change compared to control ; n = 60 - 80 ; mean ± s . e . m . of three independent experiments * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison .

O O O B-MEASURED_VAR O O O O O 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-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of Sec24C membrane association after CDK inhibitor treatment. Digitonin-permeabilized cells treated with vehicle (CTRL) or the indicated CDK inhibitor (1 µM, 3 h) were immunostained for Sec24C. The mean intensity of Sec24C in the perinuclear area normalized for the cytosolic Sec24C signal in drug-treated cells is expressed as fold change compared to the control; n= 60-80; mean ± s.e.m. of three independent experiments ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


D . HeLa transfected with HA - hnRNPK were left untreated ( Steady state ) or treated with SA ( 300µM , 30 min ) or SA + Dinaciclib ( 10 µM , 150 min ) then immunostained with anti - HA anti - G3BP antibodies . Data information Scale bars , 10 µm .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O

D. HeLa cells transfected with HA-hnRNPK were left untreated (Steady state) or treated with SA (300µM, 30 min) or SA+Dinaciclib (10 µM, 150 min) and then immunostained with anti-HA and anti-G3BP antibodies. Data information Scale bars, 10 µm.


E . TRAPPC2 interacts with hnRNPK . ( Top ) SDS - PAGE Western blot of endogenous hnRNPK after immunoprecipitation with an anti - TRAPPC2 Ab or IgG , ( bottom ) after immunoprecipitation with an anti - HA Ab from transfected ( + ) or not ( - ) with hnRNPK - HA detected with indicated antibodies .

O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O 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-CONTROLLED_VAR O O O O O O O

E. TRAPPC2 interacts with hnRNPK. (Top) SDS-PAGE and Western blot analysis of endogenous hnRNPK after immunoprecipitation with an anti-TRAPPC2 Ab or IgG, and (bottom) after immunoprecipitation with an anti-HA Ab from cells transfected (+) or not (-) with hnRNPK-HA detected with the indicated antibodies.


F . Mock or hnRNPK KD treated with SA were immunostained for TRAPPC2 eIF3 . Data information : Scale bars , 10 µm .

O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

F. Mock or hnRNPK KD cells treated with SA were immunostained for TRAPPC2 and eIF3. Data information: Scale bars, 10 µm.


G . Mock or hnRNPK KD treated with SA were immunostained for Sec24C G3BP . Data information : Scale bars , 10 µm .

G. Mock or hnRNPK KD cells treated with SA were immunostained for Sec24C and G3BP. Data information: Scale bars, 10 µm.


H . Graphs show of TRAPPC2 Sec24C redistribution to SGs in mock treated ( CTRL ) hnRNPK KD . n > 100 , N = 3 mean ± s . d . * * * p < 0 . 001 . * * * * p < 0 . 0001 , Student ' s unpaired two - t - .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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. Graphs show quantification of TRAPPC2 and Sec24C redistribution to SGs in mock treated (CTRL) and hnRNPK KD cells. n>100, N=3 mean ± s.d. ***p<0.001.****p<0.0001, Student's unpaired two-tailed t-test.


A . HeLa were starved for 8 h with HBSS subsequently exposed to SA ( 30 min , 300 µM ) , immunostained for Sec24C or TRAPPC2 eIF3 . Scale bar 10 µm .

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-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O

A. HeLa cells were starved for 8 h with HBSS and subsequently exposed to SA (30 min, 300 µM), and immunostained for Sec24C or TRAPPC2 and eIF3. Scale bar 10 µm.


B . of proliferation status of after 8 h starvation in HBSS . Top , representative of starved non - starved ( CTRL ) using EdU incorporation ( see Materials Methods ) . Scale bar 100 µm . Bottom , of EdU incorporation in starved as a of incorporation in control fed , N = 3 , mean ± s . d .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of the proliferation status of the cells after 8 h starvation in HBSS. Top, representative images of starved and non-starved (CTRL) cells using EdU incorporation (see Materials and Methods). Scale bar 100 µm. Bottom, quantification of EdU incorporation in starved cells as a percentage of incorporation in control fed cells, N=3, mean ± s.d.


C . HeLa were seeded at different confluency , treated with SA stained for Sec24C G3BP as an SG marker . Scale bar , 10 µm . Flow cytometry ( FACS ) ( right panels ) was performed to evaluate distribution of cycle phases in HeLa populations seeded at different confluency . graph shows of Sec24C mean in SG ( normalized for Sec24C ) at indicated confluency . Mean ± s . e . m . of a representative experiment out of 5 biological replicates . n = 50 - 80 . ns : not significant ; * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HeLa cells were seeded at different confluency, treated with SA and stained for Sec24C and G3BP as an SG marker. Scale bar, 10 µm. Flow cytometry (FACS) analysis (right panels) was performed to evaluate the distribution of cell cycle phases in HeLa cell populations seeded at different confluency. The graph shows quantification of Sec24C mean intensity in SG puncta (normalized for the cytosolic Sec24C) at the indicated cell confluency. Mean ± s.e.m. of a representative experiment out of 5 biological replicates. n= 50-80. ns: not significant; ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


D . Differentiation of podocytes . Western blot of growing differentiated podocytes . Synaptopodin ( SYNPO ) was used as a differentiation marker , GAPDH as a loading control .

O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

D. Differentiation of podocytes. Western blot of growing and differentiated podocytes. Synaptopodin (SYNPO) was used as a differentiation marker, GAPDH as a loading control.


E . Growing differentiated podocytes were treated with SA ( 300 µM , 30 min ) stained for TRAPPC2 or Sec24C eIF3 . Scale bar , 10 µm . graphs show of TRAPPC2 Sec24C ( mean ) in SGs . Mean ± s . e . m . of three independents experiments . * * * * p < 0 . 0001 , Student ' s unpaired two - t - .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O 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. Growing and differentiated podocytes were treated with SA (300 µM, 30 min) and stained for TRAPPC2 or Sec24C and eIF3. Scale bar, 10 µm. The graphs show quantification of TRAPPC2 and Sec24C (mean intensity) in SGs. Mean ± s.e.m. of three independents experiments. ****p<0.0001, Student's unpaired two-tailed t-test.


F . Western blot of total ( RB ) phosphorylated ( p - RB ) retinoblastoma in growing ( non - differentiated ) versus differentiated podocytes . ß - actin was used as loading control .

O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR 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 total (RB) and phosphorylated (p-RB) retinoblastoma in growing (non-differentiated) versus differentiated podocytes. ß-actin was used as loading control.


G . CDK kinase activity in growing differentiated podocytes . Western blot using a specific antibody recognizing phosphoSer - CDK substrates was used on total lysates .

O O O 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. CDK kinase activity in growing and differentiated podocytes. Western blot using a specific antibody recognizing phosphoSer-CDK substrates was used on total cell lysates.


A . SG in mock , TRAPPC2 TRAPPC3 , TRAPPC8 , TRAPPC9 - depleted treated with SA . Data are shown in box - - whisker plots : Box - plot central , median ; box limits , upper lower quartiles ; whiskers show minimum to maximum . N = 3 . * * * p < 0 . 001 , * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SG area in mock, TRAPPC2 TRAPPC3, TRAPPC8, and TRAPPC9-depleted cells treated with SA. Data are shown in box-and-whisker plots: Box-plot central line, median; box limits, upper and lower quartiles; whiskers show the minimum to maximum. N=3. ***p<0.001, ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


B . HeLa microinjected with control IgG or a TRAPPC3 - specific antibody ( right panels in green ) were treated with SA . TRAPPC3 Ab disrupts Golgi ( Yu et al , 2006 ) , monitored using an anti - TGN46 Ab . Anti - G3BP was used to stain SGs . graph shows of SG . Mean ± s . e . m . three independent experiments ; n > 80 . * * * * p < 0 . 0001 , Student ' s unpaired two - t - . Scale bar , 10 µm .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HeLa cells microinjected with control IgG or a TRAPPC3-specific antibody (right panels in green) were treated with SA. The TRAPPC3 Ab disrupts the Golgi (Yu et al, 2006), monitored using an anti-TGN46 Ab. Anti-G3BP was used to stain SGs. The graph shows quantification of the SG area. Mean ± s.e.m. three independent experiments; n>80. ****p<0.0001, Student's unpaired two-tailed t-test. Scale bar, 10 µm.


C . Structured Illumination Microscopy ( SIM ) - Super resolution ( SR ) of endogenous TRAPPC2 GFP - Sec23 localizing at SGs , stained for G3BP . Scale bar 5 µm . Right , magnification of boxed . Scale bar , 0 . 5 µm .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

C. Structured Illumination Microscopy (SIM)-Super resolution (SR) images of endogenous TRAPPC2 and GFP-Sec23 localizing at SGs, stained for G3BP. Scale bar 5 µm. Right, magnification of boxed area. Scale bar, 0.5 µm.


Localization of Raptor ( D ) in mock , TRAPPC3 - KD TRAPPC2 - KD HeLa treated with SA . G3BP was used to stain SGs . Scale bar , 10 µm . Each graph shows ( mean ) of respective in SG spots expressed as a of mock ( TRL ) . Mean ± S . D . three independent replicates . * p < 0 . 02 ; * * p < 0 . 009 in ( D ) , One - way ANOVA with Dunnett ' s multiple comparison .

O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Localization of Raptor (D) in mock, TRAPPC3-KD and TRAPPC2-KD HeLa cells treated with SA. G3BP was used to stain SGs. Scale bar, 10 µm. Each graph shows the quantification (mean intensity) of the respective protein in SG spots expressed as a percentage of the mock (TRL). Mean ± S.D. three independent replicates. *p<0.02; **p<0.009 in (D) , One-way ANOVA with Dunnett's multiple comparison test.


Localization of RACK1 ( E ) in mock , TRAPPC3 - KD TRAPPC2 - KD HeLa treated with SA . G3BP was used to stain SGs . Scale bar , 10 µm . Each graph shows ( mean ) of respective in SG spots expressed as a of mock ( TRL ) . Mean ± S . D . independent replicates . , * p < 0 . 05 ; * * * * p < 0 . 0001 in ( E ) , One - way ANOVA with Dunnett ' s multiple comparison .

O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Localization of RACK1 (E) in mock, TRAPPC3-KD and TRAPPC2-KD HeLa cells treated with SA. G3BP was used to stain SGs. Scale bar, 10 µm. Each graph shows the quantification (mean intensity) of the respective protein in SG spots expressed as a percentage of the mock (TRL). Mean ± S.D. independent replicates. , *p<0.05; ****p<0.0001 in (E), One-way ANOVA with Dunnett's multiple comparison test.


Immunoprecipitation ( IP ) with an anti - TRAPPC2 Ab or IgG from with or without SA treatment , as indicated , followed by SDS - PAGE Western blot with ant - Raptor ( F ) Abs . Input : lysate ( 40 µg for TRAPPC2 , 10 µg for Raptor ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O

Immunoprecipitation (IP) with an anti-TRAPPC2 Ab or IgG from cells with or without SA treatment, as indicated, followed by SDS-PAGE and Western blot with ant-Raptor (F) Abs. Input: cell lysate (40 µg for TRAPPC2, 10 µg for Raptor).


Immunoprecipitation ( IP ) with an anti - TRAPPC2 Ab or IgG from with or without SA treatment , as indicated , followed by SDS - PAGE Western blot with anti - RACK1 ( G ) Abs . Input : lysate ( 40 µg for TRAPPC2 , 10 µg for

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O

Immunoprecipitation (IP) with an anti-TRAPPC2 Ab or IgG from cells with or without SA treatment, as indicated, followed by SDS-PAGE and Western blot with anti-RACK1 (G) Abs. Input: cell lysate (40 µg for TRAPPC2, 10 µg for RACK1


Localization of Raptor ( H ) untreated or pre - treated with indicated CDK inhibitor ( 1 µM , 150 min ) then with SA ( 300 µM , 30 min ) . G3BP was used to stain SGs . Scale bar , 10 µm . Graphs show of localization of respective with SGs , expressed as a of control . Mean ± s . e . m . of one representative experiment out of three independent replicates , n = 60 - 80 . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison .

O O B-MEASURED_VAR O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Localization of Raptor (H) untreated cells or cells pre-treated with the indicated CDK inhibitor (1 µM, 150 min) and then with SA (300 µM, 30 min). G3BP was used to stain SGs. Scale bar, 10 µm. Graphs show quantification of the localization of the respective protein with SGs, expressed as a percentage of the control. Mean ± s.e.m. of one representative experiment out of three independent replicates, n=60-80. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


Localization of RACK1 ( I ) in untreated or pre - treated with indicated CDK inhibitor ( 1 µM , 150 min ) then with SA ( 300 µM , 30 min ) . G3BP was used to stain SGs . Scale bar , 10 µm . Graphs show of localization of respective with SGs , expressed as a of control . Mean ± s . e . m . of one representative experiment out of three independent replicates , n = 60 - 80 . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison .

O O B-MEASURED_VAR O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Localization of RACK1 (I) in untreated cells or cells pre-treated with the indicated CDK inhibitor (1 µM, 150 min) and then with SA (300 µM, 30 min). G3BP was used to stain SGs. Scale bar, 10 µm. Graphs show quantification of the localization of the respective protein with SGs, expressed as a percentage of the control. Mean ± s.e.m. of one representative experiment out of three independent replicates, n=60-80. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


J . of after overnight recovery of HeLa treated or untreated with CDKi ( SNS - 032 ; Flavopiridol or Dinaciclib , 1 µM , 150 min ) then treated or not with SA ( 500 µM , 3 h ) . were acquired automatically by OPERETTA microscope . Values indicate of total number of nuclei ( stained with DAPI ) positive for BoBo - 3 . Mean ± s . d . of one representative experiment out of three independent replicates . ns : not significant , * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison .

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

J. Analysis of cell death after overnight recovery of HeLa cells treated or untreated with CDKi (SNS-032; Flavopiridol or Dinaciclib, 1 µM, 150 min) and then treated or not with SA (500 µM, 3 h). Images were acquired automatically by OPERETTA microscope. Values indicate the percentage of the total number of nuclei (stained with DAPI) positive for BoBo-3 staining. Mean ± s.d. of one representative experiment out of three independent replicates. ns: not significant, ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


K . of in HeLa that were left untreated , treated with non - targeting siRNAs or siRNAs against TRAPPC2 , in two clones knocked out for TRAPPC2 , with without SA treatment . Mean ± s . d . of three independent replicates .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

K. Analysis of cell death in HeLa cells that were left untreated, treated with non-targeting siRNAs or siRNAs against TRAPPC2, and in two cell clones knocked out for TRAPPC2, with and without SA treatment. Mean ± s.d. of three independent replicates.


A . Left , control or SA - treated ( 500 µM , 120 min ) human fibroblasts ( HFs ) were incubated at 40 ° C for 180 min stained for eIF3 , PCI , Sec24C . Right , were imaged 10 min after shifting from 40 ° C to 32 ° C stained for eIF3 , PCI , Giantin ( to label Golgi ) . Data information Scale bars , 10 µm .

O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

A. Left, control or SA-treated (500 µM, 120 min) human fibroblasts (HFs) were incubated at 40°C for 180 min and stained for eIF3, PCI, and Sec24C. Right, the cells were imaged 10 min after shifting the temperature from 40°C to 32°C and stained for eIF3, PCI, and Giantin (to label the Golgi). Data information Scale bars, 10 µm.


B . HeLa were exposed to SA ( 200 µM ) for indicated time , alone or in combination with ISRIB SGs were analyzed . of SG ( µm ) of three independent experiments . Mean ± s . e . m . n = 60 - 80 per experiment , N = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HeLa cells were exposed to SA (200 µM) for the indicated time, alone or in combination with ISRIB and SGs were analyzed. Quantification of SG area (µm) of three independent experiments. Mean ± s.e.m. n = 60-80 cells per experiment, N = 3.


C . of Sec24C localization at SGs over time ( between Sec24C ( mean ) in SG Sec24C ) . Mean ± SD . n = 60 - 80 per experiment , N = 3 .

O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

C. Quantification of Sec24C localization at SGs over time (the ratio between Sec24C (mean fluorescence intensity) in SG puncta and cytosolic Sec24C). Mean ± SD. n = 60-80 cells per experiment, N = 3.


D . HeLa expressing VSV - G - UVR8 mEOS were left untreated or treated with SA alone or in combination with ISRIB ( 1 µM ) ( 120 min ISRIB pre - treatment 30 min SA + ISRIB ) then pulsed with blue . were taken 10 min after UV processed for with indicated markers . E . were treated with SA for 30 min , SA was washed out ( WO ) , were left to recover for 120 min in presence of cycloheximide , followed by a blue processing for as described in ( D ) . F . of VSV - G ( mean ) in Golgi to total VSV - G per under conditions described in ( D , E ) . Data are expressed as of control . Mean ± s . e . m . of three independent experiment . * * * * p < 0 . 0001 ; ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison . Data information : Scale bars , 10 µm .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HeLa cells expressing VSV-G-UVR8 mEOS were left untreated or treated with SA alone or in combination with ISRIB (1 µM) (120 min ISRIB pre-treatment and 30 min SA+ISRIB) and then pulsed with blue light. Images were taken 10 min after the UV pulse and processed for staining with the indicated markers. E. Cells were treated with SA for 30 min, the SA was washed out (WO), and cells were left to recover for 120 min in the presence of cycloheximide, followed by a blue light pulse and processing for staining as described in (D). F. Quantification of VSV-G (mean intensity) in the Golgi area to the total VSV-G per cell under the conditions described in (D,E). Data are expressed as percentage of the control. Mean ± s.e.m. of three independent experiment. ****p<0.0001; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test. Data information: Scale bars, 10 µm.


A . AiryScan of HeLa treated with SA for indicated times stained with GM130 ( gray ) . Scale bar , 10 µm . graph shows of Golgi objects in SA - treated . Mean ± s . e . m . of three independent experiments . n = 90 - 100 . * * * * p < 0 . 0001 ; ns : not significant , Student ' s unpaired two - t - .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. AiryScan images of HeLa cells treated with SA for the indicated times and stained with GM130 (gray). Scale bar, 10 µm. The graph shows quantification of Golgi objects in SA-treated cells. Mean ± s.e.m. of three independent experiments. n=90-100. ****p<0.0001; ns: not significant, Student's unpaired two-tailed t-test.


B . Control treated with Dinaciclib ( CDKi ) were treated with SA stained for GM130 ( in gray ) . Insets , eIF3 . Blue , DAPI . graph shows of Golgi objects in HeLa untreated or treated with SNS - 032 , Flavopiridol , or Dinaciclib for 150 min then treated with SA ( 300 µM , 30 min ) . Mean ± s . e . m . of three independent experiments . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Control cells and cells treated with Dinaciclib (CDKi) were treated with SA and stained for GM130 (in gray). Insets, eIF3. Blue, nuclear DAPI staining. The graph shows quantification of Golgi objects in HeLa cells untreated or treated with SNS-032, Flavopiridol, or Dinaciclib for 150 min and then treated with SA (300 µM, 30 min). Mean ± s.e.m. of three independent experiments. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


C . of Golgi objects in plated at different of confluency treated or untreated with SA . Mean ± s . e . m . of three independent experiments . * * p < 0 . 001 ; * * * p < 0 . 0002 ; ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of Golgi objects in cells plated at different percentage of confluency and treated or untreated with SA. Mean ± s.e.m. of three independent experiments. **p<0.001; ***p<0.0002; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.


D . of Golgi objects in pre - treated with ISRIB ( 1 µM ) for 3 h then exposed to SA ( 30 min ) in presence of ISRIB . * * * p < 0 . 002 , Student ' s unpaired two - t - .

O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of Golgi objects in cells pre-treated with ISRIB (1 µM) for 3 h and then exposed to SA (30 min) in the presence of ISRIB. ***p<0.002, Student's unpaired two-tailed t-test.


E . Electron microscopy of treated ( 300 µM , 30 min ) or untreated with SA . arrow indicates Golgi . Scale bar , 200 nm .

O O O O 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. Electron microscopy images of cells treated (300 µM, 30 min) or untreated with SA. The arrow indicates the Golgi complex. Scale bar, 200 nm.


F . AiryScan of HeLa at steady state or exposed to SA for indicated times . A Rab1 - GTP specific antibody was used to monitor pool of active Rab1 , GM130 to stain Golgi . Insets , eIF3 . Scale bar , 10 µm . graph shows of Rab1 - GTP at Golgi expressed as a of control . Mean ± s . e . m . of three independent experiments . * p < 0 . 011 , * * p < 0 . 002 , Student ' s unpaired two - t - .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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. AiryScan images of HeLa cells at steady state or exposed to SA for the indicated times. A Rab1-GTP specific antibody was used to monitor the pool of active Rab1, and GM130 to stain the Golgi complex. Insets, eIF3. Scale bar, 10 µm. The graph shows quantification of Rab1-GTP at the Golgi complex expressed as a percentage of the control. Mean ± s.e.m. of three independent experiments. *p<0.011, **p<0.002, Student's unpaired two-tailed t-test.


G . HeLa transfected or not with WT GFP - Rab1B were left untreated or treated with SA stained for GM130 G3BP to monitor SGs . Dashed , WT GFP - Rab1B transfected . Scale bar , 10 µm . graph shows of Golgi objects in different conditions . NT : non - transfected . Mean ± s . e . m . of three independent experiments . * * * * p < 0 . 0001 , Student ' s unpaired two - t - .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HeLa cells transfected or not with WT GFP-Rab1B were left untreated or treated with SA and stained for GM130 and G3BP to monitor SGs. Dashed white line, WT GFP-Rab1B transfected cells. Scale bar, 10 µm. The graph shows quantification of Golgi objects in the different conditions. NT: non-transfected. Mean ± s.e.m. of three independent experiments. ****p<0.0001, Student's unpaired two-tailed t-test.


( A , B ) Immunoblot of ATG5 - ATG12 in cortical brain lysates of 13 - week - old Atg5flox : CamKIIα - Cre WT / KO mice ( WT set to 100 % , KO : 28 . 13 ± 8 . 18 % ; p = 0 . 0005 , nWT = 3 , nKO = 3 , one - unpaired t - ) .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of ATG5-ATG12 protein levels in cortical brain lysates of 13-week-old Atg5flox:CamKIIα-Cre WT/KO mice (WT set to 100%, KO: 28.13±8.18%; p=0.0005, nWT=3, nKO=3, one-tailed unpaired t-test).


( C , D ) Immunoblot of ATG5 - ATG12 level in striatal brain lysates of 13 - week - old Atg5flox : Slc32a1 - Cre WT / KO mice ( WT set to 100 % , KO : 21 . 71 ± 0 . 41 % ; p < 0 . 0001 , nWT = 3 , nKO = 3 , one - unpaired t - ) .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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,D) Immunoblot analysis of ATG5-ATG12 protein level in striatal brain lysates of 13-week-old Atg5flox:Slc32a1-Cre WT/KO mice (WT set to 100%, KO: 21.71±0.41%; p<0.0001, nWT=3, nKO=3, one-tailed unpaired t-test).


of p62 are significantly increased in cortical lysates of Atg5flox : CamKIIα - Cre KO mice compared to WT set to 100 % ( KO : 4855 ± 587 . 4 % ; p < 0 . 0001 , n = 4 , one - unpaired t - ) .

O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Protein levels of p62 are significantly increased in cortical lysates of Atg5flox:CamKIIα-Cre KO mice compared to the WT set to 100% (KO: 4855± 587.4%; p<0.0001, n=4, one-tailed unpaired t-test).


( G , H ) of p62 are significantly increased in striatal lysates of Atg5flox : Slc32a1 - Cre KO mice compared to WT set to 100 % ( KO : 2956 ± 839 . 3 % ; p = 0 . 0072 , n = 4 , one - unpaired t - ) .

O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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, H) Protein levels of p62 are significantly increased in striatal lysates of Atg5flox:Slc32a1-Cre KO mice compared to the WT set to 100% (KO: 2956± 839.3%; p=0.0072, n=4, one-tailed unpaired t-test).


( F , G ) PKA R1α ( F ) PKA R1β ( G ) are significantly increased in cortical brain lysates from 13 - week - old Atg5flox : CamKIIα - Cre KO mice compared to WT set to 100 % ( KOPKA R1α : 286 . 1 ± 50 . 53 % , p = 0 . 005 , KOPKA R1β :

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

(F, G) PKA R1α (F) and PKA R1β (G) protein levels are significantly increased in cortical brain lysates from 13-week-old Atg5flox:CamKIIα-Cre KO mice compared to the WT set to 100% (KOPKA R1α: 286.1± 50.53%, p=0.005, KOPKA R1β:


( H , I ) PKA R1α ( H ) PKA R1β ( I ) are significantly increased in striatal lysates of Atg5flox : Slc32a1 - Cre KO mice compared to WT set to 100 % ( KOPKA R1α : 524 . 1 ± 8 . 35 % , p < 0 . 0001 , KOPKA R1β :

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

(H, I) PKA R1α (H) and PKA R1β (I) protein levels are significantly increased in striatal lysates of Atg5flox:Slc32a1-Cre KO mice compared to the WT set to 100% (KOPKA R1α: 524.1± 8.35%, p<0.0001, KOPKA R1β:


( M , N ) Immunohistochemistry of PKA R1α in NeuN - posititve neurons in hippocampus of Atg5flox : CamKIIα - Cre WT / KO mice . Scale bar : 50µm , 20µm ( small panels ) . WT : 54 . 70 ± 0 . 693 , KO : 89 . 79 ± 1 . 831 ; p < 0 . 0001 , two - unpaired t - . nWT = 64 ; nKO = 71 neurons from n = 3 mice per genotype .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, N) Immunohistochemistry analysis of PKA R1α levels in NeuN-posititve neurons in the hippocampus of Atg5flox:CamKIIα-Cre WT/KO mice. Scale bar: 50µm, 20µm (small panels). WT: 54.70±0.693, KO: 89.79±1.831; p<0.0001, two-tailed unpaired t-test. nWT= 64; nKO= 71 neurons from n=3 mice per genotype.


Immunohistochemistry of PKA R1α in GFAP - positive in hippocampus of Atg5flox : CamKIIα - Cre WT / KO mice . Scale bar : 50µm , 30µm ( small panels ) . WT : 63 . 86 ± 2 . 477 , KO : 61 . 71 ± 2 . 653 ; p = 0 . 5549 , two - unpaired t - . nWT = 45 ; nKO = 40 from n = 3 mice per genotype .

O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 analysis of PKA R1α levels in GFAP-positive cells in the hippocampus of Atg5flox:CamKIIα-Cre WT/KO mice. Scale bar: 50µm, 30µm (small panels). WT: 63.86±2.477, KO: 61.71±2.653; p=0.5549, two-tailed unpaired t-test. nWT= 45; nKO= 40 cells from n=3 mice per genotype.


( Q , R ) Immunohistochemistry of PKA R1α in GFAP - positive in hippocampus of Atg5flox : Slc32a1 - Cre WT KO mice . Scale bar : 50µm , 30µm ( small panels ) . WT : 45 . 28 ± 1 . 018 , KO : 48 . 50 ± 1 . 440 ; p = 0 . 0636 , two - unpaired t - . nWT = 61 ; nKO = 48 from n = 3 mice per genotype .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(Q, R) Immunohistochemistry analysis of PKA R1α in GFAP-positive cells in the hippocampus of Atg5flox:Slc32a1-Cre WT and KO mice. Scale bar: 50µm, 30µm (small panels). WT: 45.28±1.018, KO: 48.50±1.440; p=0.0636, two-tailed unpaired t-test. nWT= 61; nKO= 48 cells from n=3 mice per genotype.


( G ) Immunohistochemistry for PKA R1α postsynaptic density marker PSD95 on horizontal brains sections from Atg5flox : CamKIIα - Cre WT / KO mice . rectangular boxes indicate areas magnified below with arrows indicating PKA R1α PSD95 colocalization .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O

(G) Immunohistochemistry for PKA R1α and postsynaptic density marker PSD95 on horizontal brains sections from Atg5flox:CamKIIα-Cre WT/KO mice. White rectangular boxes indicate areas magnified below with arrows indicating PKA R1α and PSD95 colocalization.


( H ) Mander ' s overlap coefficient of PKA R1α PSD95 colocalization in Atg5flox : CamKIIα - Cre WT / KO hippocampus ( WT : 0 . 29 ± 0 . 009 , KO : 0 . 40 ± 0 . 012 ; p < 0 . 0001 , two - unpaired t - ) . nWT = 31 ; nKO = 38 from n = 3 mice .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mander's overlap coefficient of PKA R1α and PSD95 colocalization in Atg5flox:CamKIIα-Cre WT/KO hippocampus (WT: 0.29±0.009, KO: 0.40±0.012; p<0.0001, two-tailed unpaired t-test). nWT= 31; nKO= 38 from n=3 mice.


( I ) Electron microscopy - of immunogold - labeled PKA R1α / β on Tokuyasu cryosections of CA1 neuropil of hippocampus of Atg5flox : CamKIIα - Cre KO mice . No immunogold labelling was detected in samples where PKA R1α / β antibody were omitted ( negative control ) . Scale bar : upper row 50 nm , 500 nm lower picture .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(I) Electron microscopy-based analysis of immunogold-labeled PKA R1α/β on Tokuyasu cryosections of the CA1 neuropil area of the hippocampus of Atg5flox:CamKIIα-Cre KO mice. No immunogold labelling was detected in samples where the PKA R1α/β antibody were omitted (negative control). Scale bar: upper row 50 nm, 500 nm lower picture.


( J ) PKA R1α / β is enriched at mitochondria ( WT : 1 . 10 ± 0 . 266 , KO : 2 . 73 ± 0 . 515 ; p = 0 . 0082 , two - unpaired t - ) synapses ( WT : 0 . 52 ± 0 . 131 , KO : 1 . 14 ± 0 . 221 ; p = 0 . 0234 , two - unpaired t - ) of KO mice . Values display mean number of gold particles counted per structure in nWT = 21 n KO = 22 pictures per genotype .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) PKA R1α/β is enriched at mitochondria (WT: 1.10± 0.266, KO: 2.73± 0.515; p=0.0082, two-tailed unpaired t-test) and synapses (WT: 0.52± 0.131, KO: 1.14± 0.221; p=0.0234, two-tailed unpaired t-test) of KO mice. Values display the mean number of gold particles counted per structure in nWT=21 and n KO=22 pictures per genotype.


( A ) Western Blot of PKA R1α in NSC34 , treated with 250 nM Torin1 or deprived of - serum for 16 hours . Lysosomal degradation was blocked in last 4 hours using 67 nM of Bafilomycin A ( BafA ) . ( B ) Starvation significantly reduced PKA R1α in NSC34 ( vehicle set to 100 % , starvation : 31 . 19 ± 5 . 085 % , p < 0 . 0001 , one - unpaired t - ) . n = 5 . ( C ) Application of 67 nM BafA for 4 hours before harvesting was sufficient to stabilize PKA R1α in starved NSC34 ( starvation set to 100 % , starvation + BafA : 155 . 5 ± 13 . 93 % , p = 0 . 002 , one - unpaired t - ) .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O 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) Western Blot analysis of PKA R1α protein levels in NSC34 cells, treated with 250 nM Torin1 or deprived of amino-acids and serum for 16 hours. Lysosomal degradation was blocked in the last 4 hours using 67 nM of Bafilomycin A (BafA). (B) Starvation significantly reduced PKA R1α protein levels in NSC34 cells (vehicle set to 100%, starvation: 31.19± 5.085%, p<0.0001, one-tailed unpaired t-test). n=5. (C) Application of 67 nM BafA for 4 hours before harvesting was sufficient to stabilize the PKA R1α protein levels in starved NSC34 cells (starvation set to 100%, starvation+BafA: 155.5± 13.93%, p=0.002, one-tailed unpaired t-test).


16 hours starvation significantly increased intracellular cAMP level measured as mean in MAP2 - positive cultured neurons ( Vehicle : 212 . 4 ± 5 . 619 , Starvation : 255 . 1 ± 5 . 715 ; p < 0 . 0001 , two - paired t - ) . nVehicle = 314 , nStarvation = 306 neurons from n = 3 .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

16 hours starvation significantly increased intracellular cAMP level measured as mean intensity in MAP2- positive cultured primary neurons (Vehicle: 212.4±5.619, Starvation: 255.1±5.715; p<0.0001, two-tailed paired t-test). nVehicle=314, nStarvation=306 neurons from n=3.


( F , G ) Phosphorylation state of containing PKA substrate RRXS / T motif was increased upon 16 hours of - acid serum starvation in cortico - hippocampal neurons at DIV14 was suppressed by 1 µM H89 supplementation in media ( Vehicle set to 100 % , Starvation : 326 . 60 ± 94 . 53 % , H89 : 214 . 80 ± 56 . 87 % , p = 0 . 033 , one - way ANOVA with Dunn ' s multiple comparison ) . n = 4 . pPKA substrate were normalized to total amount stained with Ponceau S .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G) Phosphorylation state of proteins containing PKA substrate RRXS/T motif was increased upon 16 hours of amino-acid and serum starvation in primary cortico-hippocampal neurons at DIV14 and was suppressed by 1 µM H89 supplementation in the media (Vehicle set to 100%, Starvation: 326.60±94.53%, H89: 214.80±56.87%, p=0.033, one-way ANOVA with Dunn's multiple comparison test). n=4. The pPKA substrate levels were normalized to the total protein amount stained with Ponceau S.


Treatment of NSC34 with BafA ( 67nM ) for 6 hours significantly increased PKA R1α compared to DMSO - treated group set to 100 % ( BafA : 198 . 9 ± 28 . 44 % , p = 0 . 003 , one - unpaired t - ) .

O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Treatment of NSC34 cells with BafA (67nM) for 6 hours significantly increased PKA R1α protein levels compared to the DMSO-treated group set to 100% (BafA: 198.9± 28.44%, p=0.003, one-tailed unpaired t-test).


Treatment of NSC34 with BafA ( 67nM ) for 6 hours significantly increased PKA R1α compared to DMSO - treated group set to 100 % ( BafA : 198 . 9 ± 28 . 44 % , p = 0 . 003 , one - unpaired t - ) . n = 6 .

O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Treatment of NSC34 cells with BafA (67nM) for 6 hours significantly increased PKA R1α protein levels compared to the DMSO-treated group set to 100% (BafA: 198.9± 28.44%, p=0.003, one-tailed unpaired t-test). n=6.


( J ) Western blot of purified autophagosomes ( AVs ) ( 50μg AVs / lane ) with without Proteinase K ( PK ) treatment . TX - 100 ( 1 % final ) was used as a positive control for activity of PK . Synaptosome lysates ( Syn , 30μg / lane ) were used as a positive control for signal of antibodies .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(J) Western blot analysis of purified autophagosomes (AVs) (50μg AVs/lane) with and without Proteinase K (PK) treatment. TX-100 (1% final) was used as a positive control for the activity of PK. Synaptosome lysates (Syn, 30μg/lane) were used as a positive control for the signal of antibodies.


( M , N ) Representative subsequent of PKA R1 - α / p62 colocalization ( Pearson ' s correlation coefficient ) in cortico - hippocampal neurons , which have undergone 16 hours - serum starvation were additionally treated with BafA ( 67nM ) to visualize lysosomes ( control : 0 . 68 ± 0 . 019 , starvation : 0 . 58 ± 0 . 015 , starvation + BafA : 0 . 83 ± 0 . 014 ; pcontrol vs starvation < 0 . 0001 , pcontrol vs starvation + BafA < 0 . 0001 , pstarvation vs starvation + BafA < 0 . 0001 Ordinary one - way ANOVA with Tukey ' s multiple comparison ) . ncontrol = 36 , nstarvation = 38 , nstarvation + BafA = 36 neurons from n = 3 .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, N) Representative fluorescent images and subsequent analysis of PKA R1- α/p62 colocalization (Pearson's correlation coefficient) in primary cortico-hippocampal neurons, which have undergone 16 hours amino-acids and serum starvation and were additionally treated with BafA (67nM) to visualize the lysosomes (control: 0.68± 0.019, starvation: 0.58± 0.015, starvation+BafA: 0.83± 0.014; pcontrol vs starvation<0.0001, pcontrol vs starvation+BafA<0.0001, pstarvation vs starvation+BafA<0.0001 Ordinary one-way ANOVA with Tukey's multiple comparison test). ncontrol=36, nstarvation=38, nstarvation+BafA=36 neurons from n=3.


Co - immunoprecipitation of endogenous PKA R1 - β with PKA - Cα from Atg5flox : CamKIIα - Cre WT / KO mouse brain lysates ( WT set to 100 % , KO : 170 . 3 ± 14 . 88 % , p = 0 . 005 , one - unpaired t - , n = 3 ) . Input , 1 . 5 % of total lysate was added to .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Co-immunoprecipitation of endogenous PKA R1-β with PKA-Cα from Atg5flox:CamKIIα-Cre WT/KO mouse brain lysates (WT set to 100%, KO: 170.3± 14.88%, p=0.005, one-tailed unpaired t-test, n=3). Input, 1.5% of the total lysate was added to the assay.


( A - B ) Western Blot of pCREB in Atg5flox : CAG - CreTmx WT / KO cultured neurons at DIV14 , treated with forskolin ( WTDMSO set to 100 % , WTForskolin : 214 . 17 ± 48 . 87 % , KODMSO : 63 . 26 ± 15 . 85 % , KOForskolin : 33 . 50 ± 10 . 84 % , pWT DMSO / WT Forskolin = 0 . 042 , pWT Forskolin / KO Forskolin = 0 . 002 , two - way ANOVA with Tukey ' s multiple comparisons ) . n = 4 . same lysates were run in parallel to detect pCREB CREB normalized to their respective loading control before calculating pCREB / CREB .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O

(A-B) Western Blot analysis of pCREB protein levels in Atg5flox:CAG-CreTmx WT/ KO cultured neurons at DIV14, treated with forskolin (WTDMSO set to 100%, WTForskolin: 214.17± 48.87%, KODMSO: 63.26± 15.85%, KOForskolin: 33.50± 10.84%, pWT DMSO/WT Forskolin=0.042, pWT Forskolin/KO Forskolin=0.002, two-way ANOVA with Tukey's multiple comparisons). n=4. The same lysates were run in parallel to detect pCREB and CREB and normalized to their respective loading control before calculating the pCREB/CREB ratio.


( I , J ) Representative subsequent of cortico - hippocampal neurons containing pCREB either under DMSO - treated condition or under condition when serum starvation was induced for 16 hours in absence or presence of 1µM of PKA inhibitor H89 . Neurons were identified by their polarized in DIC . rectangular boxes indicate panel magnified to right . In inserts , LUT gradient was added to highlight pCREB in nucleus . Scale bar : 50 µm . DMSO : 52 . 50 ± 2 . 757 , starvation : 70 . 00 ± 2 . 697 , starvation + H89 : 53 . 92 ± 3 . 147 ; pDMSO / starvation < 0 . 0001 , pstarvation / starvation & H89 = 0 . 0004 , one - way ANOVA with Tukey ' s multiple comparisons , n = 30 from n = 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, J) Representative fluorescent images and subsequent analysis of primary cortico-hippocampal neurons containing nuclear pCREB either under DMSO-treated condition or under condition when amino acids and serum starvation was induced for 16 hours in the absence or presence of 1µM of PKA inhibitor H89. Neurons were identified by their polarized morphology in DIC. White rectangular boxes indicate panel magnified to the right. In the inserts, LUT gradient was added to highlight the pCREB intensity in the nucleus. Scale bar: 50 µm. DMSO: 52.50±2.757, starvation: 70.00±2.697, starvation+H89: 53.92±3.147; pDMSO/starvation<0.0001, pstarvation/starvation&H89=0.0004, one-way ANOVA with Tukey's multiple comparisons test, n=30 images from n=3 independent experiments.


( A - B ) Western Blot of PKA substrates carrying RRXS / T motif in cortico - hippocampal Atg5flox : CAG - CreTmx WT / KO neurons at DIV14 , treated with Forskolin ( WTDMSO set to 100 % , WTForskolin : 291 . 85 ± 17 . 02 % , KODMSO : 152 . 97 ± 25 . 26 % , KOForskolin : 182 . 56 ± 31 . 89 % , pWTDMSO / WTForskolin : 0 . 001 , pWTForskolin / KOForskolin : 0 . 033 , two - way ANOVA with Tukey ' s multiple comparisons ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western Blot analysis of PKA substrates carrying RRXS/T motif in primary cortico-hippocampal Atg5flox:CAG-CreTmx WT/KO neurons at DIV14, treated with Forskolin (WTDMSO set to 100%, WTForskolin: 291.85± 17.02%, KODMSO: 152.97± 25.26%, KOForskolin: 182.56± 31.89%, pWTDMSO/WTForskolin: 0.001, pWTForskolin/KOForskolin: 0.033, two-way ANOVA with Tukey's multiple comparisons).


( K - M ) Representative STED Pearson ' s colocalization coefficient of SHANK3 ( K ) CASKIN1 ( L ) PSD95 in Atg5flox : CamKIIα - Cre : Ai9 WT / KO neurons . Scale bar : 5µm , 1µm ( small panels ) . WTSHANK3 : 0 . 324 ± 0 . 022 , KOSHANK3 : 0 . 415 ± 0 . 024 ; p = 0 . 0067 , two - unpaired t - . nWT = 46 , nKO = 46 . WTCASKIN1 : 0 . 255 ± 0 . 024 , KO CASKIN1 : 0 . 326 ± 0 . 025 ; p = 0 . 0432 , two - unpaired t - . nWT = 39 , nKO = 43 from n = 2 independent experiments .

O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K-M) Representative STED images and Pearson's colocalization coefficient analysis of SHANK3 (K) and CASKIN1 (L) and PSD95 in Atg5flox:CamKIIα-Cre:Ai9 WT/KO neurons. Scale bar: 5µm, 1µm (small panels). WTSHANK3: 0.324±0.022, KOSHANK3: 0.415±0.024; p=0.0067, two-tailed unpaired t-test. nWT=46, nKO=46. WTCASKIN1: 0.255±0.024, KO CASKIN1: 0.326±0.025; p=0.0432, two-tailed unpaired t-test. nWT=39, nKO=43 from n=2 independent experiments.


( C , D ) of GLUR1 at spines of 10 - 11 week - old Atg5wt / wt : Ai9 Atg5flox / flox : Ai9 mice injected with CamKIIα - Cre AAV into hippocampus . Scale bar : 5µm . GLUR1 level at PSD95 - positve spines were assessed measuring mean gray values after background subtraction ( WT : 64 . 55 ± 7 . 988 % , KO : 99 . 14 ± 6 . 863 % ; p = 0 . 0029 , two - unpaired t - ) . nWT = 13 , nKO = 14 from n = 3 mice ( > 31 dendritic spines analyzed per each mouse ) . sp , spine .

O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,D) Analysis of GLUR1 fluorescent levels at the spines of 10-11 week-old Atg5wt/wt:Ai9 and Atg5flox/flox:Ai9 mice injected with CamKIIα-Cre AAV into the hippocampus. Scale bar: 5µm. GLUR1 level at PSD95-positve spines were assessed measuring mean gray values after background subtraction (WT: 64.55±7.988%, KO: 99.14±6.863%; p=0.0029, two-tailed unpaired t-test). nWT=13, nKO=14 images from n=3 mice (>31 dendritic spines analyzed per each mouse). sp, spine.


( G ) Representative of Atg5flox : CAG - CreTmx WT / KO neurons transfected with eGFP or eGFP - ATG5 immunostained for GLUR1 , PSD95 GFP . ( H ) overexpression of eGFP - ATG5 in Atg5flox : CAG - CreTmx KO neurons diminished of GLUR1 / PSD95 colocalization compared to eGFP - overexpressing KO neurons ( KOeGFP : 70 . 07 ± 1 . 473 % , KOeGFP - ATG5 : 65 . 42 ± 1 . 721 % , ; p = 0 . 048 , two - unpaired t - ) . nKO + GFP = 42 , nKO + GFP - ATG5 = 50 from n = 5 . ( I , J ) coexpression of eGFP - PKA R1α eGFP - PKA R1β in cultured Atg5flox : CAG - CreTmx WT neurons results in significantly increased colocalization between GLUR1 PSD95 compared to eGFP - expressing neurons ( WT : 42 . 30 ± 2 . 453 % , KO : 51 . 32 ± 2 . 624 % , p = 0 . 014 , two - unpaired t - ) . nWT + GFP = 46 , nWTGFP + PKA R1 OE = 46 from n = 3 . in H , J is performed using a unbiased custom - written Plugin for ImageJ .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative fluorescence images of Atg5flox:CAG-CreTmx WT/KO neurons transfected with eGFP or eGFP-ATG5 and immunostained for GLUR1, PSD95 and GFP. (H) The overexpression of eGFP-ATG5 in Atg5flox:CAG-CreTmx KO neurons diminished the of GLUR1/PSD95 colocalization compared to the eGFP-overexpressing KO neurons (KOeGFP: 70.07±1.473%, KOeGFP-ATG5: 65.42±1.721%,; p=0.048, two-tailed unpaired t-test). nKO+GFP=42, nKO+GFP-ATG5=50 images from n=5. (I, J) The coexpression of eGFP-PKA R1α and eGFP-PKA R1β in cultured Atg5flox:CAG-CreTmx WT neurons results in significantly increased colocalization between GLUR1 and PSD95 compared to the eGFP-expressing neurons (WT: 42.30±2.453%, KO: 51.32±2.624%, p=0.014, two-tailed unpaired t-test). nWT+GFP=46, nWTGFP+PKA R1 OE=46 images from n=3. Analysis in H, J is performed using a unbiased custom-written Plugin for ImageJ.


( K - M ) GCAMP7f responses to tetanic stimulation ( four tetani , 100 APs at 100Hz , 3s interval ) in bodies of CamKIIα - Cre - transduced cortico - hippocampal Atg5wt : wt / Ai9 ( WT ) Atg5flox : flox / Ai9 ( KO ) neurons expressing mCamKIIα - jGCaMP7f treated either with DMSO ( K ) or with 10 µM CNQX for 5 min ( L ) . Autophagy deficient showed significantly increased facilitation of GCAMP7f signal to electrical stimulation ( ΔF / Fpeak1 ) compared to WT , a phenotype which was blocked by application of CNQX ( WTDMSO : 0 . 28 ± 0 . 12 , WTCNQX : - 0 . 07 ± 0 . 03 , KODMSO : 3 . 74 ± 0 . 86 , KOCNQX : - 0 . 014 ± 0 . 13 , p WT DMSO / KO DMSO < 0 . 0001 , pKO DMSO / KO CNQX < 0 . 0001 , two - way ANOVA with Tukey ' s multiple comparisons ) . nWT DMSO = 25 , nWT CNQX = 27 , nKO DMSO = 33 , nKO CNQX = 30 neurons from n = 3 .

O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K-M) GCAMP7f responses to tetanic stimulation (four tetani, 100 APs at 100Hz, 3s interval) in cell bodies of CamKIIα-Cre-transduced primary cortico-hippocampal Atg5wt:wt/Ai9 (WT) and Atg5flox:flox/Ai9 (KO) neurons expressing mCamKIIα-jGCaMP7f and treated either with DMSO (K) or with 10 µM CNQX for 5 min (L). Autophagy deficient cells showed significantly increased facilitation of GCAMP7f signal to electrical stimulation (ΔF/Fpeak1) compared to the WT, a phenotype which was blocked by the application of CNQX (WTDMSO: 0.28± 0.12, WTCNQX: -0.07± 0.03, KODMSO: 3.74± 0.86, KOCNQX: -0.014± 0.13, p WT DMSO/ KO DMSO<0.0001, pKO DMSO/KO CNQX<0.0001, two-way ANOVA with Tukey's multiple comparisons test). nWT DMSO= 25, nWT CNQX= 27, nKO DMSO= 33, nKO CNQX= 30 neurons from n=3.


Schematic overview of sample collection procedure for single - PCR ( scPCR ) . Single cytoplasms from acutely dissected organs of Corti of C57BL / 6J ( Wt ) mice after hearing onset were aspirated processed for scPCR as depicted . ( B ' ) Expression of endophilins - A1 - 3 from individually isolated cytoplasms using RT - PCR from a representative experimental run . Please note that for these experiments , negative bath control samples from before after isolation procedure were an essential requirement to ensure lack of contamination from debris in bath solution . HPRT was used as a housekeeping gene .

O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Schematic overview of the sample collection procedure for single-cell PCR (scPCR). Single IHC cytoplasms from acutely dissected organs of Corti of C57BL/6J (Wt) mice after hearing onset were aspirated and processed for scPCR as depicted. (B') Expression analysis of endophilins-A1-3 from individually isolated IHC cytoplasms using RT-PCR from a representative experimental run. Please note that for these experiments, negative bath control samples from before and after the isolation procedure were an essential requirement to ensure lack of contamination from cellular debris in bath solution. HPRT was used as a housekeeping gene.


Immunoblotting of tissue lysates from p15 Wt ( C57BL / 6J ) , 1 - SKO , 1 / 2 - DKO 1 / 3 - DKO revealed expression of endophilins - A1 - A2 in murine cochlea ensured antibody specificity . Unfortunately , none of commercially available endophilin - A3 antibodies we tested in these experiments gave a specific signal for A3 in cochlear extracts , but rather appeared to ( also ) detect A1 . γ - adaptin was used as loading control . antibody epitopes localize to distinct C - terminal regions of different endophilin - A family members . Hi , hippocampus ; Ce , cerebellum ; Co , pooled cochleae from a single of indicated genotype ; Md , modioli ( micro - dissected pooled from 10 Wt ) ; OC , organs of Corti ( micro - dissected pooled from 10 Wt ) .

O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of tissue lysates from p15 Wt (C57BL/6J), 1-SKO, 1/2-DKO and 1/3-DKO revealed protein expression of endophilins-A1 and -A2 in the murine cochlea and ensured antibody specificity. Unfortunately, none of the commercially available endophilin-A3 antibodies we tested in these experiments gave a specific signal for A3 in cochlear extracts, but rather appeared to (also) detect A1. γ-adaptin was used as loading control. All antibody epitopes localize to the distinct C-terminal regions of the different endophilin-A family members. Hi, hippocampus; Ce, cerebellum; Co, pooled cochleae from a single individual of the indicated genotype; Md, modioli (micro-dissected and pooled from 10 Wt animals); OC, organs of Corti (micro-dissected and pooled from 10 Wt animals).


Ca2 + - current - voltage relationships evoked by incremental 15 ms step depolarizations from - 87 mV to + 58 mV revealed a ~ 25 % reduction of ICa in 1 / 3 DKO TKO mice . ( A ' ) Due to perinatal lethality of TKO mice , detailed electrophysiological characterization of TKO IHCs had to be performed on organotypically - cultured organs of Corti after 7 days in vitro ( DIV ) . C57BL / 6J ( Wt ) 1 / 3 - DKO served as controls . Please note that recordings from cultured IHCs were performed at [ Ca2 + ] e of 10 mM to maximize exocytic performance . ( A " ) statistical of maximum ICa amplitudes ( Imax ) of respective genotypes revealed a significant reduction in Imax in both endophilin mutant genotypes . ( * p = 0 . 046 , one - way ANOVA with post - hoc Tukey ' s ) .

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

Ca2+-current-voltage relationships evoked by incremental 15 ms step depolarizations from -87 mV to +58 mV revealed a ~25% reduction of ICa in 1/3 DKO and TKO mice. (A') Due to the perinatal lethality of TKO mice, detailed electrophysiological characterization of TKO IHCs had to be performed on organotypically-cultured organs of Corti after 7 days in vitro (DIV). C57BL/6J (Wt) and 1/3-DKO served as controls. Please note that all recordings from cultured IHCs were performed at [Ca2+]e of 10 mM to maximize IHC exocytic performance. (A") Quantification and statistical analysis of individual maximum ICa amplitudes (Imax) of the respective genotypes revealed a significant reduction in Imax in both endophilin mutant genotypes. (*p = 0.046, one-way ANOVA with post-hoc Tukey's).


Ca2 + - current inactivation was probed by pulses of 100 ms to Imax potential revealed a significantly stronger inactivation phenotype in TKO IHCs when directly compared to Wt 1 / 3 - DKO . ( B ' ) statistical of residual current at end of . ( * p = 0 . 046 , one - way ANOVA with post - hoc Tukey ' s ) .

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

Ca2+-current inactivation was probed by test pulses of 100 ms to the Imax potential and revealed a significantly stronger inactivation phenotype in TKO IHCs when directly compared to Wt and 1/3-DKO cells. (B') Quantification and statistical analysis of the residual current at the end of the test pulse. (*p = 0.046, one-way ANOVA with post-hoc Tukey's).


Representative ICa ( upper panel ) Cm ( lower panel ) in response to a 50 ms depolarizing step to potential eliciting Imax . ( C ' ) Exocytic ∆ Cm corresponding QCa elicited by depolarizations of stimulus durations from 2 - 100 ms for respective genotypes at [ Ca2 + ] e = 6 mM for a second set of recordings from wild - type IHCs to experimentally approximate decreased ICa observed in endophilin mutants . ( C " ) Magnification of initial , short depolarizing steps ( 2 - 20 ms ) for clarity . Exocytic ∆ Cm of cultured endophilin - deficient IHCs appears strongly reduced . ( * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA with post - hoc Tukey ' s or non - parametric K - W with post - hoc Dunn ' s ; please also refer to Appendix Tables S2 S3 for detailed statistical ) . reduced Ca2 + - efficiency of exocytosis ( ∆ Cm / QCa ) in endophilin - deficient IHCs indicates that diminished Ca2 + - influx cannot fully account for reduction of exocytosis . ( * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA with post - hoc Tukey ' s or non - parametric K - W with post - hoc Dunn ' s ; please also refer to Appendix Table S4 for detailed statistical ) .

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

Representative ICa (upper panel) and Cm (lower panel) in response to a 50 ms depolarizing step to the potential eliciting Imax. (C') Exocytic ∆Cm and corresponding QCa elicited by depolarizations of stimulus durations from 2-100 ms for all respective genotypes and at [Ca2+]e = 6 mM for a second set of recordings from wild-type IHCs to experimentally approximate the decreased ICa observed in the endophilin mutants. (C") Magnification of the initial, short depolarizing steps (2-20 ms) for clarity. Exocytic ∆Cm of cultured endophilin-deficient IHCs appears strongly reduced. (*p < 0.05; **p < 0.01; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's; please also refer to Appendix Tables S2 and S3 for detailed statistical analysis). The reduced Ca2+-efficiency of exocytosis (∆Cm/QCa) in endophilin-deficient IHCs indicates that diminished Ca2+-influx cannot fully account for the reduction of exocytosis. (*p < 0.05; **p < 0.01; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's; please also refer to Appendix Table S4 for detailed statistical analysis).


Ca2 + - current - voltage relationships in response to 10 ms step - depolarizations . ( A ' ) peak of Ca2 + - influx was significantly reduced in endophilin 1 - SKO ( * * p = 0 . 0024 ) 1 / 3 - DKO ( * * * p < 0 . 0001 ) when compared to Wt . ( Wt n = 30 / N = 20 ; 1 - SKO n = 15 / N = 9 ; 1 / 3 - DKO n = 39 / N = 20 ; Non - parametric K - W with post - hoc Dunn ' s correction ) .

O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ca2+-current-voltage relationships in response to 10 ms step-depolarizations. (A') The peak of Ca2+-influx was significantly reduced in endophilin 1-SKO (**p = 0.0024) and 1/3-DKO (***p < 0.0001) when compared to Wt. (Wt n = 30/ N = 20; 1-SKO n = 15/ N = 9; 1/3-DKO n = 39/ N = 20; Non-parametric K-W with post-hoc Dunn's correction).


of CtBP2 - labeled ribbons revealed a number of approximately 14 - 15 ribbon synapses per in groups , that remained comparable across genotypes ( Wt n = 12 / N = 9 ; 1 - SKO n = 6 / N = 4 ; 1 / 3 - DKO n = 8 / N = 6 ; one - way ANOVA , p = 0 . 717 ) .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of CtBP2-labeled ribbons revealed a number of approximately 14-15 ribbon synapses per IHC in all groups, that remained comparable across all genotypes (Wt n = 12/ N = 9; 1-SKO n = 6/ N = 4; 1/3-DKO n = 8/ N = 6; one-way ANOVA, p = 0.717).


of cross - sectional of CaV1 . 3 immunofluorescence revealed approx . 34 % smaller clusters in 1 - SKOs 24 % smaller clusters in 1 / 3 - DKOs . ( Wt n = 153 / N = 2 ; 1 - SKO n = 102 / N = 2 ; 1 / 3 - DKO n = 441 / N = 4 ; * * * p < 0 . 0001 ; K - W Dunn ' s post - hoc ) .

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

Quantification of the cross-sectional area of CaV1.3 immunofluorescence revealed approx. 34 % smaller clusters in 1-SKOs and 24 % smaller clusters in 1/3-DKOs. (Wt n = 153/ N = 2; 1-SKO n = 102/ N = 2; 1/3-DKO n = 441/ N = 4; ***p < 0.0001; K-W and Dunn's post-hoc test).


Representative maximum projections of confocal sections from organs of Corti of p15 Wt , 1 - SKO , 1 / 3 - DKO mice stained for CtBP2 to label synaptic ribbons . Please note that CtBP2 expression is also found in nucleus ( nuclei are highlighted by dashed circles in panels ) . Scale bars : 10 µm .

O O 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-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Representative maximum projections of confocal sections from organs of Corti of p15 Wt, 1-SKO, and 1/3-DKO mice stained for CtBP2 to label synaptic ribbons. Please note that CtBP2 expression is also found in the nucleus (nuclei are highlighted by dashed circles in the individual panels). Scale bars: 10 µm.


2D STED of ribbon synapses stained for CtBP2 ( magenta ) CaV1 . 3 ( green ) . CaV1 . 3 Ca2 + - channels remain clustered at AZs of 1 - SKO 1 / 3 - DKO IHCs . Scale bars : 1 µm .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

2D STED images of IHC ribbon synapses stained for CtBP2 (magenta) and CaV1.3 (green). CaV1.3 Ca2+-channels remain clustered at AZs of 1-SKO and 1/3-DKO IHCs. Scale bars: 1 µm.


Representative Ca2 + - currents ( upper panel ) exocytic capacitance increments ( ∆ Cm ; lower panel ) in response to a 200 ms depolarizing step to - 14 mV .

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

Representative Ca2+-currents (upper panel) and exocytic membrane capacitance increments (∆Cm; lower panel) in response to a 200 ms depolarizing step to -14 mV.


Exocytic ∆ Cm ( top ) corresponding Ca2 + - current integrals QCa ( bottom ) of Wt endophilin - deficient IHCs in response to voltage steps from - 84 to - 14 mV of variable stimulus duration ( 5 to 200 ms ) . In 1 / 3 - DKO IHCs , our data indicate reduced sustained exocytosis to a strong depolarization ( 200 ms ; * p = 0 . 0470 , K - W with Dunn ' s post - hoc ; please also refer to Appendix Tables S5 S6 for detailed statistical ) . Inset : Initial ∆ Cm showed comparable RRP exocytosis in endophilin - deficient IHCs .

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

Exocytic ∆Cm (top) and corresponding Ca2+-current integrals QCa (bottom) of Wt and endophilin-deficient IHCs in response to voltage steps from -84 to -14 mV of variable stimulus duration (5 to 200 ms). In 1/3-DKO IHCs, our data indicate reduced sustained exocytosis to a strong depolarization (200 ms; *p = 0.0470, K-W with Dunn's post-hoc test; please also refer to Appendix Tables S5 and S6 for detailed statistical analysis). Inset: Initial ∆Cm showed comparable RRP exocytosis in endophilin-deficient IHCs.


Ca2 + - efficiency to drive exocytosis was reduced for strong depolarizations ( 200 ms ) in 1 / 3 - DKO IHCs ; * p = 0 . 0323 , K - W with Dunn ' s post - hoc ; please also refer to Appendix Table S7 for detailed statistical .

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

Ca2+-efficiency to drive exocytosis was reduced for strong depolarizations (200 ms) in 1/3-DKO IHCs; *p = 0.0323, K-W with Dunn's post-hoc test; please also refer to Appendix Table S7 for detailed statistical analysis.


Confocal maximum projections of otoferlin - immunolabeled organs of Corti from p15 Wt 1 / 3 - DKO mice illustrated with an - coded look - up table where brighter colors indicate higher . Scale bars : 5 µm .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal maximum projections of otoferlin-immunolabeled organs of Corti from p15 Wt and 1/3-DKO mice illustrated with an intensity-coded look-up table where brighter colors indicate higher intensity. Scale bars: 5 µm.


Otoferlin were reduced by approx . 28 . 4 ± 3 . 5 % in 1 / 3 - DKO IHCs compared to Wt IHCs ( relative intensities normalized to Wt ; Wt n = 31 ( 229 IHCs ) / N = 5 organs of Corti ( 3 ) ; 1 / 3 - DKO n = 20 ( 196 IHCs ) / N = 5 organs of Corti ( 4 ) ; unpaired student ' s t - ; * * * p < 0 . 001 ; box plots illustrate median with interquartile range , whiskers indicate 10 - 90 % of data points , squares present respective mean value ) .

B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Otoferlin levels were reduced by approx. 28.4 ± 3.5 % in 1/3-DKO IHCs compared to Wt IHCs (relative intensities normalized to Wt; Wt n = 31 images (229 IHCs)/ N = 5 organs of Corti (3 animals); 1/3-DKO n = 20 images (196 IHCs)/ N = 5 organs of Corti (4 animals); unpaired student's t-test; ***p < 0.001; box plots illustrate the median with the interquartile range, whiskers indicate 10-90 % of data points, the squares present the respective mean value).


Normalized otoferlin profiles through single IHCs of respective genotypes at a longitudinal central plane through nucleus from apical ( 0 . 0 ) to basal ( 1 . 0 ) revealed similar distribution patterns of otoferlin in IHCs of Wt 1 / 3 - DKO an overall reduction of otoferlin throughout entire in 1 / 3 - DKOs . Traces indicate mean pixel ± ; Wt n = 123 / N = 3 ; 1 / 3 - DKO n = 124 / N = 3 .

O B-MEASURED_VAR O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Normalized otoferlin intensity line profiles through single IHCs of the respective genotypes at a longitudinal central plane through the nucleus from apical (0.0) to basal (1.0) revealed similar distribution patterns of otoferlin in IHCs of Wt and 1/3-DKO and an overall reduction of otoferlin levels throughout the entire cell in 1/3-DKOs. Traces indicate the mean pixel intensity ± SEM; Wt n = 123/ N = 3; 1/3-DKO n = 124/ N = 3.


Interaction of otoferlin endophilin - A1 detected by co - IP in HeLa co - expressing GFP - otoferlin RFP - endophilin - A1 . Otoferlin - GFP was immunoprecipitated ( IP ) by GFP - Trap beads , blots were probed with a KO - validated anti - endophilin - A1 antibody .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O

Interaction of otoferlin and endophilin-A1 detected by co-IP in HeLa cells co-expressing GFP-otoferlin and RFP-endophilin-A1. Otoferlin-GFP was immunoprecipitated (IP) by GFP-Trap beads, and blots were probed with a KO-validated anti-endophilin-A1 antibody.


Exogenously overexpressed GFP - otoferlin was immunoprecipitated using GFP - Trap beads incubated with purified endophilin - A1 ( pA1 ) . IP was then followed by immunoblotting with an anti - endophilin - A1 antibody .

O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O

Exogenously overexpressed GFP-otoferlin was immunoprecipitated using GFP-Trap beads and incubated with purified endophilin-A1 (pA1). IP was then followed by immunoblotting with an anti-endophilin-A1 antibody.


Endocytosis was assessed by determining decrease in Cm during 20 s post - depolarization in perforated patch recordings from both Wt , 1 / 2 - DKO 1 / 3 - DKO IHCs . two kinetic components are presented as average ∆ Cm recordings after ( A ) 20 ms ( Wt n = 12 / N = 8 ; 1 / 2 - DKO n = 13 / N = 7 ; 1 / 3 - DKO n = 13 / N = 10 ) ( B ) 200 ms ( Wt n = 12 / N = 8 ; 1 / 2 - DKO n = 13 / N = 6 ; 1 / 3 - DKO n = 14 / N = 10 ) depolarization stimuli .

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

Endocytosis was assessed by determining the decrease in Cm during 20 s post-depolarization in perforated patch recordings from both Wt, 1/2-DKO and 1/3-DKO IHCs. The two kinetic components are presented as average ∆Cm recordings after (A) 20 ms (Wt n = 12/ N = 8; 1/2-DKO n = 13/ N = 7; 1/3-DKO n = 13/ N = 10) and (B) 200 ms (Wt n = 12/ N = 8; 1/2-DKO n = 13/ N = 6; 1/3-DKO n = 14/ N = 10) depolarization stimuli.


Average time of return to baseline obtained by fitting a linear function to Cm - data following a 20 ms depolarization .

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

Average time of return to baseline obtained by fitting a linear function to the Cm-data following a 20 ms depolarization.