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 .

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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 the different CDKs as described by Selleckchem , and Sec24C recruitment to SGs as a percentage of control .

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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 . 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 cells treated with SA were immunostained for TRAPPC2 and eIF3 . Data information : Scale bars , 10 µm .

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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 cells treated with SA were immunostained for Sec24C and 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 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 and differentiated podocytes . Synaptopodin ( SYNPO ) was used as a differentiation marker , GAPDH as a loading control .

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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 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 .

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

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

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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

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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 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 .

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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 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 .

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 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

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 . 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 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 .

O 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 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 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 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 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 .

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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 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 .

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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 . 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 .

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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 . 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 .

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

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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 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 .

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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 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 .

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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 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 ) .

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(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 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 ) .

(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).


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 ) .

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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 ) 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 ) .

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(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 ) 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 :

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(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 ) 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 :

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(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 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 .

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(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 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 .

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

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(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 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 .

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(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 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 .

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(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 - 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 .

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(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 - 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 .

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 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 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 ) .

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 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 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

(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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

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 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 .

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

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 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 .

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 B-CONTROLLED_VAR 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

(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 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 .

O O O O 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 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 - tailed unpaired t - test , n = 3 ) . Input , 1 . 5 % of the total lysate was added to the assay .

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 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 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 .

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 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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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 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

(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 ) 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 .

O O O O O O O B-MEASURED_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 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 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 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 .

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

(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 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 .

O O O O O O O O O O O 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 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

O O O O O O O O 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 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 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

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 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 ) .

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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 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 ) .

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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 ) 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 ) .

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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 ' ) 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 ) .

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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).


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 ) .

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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).


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 ) .

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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 , 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 .

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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 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 .

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

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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 ) 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 .

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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 test ; please also refer to Appendix Table S7 for detailed statistical analysis .

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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 and 1 / 3 - DKO mice illustrated with an intensity - coded look - up table where brighter colors indicate higher intensity . Scale bars : 5 µm .

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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 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 ) .

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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 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 .

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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 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 .

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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 and incubated with purified endophilin - A1 ( pA1 ) . IP was then followed by immunoblotting with an anti - endophilin - A1 antibody .

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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 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 .

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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 the Cm - data following a 20 ms depolarization .

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


Average slope of the linear component of endocytosis for 20 ms and 200 ms depolarizing pulses , obtained from fitting Cm - data for 20 s after cessation of the 20 ms depolarizations or for the last 10 s of the Cm - recording for 200 ms depolarizations . ( * p < 0 . 05 ; one - way ANOVA with post - hoc Tukey ' s or non - parametric K - W with post - hoc Dunn ' s ) .

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Average slope of the linear component of endocytosis for 20 ms and 200 ms depolarizing pulses, obtained from fitting Cm-data for 20 s after cessation of the 20 ms depolarizations or for the last 10 s of the Cm- recording for 200 ms depolarizations. (*p < 0.05; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's).


Average amplitude of the exponential component obtained from fits to Cm - data following 200 ms depolarization ( nexp fit = 11 for Wt , 10 for 1 / 2 - DKO and 11 for 1 / 3 - DKO ; x data points in ( E ) correspond to IHCs with no exponential component in Cm ) . correspond to statistically identified outliers .

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Average amplitude of the exponential component obtained from fits to Cm-data following 200 ms depolarization (nexp fit = 11 for Wt, 10 for 1/2-DKO and 11 for 1/3-DKO; x data points in (E) correspond to IHCs with no exponential component in Cm). correspond to statistically identified outliers.


time constant of the exponential component obtained from fits to Cm - data following 200 ms

time constant of the exponential component obtained from fits to Cm-data following 200 ms depolarization


Representative electron micrograph illustrating the region of interest for analysis within a radius of r = 1 µm from the ribbon center ( exemplary for a 1 / 3 - DKO specimen ) . Graphical aids indicate small vesicles , ELVs and different subpopulations of coated structures quantified in ( B - D ) . Scale bar : 500 nm .

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Representative electron micrograph illustrating the region of interest for analysis within a radius of r = 1 µm from the ribbon center (exemplary for a 1/3-DKO specimen). Graphical aids indicate small vesicles, ELVs and different subpopulations of coated structures quantified in (B-D). Scale bar: 500 nm.


While an increased number of cytosolic vesicles can be observed at 1 - SKO AZs , reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1 / 2 - DKOs . RA - SVs were excluded from this quantification .

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While an increased number of cytosolic vesicles can be observed at 1-SKO AZs, reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1/2-DKOs. RA-SVs were excluded from this quantification.


Accumulations of ELVs occur in 1 / 2 - DKO and 1 / 3 - DKO IHCs , as measured by the relative cumulative ELV area per section .

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Accumulations of ELVs occur in 1/2-DKO and 1/3-DKO IHCs, as measured by the relative cumulative ELV area per section.


Increased overall number of coated structures in endophilin mutants . ( D ' ) Prominent accumulation of coated pits can be observed in 1 / 2 - DKO and 1 / 3 - DKO IHCs alongside a shift towards more coated vacuoles ( d > 70 nm ) in 1 / 2 - DKO IHCs and 1 / 3 - DKO IHCs .

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Increased overall number of coated structures in endophilin mutants. (D') Prominent accumulation of coated pits can be observed in 1/2-DKO and 1/3-DKO IHCs alongside a shift towards more coated vacuoles (d > 70 nm) in 1/2-DKO IHCs and 1/3-DKO IHCs.


Representative electron micrographs of random ultrathin sections from IHC ribbon synapses of p15 mutants lacking different endophilin - A alleles . Scale bars : 200 nm .

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Representative electron micrographs of random ultrathin sections from IHC ribbon synapses of p15 mutants lacking different endophilin-A alleles. Scale bars: 200 nm.


Reduced numbers of RA - SVs in IHCs of endophilin mutants with the strongest reduction observed in 1 / 2 - DKO mice . One - way ANOVA followed by Tukey ' s post - hoc test ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Reduced ratio of RA - SVs in the distal half of the ribbon over RA - SVs in its proximal half in 1 / 2 - DKO and 1 / 3 - DKO IHCs . K - W followed by Dunn ' s test ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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Reduced numbers of RA-SVs in IHCs of endophilin mutants with the strongest reduction observed in 1/2-DKO mice. One-way ANOVA followed by Tukey's post-hoc test; *p < 0.05; **p < 0.01; ***p < 0.001. Reduced ratio of RA-SVs in the distal half of the ribbon over RA-SVs in its proximal half in 1/2-DKO and 1/3-DKO IHCs. K-W followed by Dunn's test; **p < 0.01; ***p < 0.001.


Reduced numbers of MP - SVs ( D ) in IHCs of endophilin mutants with the strongest reduction observed in 1 / 2 - DKO mice . One - way ANOVA followed by Tukey ' s post - hoc test ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

Reduced numbers of MP-SVs (D) in IHCs of endophilin mutants with the strongest reduction observed in 1/2-DKO mice. One-way ANOVA followed by Tukey's post-hoc test; *p < 0.05; **p < 0.01; ***p < 0.001.


Significantly reduced numbers of RA - SVs in 1 / 2 - DKO IHCs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * p = 0 . 0229 ) .

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

Significantly reduced numbers of RA-SVs in 1/2-DKO IHCs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0229).


Significantly reduced numbers of MP - SVs in 1 / 2 - DKO and a strong trend ( p = 0 . 0511 ) towards less MP - SVs in 1 / 3 - DKO IHCs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * p = 0 . 0138 ) .

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

Significantly reduced numbers of MP-SVs in 1/2-DKO and a strong trend (p = 0.0511) towards less MP-SVs in 1/3-DKO IHCs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0138).


Increased SV diameter in 1 / 2 - DKO , but unchanged SV diameter in 1 / 3 - DKOs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * p = 0 . 0154 ) .

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

Increased SV diameter in 1/2-DKO, but unchanged SV diameter in 1/3-DKOs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0154).


Accumulation of ELVs with a minimal distance to the ribbon of less than 200 nm in 1 / 2 - DKOs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * * * p = 0 . 0002 ) .

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

Accumulation of ELVs with a minimal distance to the ribbon of less than 200 nm in 1/2-DKOs (one-way ANOVA followed by Tukey's post-hoc test; ***p = 0.0002).


Representative electron micrographs of Wt and 1 / 2 - DKO specimen indicate accumulations of ELVs and high numbers of coated pits forming from the ELVs in ribbon proximity ( radius r = 1 µm from the center of the ribbon ) in the DKO after 15 min stimulation with extracellular solution supplemented with 50 mM K + . Scale bars : 200 nm .

Representative electron micrographs of Wt and 1/2-DKO specimen indicate accumulations of ELVs and high numbers of coated pits forming from the ELVs in ribbon proximity (radius r = 1 µm from the center of the ribbon) in the DKO after 15 min stimulation with extracellular solution supplemented with 50 mM K+. Scale bars: 200 nm.


Significantly reduced numbers of RA - SVs in IHCs of 1 / 2 - DKO compared to Wt after stimulation , as quantified using random ultrathin sections of ribbon - occupied AZs from several IHCs per genotype . Mann - Whitney test ; * * * p < 0 . 001 .

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

Significantly reduced numbers of RA-SVs in IHCs of 1/2-DKO compared to Wt after stimulation, as quantified using random ultrathin sections of ribbon-occupied AZs from several IHCs per genotype. Mann-Whitney test; ***p < 0.001.


Numbers of MP - SVs were slightly reduced in stimulated Wt compared to stimulated 1 / 2 - DKOs , where SV numbers closely matched the ones of unstimulated 1 / 2 - DKO . Mann - Whitney test ; * p < 0 . 05 .

Numbers of MP-SVs were slightly reduced in stimulated Wt compared to stimulated 1/2-DKOs, where SV numbers closely matched the ones of unstimulated 1/2-DKO. Mann-Whitney test; *p < 0.05.


Reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1 / 2 - DKOs compared to Wt after stimulation . RA - SVs were excluded from this quantification . Student ' s t - test ; * * * p < 0 . 001 .

Reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1/2-DKOs compared to Wt after stimulation. RA-SVs were excluded from this quantification. Student's t-test; ***p < 0.001.


Stimulated 1 / 2 - DKOs exhibited accumulations of ELVs in comparison to stimulated Wt . Mann - Whitney test ; * * * p < 0 . 001 .

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

Stimulated 1/2-DKOs exhibited accumulations of ELVs in comparison to stimulated Wt. Mann-Whitney test; ***p < 0.001.