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 .

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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 ) of endogenous TRAPPC2 GFP - Sec23 localizing at SGs , stained for G3BP . Scale bar 5 µm . Right , magnification of boxed . 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 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 .

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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 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 .

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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 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 ) .

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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 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 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 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 .

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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 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 B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 . of Sec24C localization at SGs over time ( between Sec24C ( mean ) in SG Sec24C ) . Mean ± SD . n = 60 - 80 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 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR 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 - .

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

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 .

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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 . 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 - .

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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 of treated ( 300 µM , 30 min ) or untreated with SA . arrow indicates Golgi . 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 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 - .

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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 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 - .

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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 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 - ) .

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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 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 - ) .

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(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 - ) .

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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 ) 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 :

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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 ) 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 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 I-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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 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

(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 .

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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 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 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 - ) .

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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 B-CONTROLLED_VAR 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 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 .

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(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 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 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 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 B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_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 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 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 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 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 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

(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 .

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(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 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 O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR 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 .

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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 ) .

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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 ) .

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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 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 ) .

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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 ) 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 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 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 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

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 ) .

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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).


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 ) .

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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).


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 ) .

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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 , 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 .

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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 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 .

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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 ) exocytic 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 ) 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 .

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

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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 1 / 3 - DKO mice illustrated with an - coded look - up table where brighter colors indicate higher . 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 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 ) .

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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 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 .

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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 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 .

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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 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 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 .

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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 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 linear component of endocytosis for 20 ms 200 ms depolarizing pulses , obtained from fitting Cm - data for 20 s after cessation of 20 ms depolarizations or for last 10 s of 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 ) .

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

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

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

Average 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 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 region of interest for within a radius of r = 1 µm from ribbon center ( exemplary for a 1 / 3 - DKO specimen ) . Graphical aids indicate small vesicles , ELVs different subpopulations of coated structures quantified in ( B - D ) . Scale bar : 500 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 O O O O O 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 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 vesicles can be observed at 1 - SKO AZs , reduced numbers of small vesicles are present in a 1 µm radius around ribbon of 1 / 2 - DKOs . RA - SVs were excluded from this .

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

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 1 / 3 - DKO IHCs , as measured by relative cumulative ELV per section .

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

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 1 / 3 - DKO IHCs alongside a shift towards more coated vacuoles ( d > 70 nm ) in 1 / 2 - DKO IHCs 1 / 3 - DKO 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

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 of random ultrathin sections from ribbon synapses of p15 mutants lacking different endophilin - A alleles . Scale bars : 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

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

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 strongest reduction observed in 1 / 2 - DKO mice . One - way ANOVA followed by Tukey ' s post - hoc ; * 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 ; * 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

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

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 ; * 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

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 to ribbon of less than 200 nm in 1 / 2 - DKOs ( one - way ANOVA followed by Tukey ' s post - hoc ; * * * 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

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

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 ones of unstimulated 1 / 2 - DKO . Mann - Whitney ; * p < 0 . 05 .

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

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 ribbon of 1 / 2 - DKOs compared to Wt after stimulation . RA - SVs were excluded from this . Student ' s t - ; * * * 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

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 ; * * * 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

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


Increased overall number of coated structures in stimulated endophilin 1 / 2 - DKOs . ( F ' ) Both , coated vesicles coated pits forming at ELVs were significantly increased in 1 / 2 - DKO after stimulation . Mann - Whitney ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

Increased overall number of coated structures in stimulated endophilin 1/2-DKOs. (F') Both, coated vesicles and coated pits forming at ELVs were significantly increased in 1/2-DKO after stimulation. Mann-Whitney test; **p < 0.01; ***p < 0.001.


( A ) CHO were grown in a control medium ( see Materials methods ) or starved for 3 or 13 h , after which they were incubated in 50 μM DHE visualized as detailed in Materials methods .

O O O O O O O O O O O O O O O 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) CHO cells were grown in a control medium (see Materials and methods) or starved for 3 or 13 h, after which they were incubated in 50 μM DHE and visualized as detailed in Materials and methods.


( B ) CHO were grown as in ( A ) , treated with 5 nM MitoTracker Red for 30 min at 37 ° C , washed , treated with 30 μM DCFDA visualized as detailed in Materials methods .

O O O O O O O O O O O O O O O O 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) CHO cells were grown as in (A), treated with 5 nM MitoTracker Red for 30 min at 37°C, washed, treated with 30 μM DCFDA and visualized as detailed in Materials and methods.


( C ) HeLa grown in 96 ‐ well plates were deprived of serum ( αMEM ) , completely starved ( EBSS ) or maintained in a control medium ( α - MEM , 10 % FCS ) for 2 h , after which they were treated with DCFDA as in ( B ) subsequently analyzed in a fluorimeter , as explained in Materials methods .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 grown in 96‐well plates were deprived of serum (αMEM), completely starved (EBSS) or maintained in a control medium (α-MEM, 10% FCS) for 2 h, after which they were treated with DCFDA as in (B) and subsequently analyzed in a fluorimeter, as explained in Materials and methods.


( D ) Data collected from fluorometric measurements were analyzed as detailed in Materials methods .

(D) Data collected from the fluorometric measurements were analyzed as detailed in Materials and methods.


( A ) Left panel : HeLa were starved for 2 h in presence or absence of either 100 nM wortmannin or 10 mM 3MA , after which they were treated with DCFDA analyzed by a fluorimeter as described above . Right panel : rate of degradation of long ‐ lived was measured in incubated in either α ‐ MEM medium or EBSS medium in absence or presence of 100 nM wortmannin or 10 mM 3MA . Values are represented as means ± s . d . of three separate determinations .

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

(A) Left panel: HeLa cells were starved for 2 h in the presence or absence of either 100 nM wortmannin or 10 mM 3MA, after which they were treated with DCFDA and analyzed by a fluorimeter as described above. Right panel: the rate of degradation of long‐lived proteins was measured in cells incubated in either α‐MEM medium or EBSS medium in the absence or presence of 100 nM wortmannin or 10 mM 3MA. Values are represented as the means±s.d. of three separate determinations.


( B ) WT MEFs or Atg5 ( − / − ) MEFs from two separate clones were starved for 2 h before treatment with DCFDA visualization or fluorometric .

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

(B) WT MEFs or Atg5 (−/−) MEFs from two separate clones were starved for 2 h before treatment with DCFDA and visualization or fluorometric analysis.


( A ) Upper panel : CHO stably transfected with GFP ‐ GATE ‐ 16 were preincubated in presence or absence of 10 mM NAC or 1000 μ / ml catalase for 10 min before starvation for 2 h in presence or absence of these drugs , or grown in a control medium containing drugs for 2 h . were then fixed , permeabilized stained with anti ‐ GFP monoclonal antibodies . Representative are shown . Lower panel : HEK 293 were transfected with GFP ‐ GATE ‐ 16 . At 24 h post ‐ transfection , were treated with NAC or catalase starved as explained above , lysed in Ripa buffer 100 ‐ g of each lysate was separated on 10 % SDS - PAGE subsequently analyzed with anti ‐ GFP antibodies to detect transfected GATE ‐ 16 anti ‐ tubulin antibodies as control . data were quantified using NIH image program are depicted as of lipidated from total GATE ‐ 16 . ( * ) indicates non ‐ lipidated ( * * ) indicates lipidated GFP ‐ GATE ‐ 16 .

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-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 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 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

(A) Upper panel: CHO cells stably transfected with GFP‐GATE‐16 were preincubated in the presence or absence of 10 mM NAC or 1000 μ/ml catalase for 10 min before starvation for 2 h in the presence or absence of these drugs, or grown in a control medium containing the drugs for 2 h. The cells were then fixed, permeabilized and stained with anti‐GFP monoclonal antibodies. Representative images are shown. Lower panel: HEK 293 cells were transfected with GFP‐GATE‐16. At 24 h post‐transfection, the cells were treated with NAC or catalase and starved as explained above, lysed in Ripa buffer and 100 ‐g of each lysate was separated on 10% SDS-PAGE and subsequently analyzed with anti‐GFP antibodies to detect the transfected GATE‐16 and anti‐tubulin antibodies as control. The data were quantified using NIH image program and are depicted as the percentage of lipidated protein from the total GATE‐16. (*) indicates non‐lipidated and (**) indicates lipidated GFP‐GATE‐16.


( B ) CHO treated with NAC or catalase starved as detailed in ( A ) were incubated with DCFDA visualized by a confocal microscope or analyzed by a fluorimeter as in Figure 1 .

O O O O O 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

(B) CHO cells treated with NAC or catalase and starved as detailed in (A) were incubated with DCFDA and visualized by a confocal microscope or analyzed by a fluorimeter as in Figure 1.


( C ) rate of degradation of long ‐ lived was measured in CHO incubated in either α ‐ MEM medium or EBSS medium , or following pretreatment with 10 mM NAC for 10 min or with 1000 μ / ml catalase overnight . A representative experiment is shown .

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

(C) The rate of degradation of long‐lived proteins was measured in CHO cells incubated in either α‐MEM medium or EBSS medium, or following pretreatment with 10 mM NAC for 10 min or with 1000 μ/ml catalase overnight. A representative experiment is shown.


( A ) CHO ( for mammalian Atg8s ) or of S . cerevisiae ( for ScAtg8 ) were incubated in starvation medium ( EBSS or SD ‐ N , respectively ) for different time periods as indicated before isolation of by Tri ‐ reagent as explained in Materials methods . For each gene , data obtained from non ‐ starved were set to an arbitrary value of 1 , results from corresponding starved were normalized accordingly . Results represent means ± s . d . of three separate experiments .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CHO cells (for mammalian Atg8s) or cells of S. cerevisiae (for ScAtg8) were incubated in starvation medium (EBSS or SD‐N, respectively) for different time periods as indicated before isolation of RNA by the Tri‐reagent as explained in Materials and methods. For each gene, data obtained from non‐starved cells were set to an arbitrary value of 1, and results from the corresponding starved cells were normalized accordingly. Results represent the means±s.d. of three separate experiments.


( B ) HEK 293 were grown in control medium or starved for 2 h in EBSS in absence or presence of 100 nM bafilomycin A1 . Lysates ( 100 μg ) obtained in Ripa buffer were run on 12 % SDS - PAGE analyzed by Western blot , using anti ‐ GATE ‐ 16 or anti ‐ tubulin antibodies .

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

(B) HEK 293 cells were grown in control medium or starved for 2 h in EBSS in the absence or presence of 100 nM bafilomycin A1. Lysates (100 μg) obtained in Ripa buffer were run on 12% SDS-PAGE and analyzed by Western blot, using anti‐GATE‐16 or anti‐tubulin antibodies.


( C ) CHO were grown in a control medium in presence or absence of 1 mM H2O2 for 1 h , or starved for 3 or 13 h . Lysates ( 10 μg ) obtained in Ripa buffer were incubated with recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) in 50 KT reaction buffer ( 25 mM Tris , pH 7 . 4 , 50 mM KCl ) at 30 ° C for 45 min , in presence or absence of 1 mM DTT . reaction was stopped by addition of sample buffer boiling , after which samples were resolved on 15 % SDS - PAGE subsequently analyzed by Western blot , using anti ‐ His monoclonal antibodies . experiment was repeated six times ; a representative blot is shown .

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 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) CHO cells were grown in a control medium in the presence or absence of 1 mM H2O2 for 1 h, or starved for 3 or 13 h. Lysates (10 μg) obtained in Ripa buffer were incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer (25 mM Tris, pH 7.4, 50 mM KCl) at 30°C for 45 min, in the presence or absence of 1 mM DTT. The reaction was stopped by addition of sample buffer and boiling, after which the samples were resolved on 15% SDS-PAGE and subsequently analyzed by Western blot, using anti‐His monoclonal antibodies. The experiment was repeated six times; a representative blot is shown.


( D ) Left panel : HeLa transfected with Myc ‐ GATE ‐ 16 ‐ HA or with an empty vector as control were labeled with [ 35S ] methionine for 10 min lysed immediately or chased for 1 h before lysis in Ripa buffer . Lysates were immunoprecipitated using anti ‐ Myc antibodies immunoprecipitates were resolved on 15 % SDS - PAGE . Middle panel : HeLa transfected with Myc ‐ GATE ‐ 16 ‐ HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [ 35S ] methionine for 10 min , immediate lysis , by SDS - PAGE ( right panel ) using NIH image program . Values are presented as average of unprimed form out of total GATE ‐ 16 . ( * ) in sections of this figure indicates non ‐ His6 ‐ GATE ‐ 16 ‐ HA or Myc ‐ GATE ‐ 16 ‐ HA ( * * ) indicates His6 ‐ GATE ‐ 16 or Myc ‐ GATE ‐ 16 .

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

(D) Left panel: HeLa cells transfected with Myc‐GATE‐16‐HA or with an empty vector as control were labeled with [35S]methionine for 10 min and lysed immediately or chased for 1 h before lysis in Ripa buffer. Lysates were immunoprecipitated using anti‐Myc antibodies and the immunoprecipitates were resolved on 15% SDS-PAGE. Middle panel: HeLa cells transfected with Myc‐GATE‐16‐HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [35S]methionine for 10 min, immediate lysis, analysis by SDS-PAGE and quantification (right panel) using NIH image program. Values are presented as the average percentage of unprimed form out of the total GATE‐16. (*) in all sections of this figure indicates non‐cleaved His6‐GATE‐16‐HA or Myc‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16 or Myc‐GATE‐16.


( A ) Cleavage activity was tested by incubation of recombinant His6 ‐ HsAtg4A ( 0 . 1 μg ) His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) in 50 KT reaction buffer at 30 ° C for 45 min in presence of indicated concentrations of DTT followed by Western blot , using anti ‐ His monoclonal antibodies . resulting bands from three separate experiments were quantified with a densitometer using Bio ‐ Rad Multi ‐ Analyst program are presented as average of form out of total GATE ‐ 16 . ( * ) indicates non ‐ His6 ‐ GATE ‐ 16 ‐ HA ( * * ) indicates His6 ‐ GATE ‐ 16 .

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

(A) Cleavage activity was tested by incubation of recombinant His6‐HsAtg4A (0.1 μg) and His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence of indicated concentrations of DTT followed by Western blot analysis, using anti‐His monoclonal antibodies. The resulting bands from three separate experiments were quantified with a densitometer using the Bio‐Rad Multi‐Analyst program and are presented as the average percentage of cleaved form out of the total GATE‐16. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16.


( B ) His6 ‐ HsAtg4A was incubated in presence of 200 μM DTT at 4 ° C for 10 min . Reduced His6 ‐ HsAtg4A ( 0 . 1 μg ) was then incubated in 50 KT ( to obtain 15 μM DTT ) with indicated concentrations of H2O2 at 25 ° C for 5 min , after which recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) was added incubation proceeded at 30 ° C for 45 min . Reaction mixtures from three separate experiments were analyzed are presented as explained in ( A ) .

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

(B) His6‐HsAtg4A was incubated in the presence of 200 μM DTT at 4°C for 10 min. Reduced His6‐HsAtg4A (0.1 μg) was then incubated in 50 KT (to obtain 15 μM DTT) with the indicated concentrations of H2O2 at 25°C for 5 min, after which recombinant His6‐GATE‐16‐HA (0.3 μg) was added and incubation proceeded at 30°C for 45 min. Reaction mixtures from three separate experiments were analyzed and are presented as explained in (A).


( C ) His6 ‐ HsAtg4A ( 0 . 1 μg ) was incubated with recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) after following procedures : no treatment ( lane 1 ) ; pretreatment with 200 μM DTT at 25 ° C for 5 min ( lane 2 ) ; treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25 ° C for 5 min ( lane 3 ) ; treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min then 2 mM DTT ( lane 4 ) . Reaction mixtures were analyzed by Western blot using anti ‐ His monoclonal antibodies .

O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O B-CONTROLLED_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

(C) His6‐HsAtg4A (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) after the following procedures: no treatment (lane 1); pretreatment with 200 μM DTT at 25°C for 5 min (lane 2); treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25°C for 5 min (lane 3); and treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min and then 2 mM DTT (lane 4). Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies.


( A ) Recombinant His6 ‐ HsAtg4AWT , His6 ‐ HsAtg4AC77A or His6 ‐ HsAtg4AC81S ( 0 . 1 μg ) was incubated with His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) in 50 KT reaction buffer at 30 ° C for 45 min in presence or absence of 1 mM DTT . Reaction mixtures were analyzed by Western blot using anti ‐ His monoclonal antibodies . ( * ) indicates non ‐ His6 ‐ GATE ‐ 16 ‐ HA ( * * ) indicates His6 ‐ GATE ‐ 16 .

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

(A) Recombinant His6‐HsAtg4AWT, His6‐HsAtg4AC77A or His6‐HsAtg4AC81S (0.1 μg) was incubated with His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence or absence of 1 mM DTT. Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16.


( B ) His6 ‐ HsAtg4AC81S ( 0 . 1 μg ) was incubated with recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) following same procedure as detailed in Figure 5C . Reaction mixtures were analyzed are presented as explained in ( A ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) His6‐HsAtg4AC81S (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) following the same procedure as detailed in Figure 5C. Reaction mixtures were analyzed and are presented as explained in (A).


( A ) CHO stably expressing GFP ‐ GATE ‐ 16 were transiently transfected with HsAtg4AWT ‐ Myc ‐ His6 or HsAtg4AC81S ‐ Myc ‐ His6 . At 24 h post ‐ transfection , were starved for 2 . 5 h after which they were fixed , permeabilized incubated with anti ‐ Myc monoclonal antibodies . Upper panel : typical of transfected with above constructs , as visualized using a confocal microscope ; representative autophagosomes are indicated by arrows . Lower panel : of average number of autophagosomes per in different transfectants . of fixed were visualized using a Nikon eclipse TE300 microscope used for of number of autophagosomes per . results presented are means ± s . d . of a total of 100 from three separate experiments . ( * ) indicates significance at P0 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CHO cells stably expressing GFP‐GATE‐16 were transiently transfected with HsAtg4AWT‐Myc‐His6 or HsAtg4AC81S‐Myc‐His6. At 24 h post‐transfection, cells were starved for 2.5 h after which they were fixed, permeabilized and incubated with anti‐Myc monoclonal antibodies. Upper panel: typical images of cells transfected with the above constructs, as visualized using a confocal microscope; representative autophagosomes are indicated by arrows. Lower panel: quantification of the average number of autophagosomes per cell in the different transfectants. Images of the fixed cells were visualized using a Nikon eclipse TE300 fluorescent microscope and used for quantification of the number of autophagosomes per cell. The results presented are the means±s.d. of a total of 100 cells from three separate experiments. (*) indicates significance at P0.001.