( g , h ) Immunostaining for Atg16 in Cx43 knocked down ( KD ) ( g ) or knocked out ( − / − ) ( h ) cells . Right : quantification of the average of cytosolic Atg16 positive puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

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(g,h) Immunostaining for Atg16 in Cx43 knocked down (KD) (g) or knocked out (−/−) (h) cells. Right: quantification of the average of cytosolic Atg16 positive puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).


( i ) Immunoblot for Atg16 and Atg14 in immunoprecipitates ( IP ) of endogenous Cx43 from WT MEFs maintained in the absence of serum ( 4 h ) or incubated in the presence of tamoxifen or lindane . I : input ; FT : flow through . All values are mean + s . e . m and differences from untreated cells are significant for * P 0 . 01 . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

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(i) Immunoblot for Atg16 and Atg14 in immunoprecipitates (IP) of endogenous Cx43 from WT MEFs maintained in the absence of serum (4 h) or incubated in the presence of tamoxifen or lindane. I: input; FT: flow through. All values are mean + s.e.m and differences from untreated cells are significant for *P 0.01. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


( a - c ) Immunofluorescence for the indicated endosomal markers in NRK cells expressing GFP - Cx43 maintained in the presence of serum ( a ) , absence of serum ( b ) or treated with lindane ( c ) . Single black and white inverted channels , merged channels and higher magnification insets are shown .

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(a-c) Immunofluorescence for the indicated endosomal markers in NRK cells expressing GFP-Cx43 maintained in the presence of serum (a), absence of serum (b) or treated with lindane (c). Single black and white inverted channels, merged channels and higher magnification insets are shown.


( d ) Immunoblots for indicated proteins in isolated fractions from starved mouse liver . Hom , homogenate ; LE , late endosomes ; Cyt , cytosol ; APG , autophagosomes ; APL , autophagolysosomes .

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(d) Immunoblots for indicated proteins in isolated fractions from starved mouse liver. Hom, homogenate; LE, late endosomes; Cyt, cytosol; APG, autophagosomes; APL, autophagolysosomes.


( e ) Immunoblots for different Cx in homogenates and late endosomes isolated from fed and 6 h starved mice .

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(e) Immunoblots for different Cx in homogenates and late endosomes isolated from fed and 6 h starved mice.


( f - h ) Fluorescence images of NRK cells expressing GFP - Cx43 maintained in the presence of serum ( f ) , absence of serum ( g ) or treated with lindane ( h ) and incubated with Alexa594 - transferrin for 15 min before fixation . Top : single and merged channels and outlined area at higher magnification . Bottom : 3D reconstructions and higher magnification details of the outlined areas . Nuclei are highlighted with DAPI . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

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(f-h) Fluorescence images of NRK cells expressing GFP-Cx43 maintained in the presence of serum (f), absence of serum (g) or treated with lindane (h) and incubated with Alexa594-transferrin for 15 min before fixation. Top: single and merged channels and outlined area at higher magnification. Bottom: 3D reconstructions and higher magnification details of the outlined areas. Nuclei are highlighted with DAPI. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


( a ) Immunoblot for the indicated proteins in homogenates ( Hom ) and purified PM from fed ( F ) and 24 h starved mice ( Stv ) . Starvation - induced changes in the PM content relative to fed levels were as follows : Vps15 ( 1 . 2 ± 0 . 2 ) , Beclin - 1 ( 0 . 7 ± 0 . 5 ) , Vps34 ( 0 . 9 ± 0 . 1 ) , Atg16 ( 1 . 1 ± 0 . 3 ) , Atg14 ( 2 . 5 ± 0 . 2 ) and only Atg14 were significant for P 0 . 01 .

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(a) Immunoblot for the indicated proteins in homogenates (Hom) and purified PM from fed (F) and 24 h starved mice (Stv). Starvation-induced changes in the PM content relative to fed levels were as follows: Vps15 (1.2 ± 0.2), Beclin-1 (0.7 ± 0.5), Vps34 (0.9 ± 0.1), Atg16 (1.1 ± 0.3), Atg14 (2.5 ± 0.2) and only Atg14 were significant for P 0.01.


( b ) Immunoblot of Cx43immunoprecipitates ( IP ) in MEFs maintained in the presence or absence of serum . I , input ; FT , flow through .

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(b) Immunoblot of Cx43immunoprecipitates (IP) in MEFs maintained in the presence or absence of serum. I, input; FT, flow through.


( c ) Immunoblot of Cx43 IP in MEFs from WT or Atg5 − / − mice grown in serum - supplemented media .

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(c) Immunoblot of Cx43 IP in MEFs from WT or Atg5−/− mice grown in serum-supplemented media.


( d ) Immunoblot of Cx43 IP in WT or Atg16 knockdown ( KD ) MEFs maintained in the presence / absence of serum for 4 h .

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(d) Immunoblot of Cx43 IP in WT or Atg16 knockdown (KD) MEFs maintained in the presence/absence of serum for 4 h.


( e ) Co - staining for Atg14 and Cx43 in NRK cells maintained in the presence / absence of serum . Merged channels are shown and single channels and broad - field images are shown in Supplementary Fig . 5d . Insets : higher magnification .

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(e) Co-staining for Atg14 and Cx43 in NRK cells maintained in the presence/absence of serum. Merged channels are shown and single channels and broad-field images are shown in Supplementary Fig. 5d. Insets: higher magnification.


( f ) Maximum - intensity confocal microscopy 3D reconstruction in HeLa cells expressing EBFP2 - Cx43 ( pseudocoloured in red ) and EGFP - ATG14 followed by a single - plane time - lapse acquisition of the outlined area ; cells were incubated in serum - deprived medium for 1 h before imaging .

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(f) Maximum-intensity confocal microscopy 3D reconstruction in HeLa cells expressing EBFP2-Cx43 (pseudocoloured in red) and EGFP-ATG14 followed by a single-plane time-lapse acquisition of the outlined area; cells were incubated in serum-deprived medium for 1 h before imaging.


( g ) Immunoblot of Atg14 IP in WT or Atg5 − / − MEFs maintained in the presence of serum .

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(g) Immunoblot of Atg14 IP in WT or Atg5−/− MEFs maintained in the presence of serum.


( h ) Immunoblot in total homogenates and PM purified from WT or Cx43 − / − MOBs .

(h) Immunoblot in total homogenates and PM purified from WT or Cx43−/− MOBs.


( i ) Immunofluorescence for e - cadherin and Vps34 in NRK cells WT or knocked down ( KD ) for Cx43 . Single channel and merge ( left ) and z - stack surface reconstruction and higher magnification of the outlined region ( right ) .

(i) Immunofluorescence for e-cadherin and Vps34 in NRK cells WT or knocked down (KD) for Cx43. Single channel and merge (left) and z-stack surface reconstruction and higher magnification of the outlined region (right).


( j ) Immunostaining for Cx43 and e - cadherin in NRK cells expressing a tandem FYVE domain ( 2xFYVE ) tagged to GFP and maintained in serum - free media for 4 h . Single channels ( inverted black and white ) and the merged image of the outlined area ( colour ) . Inset : higher magnification image . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

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(j) Immunostaining for Cx43 and e-cadherin in NRK cells expressing a tandem FYVE domain (2xFYVE) tagged to GFP and maintained in serum-free media for 4 h. Single channels (inverted black and white) and the merged image of the outlined area (colour). Inset: higher magnification image. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


( a ) Co - immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum ( top ) . Single and merged channels of the 3D reconstruction of the outlined regions are shown ( bottom ) . Nuclei are highlighted with DAPI .

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(a) Co-immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum (top). Single and merged channels of the 3D reconstruction of the outlined regions are shown (bottom). Nuclei are highlighted with DAPI.


( b , c ) Immunoblot for the indicated proteins of immunoprecipitates ( IP ) of Cx43 in NRK cells maintained in the presence or absence of serum ( b ) or treated or not with 3 - methyladenine ( c ) . Atg5 is shown as the negative control for immunoprecipitation .

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(b,c) Immunoblot for the indicated proteins of immunoprecipitates (IP) of Cx43 in NRK cells maintained in the presence or absence of serum (b) or treated or not with 3-methyladenine (c). Atg5 is shown as the negative control for immunoprecipitation.


( d ) Co - staining for Atg9 and e - cadherin in control NRK cells and those knocked down for Cx43 . Single and merged channels at higher magnification areas are shown .

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(d) Co-staining for Atg9 and e-cadherin in control NRK cells and those knocked down for Cx43. Single and merged channels at higher magnification areas are shown.


( e ) Immunofluorescence for endogenous Atg9 and Cx43 in control NRK cells and those knocked down for Eps15 .

(e) Immunofluorescence for endogenous Atg9 and Cx43 in control NRK cells and those knocked down for Eps15.


( f , g ) Immunofluorescence for endogenous Cx43 ( green ) in control NRK cells or those knocked down for Atg14 ( f ) maintained in the presence or absence of serum .

(f,g) Immunofluorescence for endogenous Cx43 (green) in control NRK cells or those knocked down for Atg14 (f) maintained in the presence or absence of serum.


( f , g ) Immunofluorescence for endogenous Cx43 ( green ) in control NRK cells or those knocked down for Atg9 ( g ) maintained in the presence or absence of serum . E - cadherin staining ( red ) is shown to highlight PM in g and h .

(f,g) Immunofluorescence for endogenous Cx43 (green) in control NRK cells or those knocked down for Atg9 (g) maintained in the presence or absence of serum. E-cadherin staining (red) is shown to highlight PM in g and h.


( h ) Immunofluorescence of endogenous Cx43 in control NRK cells or those knocked down for Atg14 , treated ( or not ) with tamoxifen or lindane ( left ) . E - cadherin staining ( red ) is shown to highlight PM in g and h . Full fields are shown in Supplementary Fig . 6h . Quantification of intracytoplasmic Cx43 - positives vesicles ( right ; n = 3 wells , 3 independent experiments , > 30 cells per experiment ) . Values are mean + s . e . m and significant for * P 0 . 01 and * * P 0 . 001 using analysis of variance + Bonferroni test . Scale bars : 5 μm . Uncropped images of blots are shown in Supplementary Fig . 9 .

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(h) Immunofluorescence of endogenous Cx43 in control NRK cells or those knocked down for Atg14, treated (or not) with tamoxifen or lindane (left). E-cadherin staining (red) is shown to highlight PM in g and h. Full fields are shown in Supplementary Fig. 6h. Quantification of intracytoplasmic Cx43-positives vesicles (right; n = 3 wells, 3 independent experiments, >30 cells per experiment). Values are mean + s.e.m and significant for *P 0.01 and **P 0.001 using analysis of variance + Bonferroni test. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.


( a ) Immunoblot for the indicated proteins in control MEFs or those knocked down ( KD ) for the three different Cx .

(a) Immunoblot for the indicated proteins in control MEFs or those knocked down (KD) for the three different Cx.


( b ) Immunofluorescence of LC3 in control MEFs ( Ctr ) or those knocked down ( KD ) for three different Cx maintained in the presence or absence of serum . Left : representative fields and higher magnification insets . Nuclei are highlighted with DAPI . Right : Quantification of the average number of LC3 - positive puncta per cell ( n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

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(b) Immunofluorescence of LC3 in control MEFs (Ctr) or those knocked down (KD) for three different Cx maintained in the presence or absence of serum. Left: representative fields and higher magnification insets. Nuclei are highlighted with DAPI. Right: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment).


( c ) Immunoblot for LC3 in the same cells maintained in the presence of serum untreated ( − ) or treated with inhibitors of lysosomal proteases ( PI ) for the indicated times . GAPDH is shown as the loading control .

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(c) Immunoblot for LC3 in the same cells maintained in the presence of serum untreated (−) or treated with inhibitors of lysosomal proteases (PI) for the indicated times. GAPDH is shown as the loading control.


( d ) Representative images of control MEFs or those knocked down ( KD ) for the indicated proteins transfected with the mCherry - GFP - LC3 tandem reporter . Right : quantification of the average number per cell of puncta positive for GFP and mCherry ( autophagosomes ; APG ) and those positive only for mCherry ( autophagolysosomes ; APL ; n = 3 wells , 3 independent experiments , > 50 cells per experiment ) .

(d) Representative images of control MEFs or those knocked down (KD) for the indicated proteins transfected with the mCherry-GFP-LC3 tandem reporter. Right: quantification of the average number per cell of puncta positive for GFP and mCherry (autophagosomes; APG) and those positive only for mCherry (autophagolysosomes; APL; n = 3 wells, 3 independent experiments, >50 cells per experiment).


( e ) Electron micrographs of control MEFs or those knocked down for the indicated Cx . Insets show autophagic vesicles . Bottom : Quantification of the average number of AV ( left ) and percentage of cytosolic area ( right ) occupied by AVs ( n = 4 wells , 4 independent experiments , > 10 micrographs per well ) .

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(e) Electron micrographs of control MEFs or those knocked down for the indicated Cx. Insets show autophagic vesicles. Bottom: Quantification of the average number of AV (left) and percentage of cytosolic area (right) occupied by AVs (n = 4 wells, 4 independent experiments, >10 micrographs per well).


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


C . Evaluation of CDK kinase activity upon SA treatment . Western blot 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

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

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 .

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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 .

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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 .

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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 .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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

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 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 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

(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 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 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

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 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 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

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

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

(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 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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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