( e ) RALB ubiquitylation controls interaction between EXO84 and SEC5 . Flag - tagged SEC5 was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells overexpressing RALB - G23V or RALB - G23V - K47R . The presence of EXO84 in SEC5 complexes was analysed by immunoblotting using anti - EXO84 antibody .

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

(e) RALB ubiquitylation controls interaction between EXO84 and SEC5. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing RALB-G23V or RALB-G23V-K47R. The presence of EXO84 in SEC5 complexes was analysed by immunoblotting using anti-EXO84 antibody.


( f ) USP33 regulates EXO84 - SEC5 complex formation . Flag - tagged SEC5 was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells expressing the indicated constructs followed by immunoblotting using anti - EXO84 antibody .

O O O O O O O O O O O O O O O O O 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) USP33 regulates EXO84-SEC5 complex formation. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs followed by immunoblotting using anti-EXO84 antibody.


( g ) Suppression of SEC5 affects RALB binding to EXO84 . HA - tagged RALB - G23V was immunoprecipitated from HEK293T cells expressing the indicated constructs . The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti - EXO84 antibody . For a - c , f , g immunoblotting using antibodies specific for USP33 , anti - Flag ( M2 ) or anti - HA determined the levels of USP33 suppression or overexpression . Immunoblot analysis was performed using the indicated antibodies . Uncropped images of blots are shown in Supplementary Fig . S7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Suppression of SEC5 affects RALB binding to EXO84. HA-tagged RALB-G23V was immunoprecipitated from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-EXO84 antibody. For a-c,f,g immunoblotting using antibodies specific for USP33, anti-Flag (M2) or anti-HA determined the levels of USP33 suppression or overexpression. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) Suppression of USP33 facilitates TBK1 - SEC5 complex formation . Flag - tagged SEC5 was introduced into HEK293T cells expressing empty vector ( V ) or HA - tagged RALB - G23V together with GFP shRNA or USP33 shRNA as indicated , and then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using antibody specific for TBK1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Suppression of USP33 facilitates TBK1-SEC5 complex formation. Flag-tagged SEC5 was introduced into HEK293T cells expressing empty vector (V) or HA-tagged RALB-G23V together with GFP shRNA or USP33 shRNA as indicated, and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using antibody specific for TBK1.


( c ) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB - SEC5 complex in response to poly ( I : C ) treatment . Flag - tagged SEC5 and the indicated HA - tagged RALB mutants were introduced into 293 - hTLR3 cells . At 48 h after transfection cells were treated 100 μg ml − 1 of poly ( I : C ) for 6 h . Flag - tagged SEC5 was immunoprecipitated with anti - Flag ( M2 ) , and analysed for co - precipitation by immunoblotting using anti - HA antibody .

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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB-SEC5 complex in response to poly(I:C) treatment. Flag-tagged SEC5 and the indicated HA-tagged RALB mutants were introduced into 293-hTLR3 cells. At 48 h after transfection cells were treated 100 μg ml−1 of poly(I:C) for 6 h. Flag-tagged SEC5 was immunoprecipitated with anti-Flag (M2), and analysed for co-precipitation by immunoblotting using anti-HA antibody.


( d ) RALB ubiquitylation at Lys 47 affects activation of TBK1 and IRF3 . At 48 h after transfection with empty vector ( V ) , Flag - RALB - G23V and Flag - RALB - G23V - K47R , 293 - hTLR3 cells were treated with 10 μg ml − 1 of poly ( I : C ) for 6 h . Immunoblot analysis was performed using the indicated antibodies . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

(d) RALB ubiquitylation at Lys 47 affects activation of TBK1 and IRF3. At 48 h after transfection with empty vector (V), Flag-RALB-G23V and Flag-RALB-G23V-K47R, 293-hTLR3 cells were treated with 10 μg ml−1 of poly(I:C) for 6 h. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.


( a - d ) USP33 inhibits assembly of SEC5 - beclin - 1 complexes . The indicated proteins were immunoprecipitated from cells expressing GFP shRNA or USP33 shRNA with anti - Flag ( M2 ) , anti - HA or anti - SEC5 antibody and analysed for co - precipitation by immunoblotting using specific antibodies as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-d) USP33 inhibits assembly of SEC5-beclin-1 complexes. The indicated proteins were immunoprecipitated from cells expressing GFP shRNA or USP33 shRNA with anti-Flag (M2), anti-HA or anti-SEC5 antibody and analysed for co-precipitation by immunoblotting using specific antibodies as indicated.


( e ) USP33 potentiates the interaction between EXO84 and beclin 1 . Endogenous beclin 1 was immunoprecipitated from HEK293T cells , expressing either empty vector ( V ) , HA - USP33 wild type ( WT ) or the catalytically inactive mutant C194S - H683Q , using anti - beclin 1 antibody . Immunoprecipitates were analysed for co - precipitation by immunoblotting using anti - EXO84 or anti - beclin 1 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 O O O O O

(e) USP33 potentiates the interaction between EXO84 and beclin 1. Endogenous beclin 1 was immunoprecipitated from HEK293T cells, expressing either empty vector (V), HA-USP33 wild type (WT) or the catalytically inactive mutant C194S-H683Q, using anti-beclin 1 antibody. Immunoprecipitates were analysed for co-precipitation by immunoblotting using anti-EXO84 or anti-beclin 1 antibodies.


( f , g ) RALB but not RALA undergoes deubiquitylation under nutrient deprivation . 6xHis - tagged ubiquitin and the indicated RAL mutants were overexpressed in HEK293T cells . At 48 h after transfection cells were deprived of nutrients and incubated in Hank ' s buffered salt solution ( HBSS ) medium for 90 min . Ubiquitylated RALproteins were purified by Co2 + metal affinity chromatography and analysed by immunoblotting using anti - RALA or anti - RALB antibodies .

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

(f,g) RALB but not RALA undergoes deubiquitylation under nutrient deprivation. 6xHis-tagged ubiquitin and the indicated RAL mutants were overexpressed in HEK293T cells. At 48 h after transfection cells were deprived of nutrients and incubated in Hank's buffered salt solution (HBSS) medium for 90 min. Ubiquitylated RALproteins were purified by Co2+ metal affinity chromatography and analysed by immunoblotting using anti-RALA or anti-RALB antibodies.


( h ) Nutrient starvation results in the accumulation of USP33 . HEK TE cells were incubated in HBSS medium for the indicated periods of time . Immunoblotting was performed using anti - USP33 antibody .

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

(h) Nutrient starvation results in the accumulation of USP33. HEK TE cells were incubated in HBSS medium for the indicated periods of time. Immunoblotting was performed using anti-USP33 antibody.


( i ) USP33 co - localizes with RALB - positive vesicles in the absence of nutrients . At 48 h after co - transfection with RALB - G23V - EGFP and USP33 - mCherry , HeLa cells were incubated in HBSS medium for 90 min . Cells were imaged for EGFP and mCherry fluorescence . Scale bars , 10 μm . The outlined areas are shown at higher magnification in the bottom panels . Arrows indicate USP33 co - localization to RALB - positive vesicles .

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

(i) USP33 co-localizes with RALB-positive vesicles in the absence of nutrients. At 48 h after co-transfection with RALB-G23V-EGFP and USP33-mCherry, HeLa cells were incubated in HBSS medium for 90 min. Cells were imaged for EGFP and mCherry fluorescence. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. Arrows indicate USP33 co-localization to RALB-positive vesicles.


( j ) Beclin 1 co - localizes with USP33 . HeLa cells were transfected with beclin - 1 - HA and USP33 - mCherry . At 48 h after transfection , cells were incubated in HBSS medium for 90 minutes and treated as in i . Scale bars , 10 μm . The outlined areas are shown at higher magnification in the bottom panels . Uncropped images of blots are shown in Supplementary Fig . S7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Beclin 1 co-localizes with USP33. HeLa cells were transfected with beclin-1-HA and USP33-mCherry. At 48 h after transfection, cells were incubated in HBSS medium for 90 minutes and treated as in i. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) USP33 depletion inhibits accumulation of LC3 - lipid conjugates . HEK TE cells stably expressing GFP shRNA or USP33 shRNA were incubated with DMEM or HBSS medium for 4 h , in the presence or absence of 50 μM of chloroquine ( CQ ) . Cells were then assayed for the relative accumulation of LC3 - I and LC3 - II by immunoblotting analysis using anti - LC3 antibody . LC3 - II / GAPDH ratios have been calculated using ImageJ densitometric analysis .

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

(a) USP33 depletion inhibits accumulation of LC3-lipid conjugates. HEK TE cells stably expressing GFP shRNA or USP33 shRNA were incubated with DMEM or HBSS medium for 4 h, in the presence or absence of 50 μM of chloroquine (CQ). Cells were then assayed for the relative accumulation of LC3-I and LC3-II by immunoblotting analysis using anti-LC3 antibody. LC3-II/GAPDH ratios have been calculated using ImageJ densitometric analysis.


( b - d ) USP33 expression levels affect the accumulation of LC3punctae . Wild - type USP33 or a USP33 - shRNA - resistant USP33 mutant ( USP33R ) was introduced into HEK TE cells stably expressing GFP shRNA or USP33 shRNA . The generated cells were incubated in HBSS medium for 4 h . USP33 was analysed by immunoblotting using anti - USP33 antibody ( b ) . Representative images of LC3immunostaining are shown ( c ) . Endogenous LC3punctae were quantified using the In Cell Analyser 2000 automatic imaging system and IN Cell Investigator software . The mean distribution of LC3punctae per cell is represented as the mean of two independent experiments . Statistics source data for d can be found in Supplementary Table S1 ( d ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-d) USP33 expression levels affect the accumulation of LC3punctae. Wild-type USP33 or a USP33-shRNA-resistant USP33 mutant (USP33R) was introduced into HEK TE cells stably expressing GFP shRNA or USP33 shRNA. The generated cells were incubated in HBSS medium for 4 h. USP33 was analysed by immunoblotting using anti-USP33 antibody (b). Representative images of LC3immunostaining are shown (c). Endogenous LC3punctae were quantified using the In Cell Analyser 2000 automatic imaging system and IN Cell Investigator software. The mean distribution of LC3punctae per cell is represented as the mean of two independent experiments. Statistics source data for d can be found in Supplementary Table S1 (d).


( e ) USP33 depletion leads to increased levels of p62 . HEK TE cells expressing GFP shRNA or USP33 shRNA were incubated in HBSS medium for the indicated periods of time . Immunoblotting was performed using the indicated antibodies .

(e) USP33 depletion leads to increased levels of p62. HEK TE cells expressing GFP shRNA or USP33 shRNA were incubated in HBSS medium for the indicated periods of time. Immunoblotting was performed using the indicated antibodies.


( f ) Overexpression of the RALB - G23V - K47R mutant leads to accumulation of LC3 - lipid conjugates . HEK293T cells overexpressing the RALB - G23V or RALB - G23V - K47R mutants were assayed for the relative accumulation of LC3 - I and LC3 - II by immunoblotting using anti - LC3 antibody .

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

(f) Overexpression of the RALB-G23V-K47R mutant leads to accumulation of LC3-lipid conjugates. HEK293T cells overexpressing the RALB-G23V or RALB-G23V-K47R mutants were assayed for the relative accumulation of LC3-I and LC3-II by immunoblotting using anti-LC3 antibody.


Demonstration of translational readthrough of AGO1 using luciferase - based reporter assay . Plasmid containing in - frame AGO1 - TGA ( or GCA ) - ISR - FLuc were transfected in HEK293 cells and translational readthrough was detected as FLuc activity normalized to the activity of co - transfected renilla luciferase ( RLuc ) . FLuc mRNA levels determined by RT - PCR are shown below . * * * , P = 0 . 0001 .

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

Demonstration of translational readthrough of AGO1 using luciferase-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-FLuc were transfected in HEK293 cells and translational readthrough was detected as FLuc activity normalized to the activity of co-transfected renilla luciferase (RLuc). FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0001.


Significance of the identity of the canonical stop codon in AGO1 translational readthrough . AGO1 - ISR - FLuc constructs containing TGA or TAA or TAG were transfected in HEK293 cells and translational readthrough was quantified as described above . FLuc mRNA levels determined by RT - PCR are shown below . * * * , P = 0 . 0002 ; * * , P = 0 . 0004 .

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

Significance of the identity of the canonical stop codon in AGO1 translational readthrough. AGO1-ISR-FLuc constructs containing TGA or TAA or TAG were transfected in HEK293 cells and translational readthrough was quantified as described above. FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0002; **, P = 0.0004.


First 57 nucleotides of ISR are sufficient to drive AGO1 translational readthrough . AGO1 - TGA - FLuc constructs with different lengths of ISR ( all in - frame with AGO1 and FLuc ) were in vitro transcribed and in vitro translated using rabbit reticulocyte lysate . FLuc activity reflects readthrough activity . * * , P = 0 . 012 ; * , P = 0 . 017 .

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

First 57 nucleotides of ISR are sufficient to drive AGO1 translational readthrough. AGO1-TGA-FLuc constructs with different lengths of ISR (all in-frame with AGO1 and FLuc) were in vitro transcribed and in vitro translated using rabbit reticulocyte lysate. FLuc activity reflects readthrough activity. **, P = 0.012; *, P = 0.017.


Demonstration of translational readthrough of AGO1 using fluorescence - based reporter assay . Plasmid containing in - frame AGO1 - TGA ( or GCA ) - ISR - GFP were transfected in HEK293 cells and translational readthrough was detected as fluorescence . Scale bar , 50 um . The bar graph shows mean fluorescence intensities in cells transfected with indicated constructs . Fluorescence intensity was measured by flowcytometry . * * * , P = 0 . 007 .

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

Demonstration of translational readthrough of AGO1 using fluorescence-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-GFP were transfected in HEK293 cells and translational readthrough was detected as fluorescence. Scale bar, 50 um. The bar graph shows mean fluorescence intensities in cells transfected with indicated constructs. Fluorescence intensity was measured by flowcytometry. ***, P = 0.007.


Detection of endogenous Ago1x by Western blot in HEK293 cells using anti - Ago1x antibody and pre - immune serum .

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

Detection of endogenous Ago1x by Western blot in HEK293 cells using anti-Ago1x antibody and pre-immune serum.


Western blot of lysates from HEK293 and HeLa cells using anti - Ago1x antibody pre - treated with Ago1x - specific peptide .

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

Western blot of lysates from HEK293 and HeLa cells using anti-Ago1x antibody pre-treated with Ago1x-specific peptide.


Expression of Ago1x in HEK293 cells transfected with three AGO1 - specific siRNAs ( 1 , 2 , 3 ) . qRT - PCR analysis of AGO1 transcript expression in these cells is shown below .

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

Expression of Ago1x in HEK293 cells transfected with three AGO1-specific siRNAs (1,2,3). qRT-PCR analysis of AGO1 transcript expression in these cells is shown below.


Proportion of Ago1x isoform in HEK293 cells . To separate Ago1x and Ago1 ( ~ 3 kDa difference ) , the cell lysate was electrophoresed for 3 . 5 h in 10 % polyacrylamide gel . Proportion of Ago1x and canonical isoform ( Ago1 ) was calculated by densitometric analysis . Results from three independent experiments are shown as a bar graph ( mean ± S . E . ) .

Proportion of Ago1x isoform in HEK293 cells. To separate Ago1x and Ago1 (~ 3 kDa difference), the cell lysate was electrophoresed for 3.5 h in 10% polyacrylamide gel. Proportion of Ago1x and canonical isoform (Ago1) was calculated by densitometric analysis. Results from three independent experiments are shown as a bar graph (mean ± S.E.).


Expression of Ago1x in mouse organs.


Presence of ribosome footprint in the Inter stop codon region of AGO1 transcript in U2 - OS cells . Ribosome profiling data was taken from NCBI ' s sequence read archive ( SRA Accession no . SRR1257257 ) and ribosome footprints on AGO1 transcript were analyzed as described in Methods section . Zoomed area near the inter - stop codon region is shown in the inset . Position of canonical stop codon and the downstream in - frame stop codon are shown . Detection of Ago1x by Western blot in U2 - OS cells is shown ( right ) .

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

Presence of ribosome footprint in the Inter stop codon region of AGO1 transcript in U2-OS cells. Ribosome profiling data was taken from NCBI's sequence read archive (SRA Accession no. SRR1257257) and ribosome footprints on AGO1 transcript were analyzed as described in Methods section. Zoomed area near the inter-stop codon region is shown in the inset. Position of canonical stop codon and the downstream in-frame stop codon are shown. Detection of Ago1x by Western blot in U2-OS cells is shown (right).


Effect of let - 7a inhibitor on translational readthrough of AGO1 . let - 7a inhibitor or control inhibitor was transfected in HeLa cells and the expression of Ago1x was determined by Western blot after 48 h . Densitometric analysis of three independent experiments is shown as a bar diagram ( mean ± S . E . ) . * , P = 0 . 01 .

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

Effect of let-7a inhibitor on translational readthrough of AGO1. let-7a inhibitor or control inhibitor was transfected in HeLa cells and the expression of Ago1x was determined by Western blot after 48 h. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.01.


Effect of let - 7a overexpression on translational readthrough of AGO1 . HeLa cells were stably transfected with pri - let - 7a overexpression construct . Ago1x expression was determined by Western blot . Densitometric analysis of three independent experiments is shown as a bar diagram ( mean ± S . E . ) . * , P = 0 . 007 .

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

Effect of let-7a overexpression on translational readthrough of AGO1. HeLa cells were stably transfected with pri-let-7a overexpression construct. Ago1x expression was determined by Western blot. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.007.


let - 7a inhibitor reduces the AGO1 readthrough efficiency . HeLa cells were transfected with let - 7a inhibitor or control inhibitor along with AGO1 - TGA ( or GCA ) - ISR - FLuc construct . Readthrough was quantified by measuring FLuc activity , which was normalized to co - transfected renilla luciferase activity . FLuc mRNA expression in HeLa cells treated with miRNA inhibitors is shown ( Right ) . * , P = 0 . 027 .

let-7a inhibitor reduces the AGO1 readthrough efficiency. HeLa cells were transfected with let-7a inhibitor or control inhibitor along with AGO1-TGA (or GCA)-ISR-FLuc construct. Readthrough was quantified by measuring FLuc activity, which was normalized to co-transfected renilla luciferase activity. FLuc mRNA expression in HeLa cells treated with miRNA inhibitors is shown (Right). *, P = 0.027.


AGO1 ISR with mutated let - 7a binding site shows reduced translational readthrough activity . Assay was done as described above in HeLa cells . Mutated sequence is shown ( top ) . * * * , P = 0 . 008 .

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

AGO1 ISR with mutated let-7a binding site shows reduced translational readthrough activity. Assay was done as described above in HeLa cells. Mutated sequence is shown (top). ***, P = 0.008.


AGO1 ISR with displaced let - 7a binding site ( AGO1 - TGA - disISR - FLuc ) shows reduced translational readthrough activity . let - 7a binding site was moved 18 nucleotides downstream of the stop codon . Readthrough assay was done as described above in HeLa cells . * * * , P < 0 . 0001 .

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

AGO1 ISR with displaced let-7a binding site (AGO1-TGA-disISR-FLuc) shows reduced translational readthrough activity. let-7a binding site was moved 18 nucleotides downstream of the stop codon. Readthrough assay was done as described above in HeLa cells. ***, P < 0.0001.


AGO1 ISR can drive translational readthrough in a heterologous context of RHOA . AGO1 coding sequence in AGO1 - TGA - ISR - FLuc construct was replaced by RHOA coding sequence ( 582 nucleotides ) and the readthrough assay was done as described above in HeLa cells . 3 ′ UTRRho , 159 nucleotides in the proximal 3 ′ UTR of RHOA ( NM _ 001664 ) ; ISRAgo1 , Inter - stop codon region of AGO1 . * * * , P < 0 . 0001 .

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

AGO1 ISR can drive translational readthrough in a heterologous context of RHOA. AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RHOA coding sequence (582 nucleotides) and the readthrough assay was done as described above in HeLa cells. 3′UTRRho, 159 nucleotides in the proximal 3′UTR of RHOA (NM_001664); ISRAgo1, Inter-stop codon region of AGO1. ***, P < 0.0001.


AGO1 ISR can drive translational readthrough in a heterologous context of renilla luciferase ( RLuc ) . AGO1 coding sequence in AGO1 - TGA - ISR - FLuc construct was replaced by RLuc coding sequence and the readthrough assay was done as described above in HeLa cells ( left ) and in vitro using rabbit reticulocyte lysate system ( right ) . * * , P = 0 . 006 ; * * * , P = 0 . 0001 .

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

AGO1 ISR can drive translational readthrough in a heterologous context of renilla luciferase (RLuc). AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RLuc coding sequence and the readthrough assay was done as described above in HeLa cells (left) and in vitro using rabbit reticulocyte lysate system (right). **, P = 0.006; ***, P = 0.0001.


Tethering of N - peptide - tagged Ago1 or Ago1x can induce readthrough of AGO1 . Two BoxB elements were cloned downstream of the AGO1 and upstream of the in - frame FLuc as shown in the schematic . This construct was transfected in HeLa cells expressing N - peptide - tagged or FLAG - tagged Ago proteins as indicated . Readthrough was measured as described above . * , P = 0 . 035 ( with Welch correction ) ; * * , P = 0 . 016 .

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

Tethering of N-peptide-tagged Ago1 or Ago1x can induce readthrough of AGO1. Two BoxB elements were cloned downstream of the AGO1 and upstream of the in-frame FLuc as shown in the schematic. This construct was transfected in HeLa cells expressing N-peptide-tagged or FLAG-tagged Ago proteins as indicated. Readthrough was measured as described above. *, P = 0.035 (with Welch correction); **, P = 0.016.


Linear regression analysis of miRNAs co - immunoprecipiated with Ago1 and those co - immunoprecipiated with Ago1x in HeLa cells . Reads per million ( RPM ) values of 395 miRNAs , which were common to Ago1 and Ago1x are 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 O O O O O O O O O O O

Linear regression analysis of miRNAs co-immunoprecipiated with Ago1 and those co-immunoprecipiated with Ago1x in HeLa cells. Reads per million (RPM) values of 395 miRNAs, which were common to Ago1 and Ago1x are shown.


qRT - PCR results showing the enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa cells . Bars show mean of three experiments ± S . E .

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

qRT-PCR results showing the enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa cells. Bars show mean of three experiments ± S.E.


RT - PCR results showing PTEN and MYC mRNAs co - immunoprecipitated with Ago1 or Ago1x in HeLa cells .

RT-PCR results showing PTEN and MYC mRNAs co-immunoprecipitated with Ago1 or Ago1x in HeLa cells.


Result of co - immunoprecipitation experiments demonstrating the interaction of Dicer protein with Ago1 and Ago1x in HeLa cells .

Result of co-immunoprecipitation experiments demonstrating the interaction of Dicer protein with Ago1 and Ago1x in HeLa cells.


Result of co - immunoprecipitation experiment in HeLa cells shows that GW182 is co - immunoprecipitated with Ago1 , but not with Ago1x .

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

Result of co-immunoprecipitation experiment in HeLa cells shows that GW182 is co-immunoprecipitated with Ago1, but not with Ago1x.


Confocal immunofluoresence images of HeLa cells show co - localization of GW182 with Ago1 , but not with Ago1x . White circles indicate zoomed areas .

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

Confocal immunofluoresence images of HeLa cells show co-localization of GW182 with Ago1, but not with Ago1x. White circles indicate zoomed areas.


Confocal microscopy images of proximity ligation assay ( PLA ) in HeLa cells showing interaction of GW182 with Ago1 , but not with Ago1x . Rabbit anti - GW182 and Mouse anti - FLAG antibodies were used to achieve proximity ligation . Interaction is indicated by red dots that results from proximity ligation . Quantification of dots per cell is shown ( right ) . Each black circle represents a microscopic field . Mean ± S . E . are shown as horizontal lines ( n = 10 fields for vector control ; n = 12 fields for Ago1 ; n = 11 fields for Ago1x ) . At least 100 cells were counted for dots in each category . * * * , P < 0 . 0001 ( Mann - Whitney test ) .

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

Confocal microscopy images of proximity ligation assay (PLA) in HeLa cells showing interaction of GW182 with Ago1, but not with Ago1x. Rabbit anti-GW182 and Mouse anti-FLAG antibodies were used to achieve proximity ligation. Interaction is indicated by red dots that results from proximity ligation. Quantification of dots per cell is shown (right). Each black circle represents a microscopic field. Mean ± S.E. are shown as horizontal lines (n=10 fields for vector control; n=12 fields for Ago1; n=11 fields for Ago1x). At least 100 cells were counted for dots in each category. ***, P < 0.0001 (Mann-Whitney test).


Activity of RLuc relative to co - transfected FLuc in HeLa cells expressing N - HA - tagged or untagged Ago proteins as indicated . Bar graph ( mean ± S . E . ) is a representative of four independent experiments done in triplicates . Expression of HA - tagged Ago proteins was confirmed by Western blot ( bottom ) . * , P = 0 . 018 ; # , P = 0 . 041 ; $ , P = 0 . 013 . Numbers on the bars represent % change in relative luciferase activity .

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

Activity of RLuc relative to co-transfected FLuc in HeLa cells expressing N-HA-tagged or untagged Ago proteins as indicated. Bar graph (mean ± S.E.) is a representative of four independent experiments done in triplicates. Expression of HA-tagged Ago proteins was confirmed by Western blot (bottom). *, P = 0.018; #, P = 0.041; $, P = 0.013. Numbers on the bars represent % change in relative luciferase activity.


Expression of RLuc protein in HeLa cells expressing N - HA - tagged or untagged Ago proteins as indicated . Densitometric analysis of three independent experiments is shown as bar graph ( mean ± S . E . ) . RT - PCR analysis for RLuc mRNA level is shown ( bottom ) . * * , P = 0 . 001 .

Expression of RLuc protein in HeLa cells expressing N-HA-tagged or untagged Ago proteins as indicated. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). RT-PCR analysis for RLuc mRNA level is shown (bottom). **, P = 0.001.


Activity of RLuc relative to co - transfected FLuc in HeLa cells expressing N - HA - tagged Ago2 ( N - HA - Ago2 ) or N - HA - tagged chimeric Ago2 appended with the ISR region of Ago1x ( N - HA - Ago2 - ISRAgo1 ) as indicated . Bar graph ( mean ± S . E . ) is a representative of three independent experiments done in triplicates . Expression of HA - tagged Ago proteins was confirmed by Western blot ( bottom ) . * * * , P < 0 . 0001 ; * * , P = 0 . 0004 .

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

Activity of RLuc relative to co-transfected FLuc in HeLa cells expressing N-HA-tagged Ago2 (N-HA-Ago2) or N-HA-tagged chimeric Ago2 appended with the ISR region of Ago1x (N-HA-Ago2-ISRAgo1) as indicated. Bar graph (mean ± S.E.) is a representative of three independent experiments done in triplicates. Expression of HA-tagged Ago proteins was confirmed by Western blot (bottom). ***, P < 0.0001; **, P = 0.0004.


Ribopuromycylation assay showing global translation profile of HeLa cells overexpressing FLAG - HA - Ago1 or FLAG - HA - Ago1x . Densitometric analysis of four independent experiments is shown as bar graph ( mean ± S . E . ) . Puromycin densities were normalized to ponceau S stain densities . * , P < 0 . 029 ( Mann - Whitney test ) .

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

Ribopuromycylation assay showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of four independent experiments is shown as bar graph (mean ± S.E.). Puromycin densities were normalized to ponceau S stain densities. *, P < 0.029 (Mann-Whitney test).


[ 35S ] - methionine autoradiogram showing global translation profile of HeLa cells overexpressing FLAG - HA - Ago1 or FLAG - HA - Ago1x . Densitometric analysis of three independent experiments is shown as bar graph ( mean ± S . E . ) . Autoradiogram densities were normalized to Coomassie stain densities . * , P < 0 . 029 ( Mann - Whitney test ) .

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

[35S]-methionine autoradiogram showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). Autoradiogram densities were normalized to Coomassie stain densities. *, P < 0.029 (Mann-Whitney test).


( a ) Western blot analysis showing the replacement of endogenous Beclin 1 with Beclin 1 - EGFP in Becn1 - / - ; Becn1 - EGFP / + mice , as detected by an anti - Beclin 1 antibody .

O O O O O O O O O O O O O O O O O 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 showing the replacement of endogenous Beclin 1 with Beclin 1-EGFP in Becn1-/-;Becn1-EGFP/+ mice, as detected by an anti-Beclin 1 antibody.


( b ) Coomassie - stained SDS - PAGE showing the Beclin 1 - interacting proteins immuno - isolated from brain and liver of the ' rescued ' mice ( lanes 2 and 4 ) and of control Becn1 + / - littermates ( lanes 1 and 3 ) , using an anti - GFP antibody . Proteins in the gel bands were extracted and identified by mass spectrometry as Vps15 / p150 ( band 1 ) , Vps34 / class III PI ( 3 ) K ( band 3 ) , UVRAG ( band 4 ) , Beclin 1 - EGFP ( band 5 ) , Atg14L ( band 6 , asterisk , gi | 27369860 ) and Rubicon ( band 2 , asterisk , gi | 45708948 ) . UVRAG levels varied with different affinity - purification conditions , suggesting an unstable association of UVRAG with the complex .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Coomassie-stained SDS-PAGE showing the Beclin 1-interacting proteins immuno-isolated from brain and liver of the 'rescued' mice (lanes 2 and 4) and of control Becn1+/- littermates (lanes 1 and 3), using an anti-GFP antibody. Proteins in the gel bands were extracted and identified by mass spectrometry as Vps15/p150 (band 1), Vps34/class III PI(3)K (band 3), UVRAG (band 4), Beclin 1-EGFP (band 5), Atg14L (band 6, asterisk, gi|27369860) and Rubicon (band 2, asterisk, gi|45708948). UVRAG levels varied with different affinity-purification conditions, suggesting an unstable association of UVRAG with the complex.


( d ) Western blot analysis of Atg14L , Rubicon , Vps34 and Beclin 1 in gel filtration fractions from wild - type mouse liver extract showed co - elution of these proteins in fractions 38 - 45 . Atg14L was also eluted in later fractions 51 - 56 . The fractions for the peak elution of thyroglobulin ( 670K ) and γ - globulin ( 158K ) are indicated by arrows . Control siRNA - transfected NIH 3T3 cell lysate was loaded as a positive control ( labelled as 1 ) for the migration position of the Atg14L protein on SDS - PAGE ; Atg14L siRNA - transfected NIH 3T3 cell lysate was loaded as a negative control ( labelled as 2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of Atg14L, Rubicon, Vps34 and Beclin 1 in gel filtration fractions from wild-type mouse liver extract showed co-elution of these proteins in fractions 38-45. Atg14L was also eluted in later fractions 51-56. The fractions for the peak elution of thyroglobulin (670K) and γ-globulin (158K) are indicated by arrows. Control siRNA-transfected NIH 3T3 cell lysate was loaded as a positive control (labelled as 1) for the migration position of the Atg14L protein on SDS-PAGE; Atg14L siRNA-transfected NIH 3T3 cell lysate was loaded as a negative control (labelled as 2).


( e , f ) Co - immunoprecipitation confirmed protein - protein interaction between Atg14L and Rubicon . HEK 293 cells were co - transfected with Atg14L - EGFP and Flag - Rubicon ( e ) or Rubicon - EGFP and Flag - Atg14L ( f ) . Cell lysates were used for immunoprecipitation with an anti - GFP antibody and the resulting immunoprecipitates were blotted with an anti - Flag antibody . Our results show immunoprecipitation of Rubicon by Atg14L ( e ) and vice versa ( f ) . WCL , whole cell lysate ; IP , immunoprecipitated . See Supplementary Information , Fig . S6 for full scans of blots in a , d , e and f .

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

(e, f) Co-immunoprecipitation confirmed protein-protein interaction between Atg14L and Rubicon. HEK 293 cells were co-transfected with Atg14L-EGFP and Flag-Rubicon (e) or Rubicon-EGFP and Flag-Atg14L (f). Cell lysates were used for immunoprecipitation with an anti-GFP antibody and the resulting immunoprecipitates were blotted with an anti-Flag antibody. Our results show immunoprecipitation of Rubicon by Atg14L (e) and vice versa (f). WCL, whole cell lysate; IP, immunoprecipitated. See Supplementary Information, Fig. S6 for full scans of blots in a, d, e and f.


( a ) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62 / SQSTM1 and LC3 II levels under normal and nutrient - starvation conditions in NIH 3T3 cells .

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

(a) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62/SQSTM1 and LC3 II levels under normal and nutrient-starvation conditions in NIH 3T3 cells.


( b ) Compared with control siRNA , Atg14L siRNA decreased long - lived protein degradation in NIH 3T3 cells under normal ( * P = 0 . 007 ) and starvation ( * P = 5 × 10 − 6 ) conditions ( one - tailed Student ' s t - test with equal variances , n = 4 ) . This difference was diminished when the starved cells were treated with 3 - methyladenine ( 3MA , 10 mM ) , a PI ( 3 ) K inhibitor .

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

(b) Compared with control siRNA, Atg14L siRNA decreased long-lived protein degradation in NIH 3T3 cells under normal (*P = 0.007) and starvation (*P = 5 × 10−6) conditions (one-tailed Student's t-test with equal variances, n = 4). This difference was diminished when the starved cells were treated with 3-methyladenine (3MA, 10 mM), a PI(3)K inhibitor.


( c ) Vps34 kinase assay . HEK 293T cells were co - transfected with Myc - Vps34 - Vps15 and Flag - Atg14L or Flag vector , either in the absence or in the presence of Beclin 1 - EGFP . Myc - Vps34 - Vps15 was immunoprecipitated by anti - Myc antibody for the in vitro kinase assay . The resulting radioactive PI ( 3 ) P was separated by thin - layer chromatography ( TLC ) , quantified and normalized against the amount of immunoprecipitated Myc - tagged Vps34 as measured by western blot ( upper panel ) . The quantified results ( lower panel ) show that overexpressing Atg14L significantly upregulated Vps34 kinase activity by 2 . 5 - fold , but only when Beclin 1 was also overexpressed ( * P = 0 . 04 , one - tailed Student ' s t - test with unequal variances , n = 5 ) .

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

(c) Vps34 kinase assay. HEK 293T cells were co-transfected with Myc-Vps34-Vps15 and Flag-Atg14L or Flag vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by anti-Myc antibody for the in vitro kinase assay. The resulting radioactive PI(3)P was separated by thin-layer chromatography (TLC), quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34 as measured by western blot (upper panel). The quantified results (lower panel) show that overexpressing Atg14L significantly upregulated Vps34 kinase activity by 2.5-fold, but only when Beclin 1 was also overexpressed (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 5).


( d ) Colocalization of co - expressed Atg14L - EGFP ( green ) and Beclin 1 - AsRed ( red ) in punctate structures in transiently transfected HeLa cells . Scale bar , 10 μm .

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

(d) Colocalization of co-expressed Atg14L-EGFP (green) and Beclin 1-AsRed (red) in punctate structures in transiently transfected HeLa cells. Scale bar, 10 μm.


( e ) Electron microscopy images show large structures ( asterisks ) that are often enwrapped with double membranes in the HEK 293T cells co - transfected with Atg14L - EGFP and Beclin 1 - AsRed : concentric membrane ' rings ' ( panel 1 ) ; two large structures ( 3 - 5 μm in diameter , panel 2 ) containing material with high electron density ( inset , enwrapping double membranes ) ; numerous autophagosomes ( arrows , panel 3 ) in the cytoplasm ; immuno - electron microscopy image of a Atg14L - Beclin 1 structure ( labelled with anti - GFP antibody and developed by DAB , panel 4 ) enwrapped with concentric membrane ' rings ' . M , mitochondria ; N , nucleus . Scale bar , 500 nm .

(e) Electron microscopy images show large structures (asterisks) that are often enwrapped with double membranes in the HEK 293T cells co-transfected with Atg14L-EGFP and Beclin 1-AsRed: concentric membrane 'rings' (panel 1); two large structures (3-5 μm in diameter, panel 2) containing material with high electron density (inset, enwrapping double membranes); numerous autophagosomes (arrows, panel 3) in the cytoplasm; immuno-electron microscopy image of a Atg14L-Beclin 1 structure (labelled with anti-GFP antibody and developed by DAB, panel 4) enwrapped with concentric membrane 'rings'. M, mitochondria; N, nucleus. Scale bar, 500 nm.


( f , g ) EGFP - Atg12 ( f ) or EGFP - Atg5 ( g ) ( green ) was colocalized with the large structures ( arrows ) that were labelled by Atg14L - AsRed ( red ) and Beclin 1 - Myc ( blue ) in transfected HeLa cells . Some of these structures seemed to be ' ring ' - shaped ( yellow arrows and inset ) . Scale bar , 10 μm . See Supplementary Information , Fig . S6 for full scans of blots in a and c .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) EGFP-Atg12 (f) or EGFP-Atg5 (g) (green) was colocalized with the large structures (arrows) that were labelled by Atg14L-AsRed (red) and Beclin 1-Myc (blue) in transfected HeLa cells. Some of these structures seemed to be 'ring'-shaped (yellow arrows and inset). Scale bar, 10 μm. See Supplementary Information, Fig. S6 for full scans of blots in a and c.


( a ) Rubicon siRNA treatment of the NIH 3T3 cells led to decreased levels of p62 and LC3 II under both normal and nutrient - starvation conditions .

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

(a) Rubicon siRNA treatment of the NIH 3T3 cells led to decreased levels of p62 and LC3 II under both normal and nutrient-starvation conditions.


( b ) Overexpression of Rubicon resulted in increased levels of p62 under both normal and nutrient - starved conditions in HEK 293 cells either stably expressing ( upper rows ) or transiently transfected with ( lower rows ) Rubicon - EGFP . The control cells were either stably expressing or transiently transfected with the EGFP - N3 vector .

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

(b) Overexpression of Rubicon resulted in increased levels of p62 under both normal and nutrient-starved conditions in HEK 293 cells either stably expressing (upper rows) or transiently transfected with (lower rows) Rubicon-EGFP. The control cells were either stably expressing or transiently transfected with the EGFP-N3 vector.


( c ) Vps34 kinase activity . HEK 293T cells were co - transfected with Myc - Vps34 - Vps15 and Flag - Rubicon or Flag vector , either in the absence or in the presence of Beclin 1 - EGFP . Myc - Vps34 - Vps15 was immunoprecipitated by an anti - Myc antibody and used for the in vitro kinase assay . The resulting radioactive PI ( 3 ) P was separated by TLC , quantified and normalized against the amount of immunoprecipitated Myc - tagged Vps34 , as measured by western blotting ( upper panel ) . The quantified results ( lower panel ) show that overexpressing Rubicon significantly downregulated the Vps34 kinase activity to 0 . 58 - fold , but only without Beclin 1 overexpression ( * P = 0 . 04 , one - tailed Student ' s t - test with unequal variances , n = 4 ) .

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

(c) Vps34 kinase activity. HEK 293T cells were co-transfected with Myc-Vps34-Vps15 and Flag-Rubicon or Flag vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by an anti-Myc antibody and used for the in vitro kinase assay. The resulting radioactive PI(3)P was separated by TLC, quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34, as measured by western blotting (upper panel). The quantified results (lower panel) show that overexpressing Rubicon significantly downregulated the Vps34 kinase activity to 0.58-fold, but only without Beclin 1 overexpression (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 4).


( d ) Effect of overexpressing Flag - Rubicon on autophagosome acidification , as monitored by mCherry - GFP - LC3 fluorescence . HeLa cells were transiently co - transfected with mCherry - GFP - LC3 and Flag - Rubicon ( or control Flag vector ) . Cells co - expressing mCherry - GFP - LC3 and control Flag vector contained many red - only puncta along with yellow ( indicating the presence of both red and green ) puncta , suggesting the presence of both autolysosomes and nascent autophagosomes ( upper panel ) . In contrast , cells co - expressing mCherry - GFP - LC3 and Flag - Rubicon contained primarily yellow or white puncta , suggesting the presence of only nascent autophagosomes ( lower panel , white arrows ) . Notably , some cells , which were co - transfected with mCherry - GFP - LC3 and Flag - Rubicon but expressed high levels of mCherry - GFP - LC3 and undetectable levels of FLAG - Rubicon , contained many red - only puncta ( lower panel , yellow arrows ) . ( e ) Quantification of the results in d show that overexpressing Flag - Rubicon markedly reduced the percentage of red - only puncta ( mCherry - LC3 ) from 39 % in the control Flag vector - transfected cells to 2 % in the Flag - Rubicon - transfected cells ( * P = 2 × 10 − 26 , one - tailed Student ' s t - test with unequal variances , n = 30 ) , indicating that overexpression of Rubicon blocks autophagosome acidification or maturation . See Supplementary Information , Fig . S6 for full scans of blots in a - c .

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

(d) Effect of overexpressing Flag-Rubicon on autophagosome acidification, as monitored by mCherry-GFP-LC3 fluorescence. HeLa cells were transiently co-transfected with mCherry-GFP-LC3 and Flag-Rubicon (or control Flag vector). Cells co-expressing mCherry-GFP-LC3 and control Flag vector contained many red-only puncta along with yellow (indicating the presence of both red and green) puncta, suggesting the presence of both autolysosomes and nascent autophagosomes (upper panel). In contrast, cells co-expressing mCherry-GFP-LC3 and Flag-Rubicon contained primarily yellow or white puncta, suggesting the presence of only nascent autophagosomes (lower panel, white arrows). Notably, some cells, which were co-transfected with mCherry-GFP-LC3 and Flag-Rubicon but expressed high levels of mCherry-GFP-LC3 and undetectable levels of FLAG-Rubicon, contained many red-only puncta (lower panel, yellow arrows). (e) Quantification of the results in d show that overexpressing Flag-Rubicon markedly reduced the percentage of red-only puncta (mCherry-LC3) from 39% in the control Flag vector-transfected cells to 2% in the Flag-Rubicon-transfected cells (*P = 2 × 10−26, one-tailed Student's t-test with unequal variances, n = 30), indicating that overexpression of Rubicon blocks autophagosome acidification or maturation. See Supplementary Information, Fig. S6 for full scans of blots in a-c.


( a ) Colocalization of Rubicon - EGFP - associated structures with the late endosome / lysosome marker Lamp1 ( arrows ) in HeLa cells transfected with Rubicon - EGFP . Note that some of the Rubicon - EGFP - associated structures show a ' ring ' shape ( yellow arrows ) . Scale bar , 10 μm .

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

(a) Colocalization of Rubicon-EGFP-associated structures with the late endosome/lysosome marker Lamp1 (arrows) in HeLa cells transfected with Rubicon-EGFP. Note that some of the Rubicon-EGFP-associated structures show a 'ring' shape (yellow arrows). Scale bar, 10 μm.


( b ) Partial colocalization of Rubicon - EGFP - associated structures with the MVB marker LBPA ( arrows ) in HeLa cells transfected with Rubicon - EGFP . Scale bar , 10 μm .

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

(b) Partial colocalization of Rubicon-EGFP-associated structures with the MVB marker LBPA (arrows) in HeLa cells transfected with Rubicon-EGFP. Scale bar, 10 μm.


( c ) Representative ultrastructural images show aberrant expansion of late endosomal / lysosomal structures in HEK 293T cells overexpressing Rubicon - EGFP . These abnormal organelles are large in size , with high ( orange arrows ) or low ( black arrows ) electron density . Some enclose small vesicles ( purple arrows ) and some resemble the MVB ( blue arrows ) . Scale bars , 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 O O O O O O O O O O O O O O

(c) Representative ultrastructural images show aberrant expansion of late endosomal/lysosomal structures in HEK 293T cells overexpressing Rubicon-EGFP. These abnormal organelles are large in size, with high (orange arrows) or low (black arrows) electron density. Some enclose small vesicles (purple arrows) and some resemble the MVB (blue arrows). Scale bars, 500 nm.


( d ) Representative ultrastructural images show late endosome / lysosome - like structures that are labelled with anti - GFP gold particles ( panels 3 , 4 ) in HEK 293T cells transiently transfected with Rubicon - EGFP . These structures are enwrapped by double membranes ( panel 4 inset ) and co - labelled by anti - GFP ( developed by DAB ) and anti - Lamp1 ( gold enhanced ) ( panels 5 - 7 ) antibodies . Note that mitochondria are mostly negative for Rubicon - EGFP ( panel 4 ) . The negative controls are without antibody ( panels 1 - 2 ) . M , mitochondria . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative ultrastructural images show late endosome/lysosome-like structures that are labelled with anti-GFP gold particles (panels 3, 4) in HEK 293T cells transiently transfected with Rubicon-EGFP. These structures are enwrapped by double membranes (panel 4 inset) and co-labelled by anti-GFP (developed by DAB) and anti-Lamp1 (gold enhanced) (panels 5-7) antibodies. Note that mitochondria are mostly negative for Rubicon-EGFP (panel 4). The negative controls are without antibody (panels 1-2). M, mitochondria. Scale bars, 200 nm.


( b ) Colocalization of the PI ( 3 ) P - enriched lipid domain marker p40 ( phox ) - PX - EGFP ( green ) and Rubicon - AsRed ( red ) on large punctate structures ( arrows ) in the co - transfected HeLa cells ( upper panels ) . Treatment with the PI ( 3 ) K inhibitor wortmannin ( 75 nM ) for 1 h caused disappearance of the PI ( 3 ) P - enriched lipid domains , whereas the Rubicon - AsRed - positive structures were maintained ( lower panels ) . Scale bars , 10 μm .

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

(b) Colocalization of the PI(3)P-enriched lipid domain marker p40 (phox)-PX-EGFP (green) and Rubicon-AsRed (red) on large punctate structures (arrows) in the co-transfected HeLa cells (upper panels). Treatment with the PI(3)K inhibitor wortmannin (75 nM) for 1 h caused disappearance of the PI(3)P-enriched lipid domains, whereas the Rubicon-AsRed-positive structures were maintained (lower panels). Scale bars, 10 μm.


( c ) Subcellular localization of transiently transfected Rubicon - EGFP , RubiconΔRUN - EGFP , RubiconΔC - EGFP or RubiconΔRUNΔC - EGFP in HeLa cells . In contrast to punctate Rubicon - EGFP and RubiconΔRUN - EGFP , RubiconΔC - EGFP and RubiconΔRUNΔC - EGFP were dispersed in the cytoplasm . ΔRUN , RUN domain deletion ; ΔC , cysteine - rich domain deletion . Scale bars , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Subcellular localization of transiently transfected Rubicon-EGFP, RubiconΔRUN-EGFP, RubiconΔC-EGFP or RubiconΔRUNΔC-EGFP in HeLa cells. In contrast to punctate Rubicon-EGFP and RubiconΔRUN-EGFP, RubiconΔC-EGFP and RubiconΔRUNΔC-EGFP were dispersed in the cytoplasm. ΔRUN, RUN domain deletion; ΔC, cysteine-rich domain deletion. Scale bars, 10 μm.


( d , e ) Absence of full - length Beclin 1 ( d ) or Beclin 1 - CE mutant ( e ) ( red ) on the Rubicon - EGFP - positive structures ( green ) in HEK 293 cells stably expressing Rubicon - EGFP . These cells were transiently transfected with either Beclin 1 - AsRed ( d ) or Flag - Beclin 1 - CE ( e ; that is , the Flag - tagged Beclin 1 mutant containing both CCD and ECD , which mediate the Beclin 1 - Rubicon interaction as shown in Supplementary Information , Fig . S3h ) . Scale bars , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, e) Absence of full-length Beclin 1 (d) or Beclin 1-CE mutant (e) (red) on the Rubicon-EGFP-positive structures (green) in HEK 293 cells stably expressing Rubicon-EGFP. These cells were transiently transfected with either Beclin 1-AsRed (d) or Flag-Beclin 1-CE (e; that is, the Flag-tagged Beclin 1 mutant containing both CCD and ECD, which mediate the Beclin 1-Rubicon interaction as shown in Supplementary Information, Fig. S3h). Scale bars, 10 μm.


( f ) The formation of the Rubicon - EGFP - positive structures was not affected by siRNA knockdown of Beclin 1 in HEK 293 cells stably expressing Rubicon - EGFP .

O O O O O O O O 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) The formation of the Rubicon-EGFP-positive structures was not affected by siRNA knockdown of Beclin 1 in HEK 293 cells stably expressing Rubicon-EGFP.


( D , E ) Scatter plot visualization of YFP vs mCherry fluorescence signals in the GCG1 and ORC2 promoter FPR screens . Each data point represents the median signal of a deletion mutant normalized to individual plate median . Highlighted data points represent the mutants of Hda1C ( purple ) , SAGA acetylation module ( blue ) , SAGA deubiquitination module ( orange ) , and cac2 ∆ ( yellow ) . The regression line is marked in black . The mutants favoring DNC transcription are found above the regression line . The mutants favoring coding transcription are below the regression line . Technical repeat = 1 , biological observations = 50 , 000 individual cells before filtering .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) Scatter plot visualization of YFP vs mCherry fluorescence signals in the GCG1 and ORC2 promoter FPR screens. Each data point represents the median signal of a deletion mutant normalized to individual plate median. Highlighted data points represent the mutants of Hda1C (purple), SAGA acetylation module (blue), SAGA deubiquitination module (orange), and cac2∆ (yellow). The regression line is marked in black. The mutants favoring DNC transcription are found above the regression line. The mutants favoring coding transcription are below the regression line. Technical repeat = 1, biological observations = 50,000 individual cells before filtering.


( C ) Flow cytometry quantification of mCherry / YFP fluorescence in the mutants with GCG1pr FPR background . The data are normalized to the signal values of the wild type ( FPR ) strain .

O O O O O O O O O O O O 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) Flow cytometry quantification of mCherry/YFP fluorescence in the mutants with GCG1pr FPR background. The data are normalized to the signal values of the wild type (FPR) strain.


( D ) Endogenous transcript analysis by RT - qPCR in the mutants . The bars represent fold gene expression ratio of coding and divergent transcript normalized to the expression of the reference gene , UBC6 .

(D) Endogenous transcript analysis by RT-qPCR in the mutants. The bars represent fold gene expression ratio of coding and divergent transcript normalized to the expression of the reference gene, UBC6.


. ( B ) NET - seq data at the YPL172C locus . The signal represents NET - seq reads showing nascent transcription at the genomic position of the divergent non - coding strand ( black ) and the coding strand ( grey ) . The bar graph depicts NET - seq reads as FPKM values for the coding and DNC transcript in the strains

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NET-seq data at the YPL172C locus. The signal represents NET-seq reads showing nascent transcription at the genomic position of the divergent non-coding strand (black) and the coding strand (grey). The bar graph depicts NET-seq reads as FPKM values for the coding and DNC transcript in the strains


. ( C ) NET - seq data at the YDR261C locus with a novel DNC transcript . Annotations as in ( B )

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) NET-seq data at the YDR261C locus with a novel DNC transcript. Annotations as in (B)


( C ) A representative image of transcription initiation observed as fluorescence spots in the recorded live - cell movie of wild type strain . The movie output using Python were analyzed and the snapshot was obtained using Image J software . The red and green channel denotes GCG1 and SUT098 transcript initiation , respectively . Images represent maximum intensity projections , and the right and bottom sidebars indicate sideviews in the yz and xz directions , respectively . Scale : 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A representative image of transcription initiation observed as fluorescence spots in the recorded live-cell movie of wild type strain. The movie output using Python were analyzed and the snapshot was obtained using Image J software. The red and green channel denotes GCG1 and SUT098 transcript initiation, respectively. Images represent maximum intensity projections, and the right and bottom sidebars indicate sideviews in the yz and xz directions, respectively. Scale: 5 µm.


( D , E ) Quantification of transcription dynamics by the duration and frequency ( time between initiation ) of transcription . Bar graphs show the transcription parameters of SUT098 in candidate mutants compared to the wild type strain . The error bars represent SEM calculated by live - cell image analysis of 179 , 135 , 60 and 129 single - cell biological repeats for WT , hda1 ∆ , hda3 ∆ and cac2 ∆ strains , respectively . Statistical significance was calculated by random sampling with replacement method using a bootstrapping algorithm in Python ( the asterisk * denotes p - value < 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) Quantification of transcription dynamics by the duration and frequency (time between initiation) of transcription. Bar graphs show the transcription parameters of SUT098 in candidate mutants compared to the wild type strain. The error bars represent SEM calculated by live-cell image analysis of 179, 135, 60 and 129 single-cell biological repeats for WT, hda1∆, hda3∆ and cac2∆ strains, respectively. Statistical significance was calculated by random sampling with replacement method using a bootstrapping algorithm in Python (the asterisk * denotes p-value <0.05).


( A ) Flow cytometry data analysis . Bars represent the mCherry / YFP fluorescence ratio of the respective yeast mutants normalized to the signal values of the H3 wild type FPR strain . The error bars show SEM calculated from biological triplicates with three technical repeats . Unpaired two - tailed student ' s t - test analysis indicates the statistical significance of the mutant compared to the respective wild type . The asterisks * , * * and * * * indicate p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow cytometry data analysis. Bars represent the mCherry/YFP fluorescence ratio of the respective yeast mutants normalized to the signal values of the H3 wild type FPR strain. The error bars show SEM calculated from biological triplicates with three technical repeats. Unpaired two-tailed student's t-test analysis indicates the statistical significance of the mutant compared to the respective wild type. The asterisks *, ** and *** indicate p-values <0.05, <0.01 and <0.001, respectively.


( b ) Cells stably expressing WT or D620N GFP - VPS35 or otherwise transiently transfected with a panel of GFP - VPS35 constructs were lysed and the respective GFP - tagged protein recovered by immunoprecipitation ( IP ) .

O O O O O O O O O O O O O O O O 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) Cells stably expressing WT or D620N GFP-VPS35 or otherwise transiently transfected with a panel of GFP-VPS35 constructs were lysed and the respective GFP-tagged protein recovered by immunoprecipitation (IP).


( c ) Untransfected HeLa cells or cells stably expressing VPS29 - GFP , GFP - VPS35 wild - type or GFP - VPS35 D620N were lysed and the lysates incubated with anti - VPS26 to IP the retromer CSC .

O O O O O O O O O O O O O O O O O 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) Untransfected HeLa cells or cells stably expressing VPS29-GFP, GFP-VPS35 wild-type or GFP-VPS35 D620N were lysed and the lysates incubated with anti-VPS26 to IP the retromer CSC.


( d ) Cells stably expressing the VPS29 - GFP V90D mutant that cannot assemble with VPS35 were transiently transfected with mCherry - tagged WT VPS35 , D620N and H675R constructs , along with an additional control of mCherry - FAM21 tail . Cells were lysed 48 h post transfection , and VPS29 - GFP V90D and associated proteins were recovered by anti - GFP native IP . Blots shown are representative of experiments replicated at least twice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells stably expressing the VPS29-GFP V90D mutant that cannot assemble with VPS35 were transiently transfected with mCherry-tagged WT VPS35, D620N and H675R constructs, along with an additional control of mCherry-FAM21 tail. Cells were lysed 48 h post transfection, and VPS29-GFP V90D and associated proteins were recovered by anti-GFP native IP. Blots shown are representative of experiments replicated at least twice.


a ) Cells expressing either WT GFP - VPS35 or the D620N mutant were treated with siRNA to abolish expression of endogenous VPS35 . Each dish of cells was lysed in either HEPES lysis buffer ( H ) or PBS lysis buffer ( P ) . Following centrifugation , equal portions of each lysate were combined to generate a mixed lysate ( H / P ) . Each lysate was incubated with anti - GFP to recover the respective GFP - tagged VPS35 protein .

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

a) Cells expressing either WT GFP-VPS35 or the D620N mutant were treated with siRNA to abolish expression of endogenous VPS35. Each dish of cells was lysed in either HEPES lysis buffer (H) or PBS lysis buffer (P). Following centrifugation, equal portions of each lysate were combined to generate a mixed lysate (H/P). Each lysate was incubated with anti-GFP to recover the respective GFP-tagged VPS35 protein.


( b ) Similar to ( a ) , but only cells expressing GFP - VPS35 wild type were analysed , either mock treated or treated with siRNA to silence FKBP15 expression .

O O O O O O O O O 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) Similar to (a), but only cells expressing GFP-VPS35 wild type were analysed, either mock treated or treated with siRNA to silence FKBP15 expression.


( c ) Cells stably expressing WT or D620N GFP - VPS35 were treated with indicated siRNAs , lysed and immunoprecipitated as 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

(c) Cells stably expressing WT or D620N GFP-VPS35 were treated with indicated siRNAs, lysed and immunoprecipitated as in (a).


( a , b ) Cells expressing either WT or D620N GFP - VPS35 were treated with siRNA to silence expression of endogenous VPS35 , fixed and labelled for GFP and FKBP15 ( a ) or FAM21 ( b ) . Cells were imaged by fluorescence microscopy . Scale bar , 20 μm .

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

(a,b) Cells expressing either WT or D620N GFP-VPS35 were treated with siRNA to silence expression of endogenous VPS35, fixed and labelled for GFP and FKBP15 (a) or FAM21 (b). Cells were imaged by fluorescence microscopy. Scale bar, 20 μm.


( c ) Cells stably expressing WT or D620N GFP - tagged VPS35 were treated with siRNA to abolish expression of endogenous VPS35 and subsequently fixed and labelled for GFP and either VPS26 , FKBP15 or FAM21 . The cells were imaged using Cellomics automated fluorescence microscopy . Approximately 250 cells per well in four wells were analysed for each cell line . The data for FKBP15 and FAM21 spot intensity were normalized to GFP - VPS35 and VPS26 signals . P0 . 0002 for both FKBP15 and FAM21 spot intensity in D620N compared with WT . Error bars indicate s . d .

(c) Cells stably expressing WT or D620N GFP-tagged VPS35 were treated with siRNA to abolish expression of endogenous VPS35 and subsequently fixed and labelled for GFP and either VPS26, FKBP15 or FAM21. The cells were imaged using Cellomics automated fluorescence microscopy. Approximately 250 cells per well in four wells were analysed for each cell line. The data for FKBP15 and FAM21 spot intensity were normalized to GFP-VPS35 and VPS26 signals. P0.0002 for both FKBP15 and FAM21 spot intensity in D620N compared with WT. Error bars indicate s.d.


( d ) Cells stably expressing either WT GFP - VPS35 or the D620N mutant were treated with siRNA to silence the expression of endogenous VPS35 . Cells were permeabilized by flash freezing followed by rapid thawing and centrifuged to separate supernatant ( S ) and membrane pellet ( P ) fractions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells stably expressing either WT GFP-VPS35 or the D620N mutant were treated with siRNA to silence the expression of endogenous VPS35. Cells were permeabilized by flash freezing followed by rapid thawing and centrifuged to separate supernatant (S) and membrane pellet (P) fractions.


( e ) The graph shows the percentage of membrane - associated FKBP15 and strumpellin and is the mean of three experiments . The error bars indicate s . e . m . A representative blot of three experiments is shown , indicating the efficacy of the knockdown of endogenous VPS35 and also further showing that membrane proteins such as the CIMPR are detected only in the pellet fraction .

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

(e) The graph shows the percentage of membrane-associated FKBP15 and strumpellin and is the mean of three experiments. The error bars indicate s.e.m. A representative blot of three experiments is shown, indicating the efficacy of the knockdown of endogenous VPS35 and also further showing that membrane proteins such as the CIMPR are detected only in the pellet fraction.


a ) HeLa cells stably expressing GFP - VPS35 WT and D620N were treated with bafilomycin A1 or DMSO vehicle control . Endogenous LC3 - II and tubulin levels were examined by western blot . A representative experiment of six experiments is shown . ( b ) Quantification of the representative experiment in triplicate shown in a , in which endogenous LC3 - II levels are normalized to tubulin and expressed as a ratio of levels in WT . * * * P = 7 . 86 × 10 − 6 ( DMSO ) and 3 . 81 × 10 − 5 ( Baf ) by 2 - tailed Student ' s t - test .

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

a) HeLa cells stably expressing GFP-VPS35 WT and D620N were treated with bafilomycin A1 or DMSO vehicle control. Endogenous LC3-II and tubulin levels were examined by western blot. A representative experiment of six experiments is shown. (b) Quantification of the representative experiment in triplicate shown in a, in which endogenous LC3-II levels are normalized to tubulin and expressed as a ratio of levels in WT. ***P=7.86 × 10−6 (DMSO) and 3.81 × 10−5 (Baf) by 2-tailed Student's t-test.


c ) HeLa cells stably expressing GFP - VPS35 WT and D620N were transfected with mRFP - LC3 . The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy . A representative experiment of three independent experiments is shown , with 344 ( WT ) and 401 ( D620N ) cells analysed . * * * P0 . 0001 by 2 - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 stably expressing GFP-VPS35 WT and D620N were transfected with mRFP-LC3. The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy. A representative experiment of three independent experiments is shown, with 344 (WT) and 401 (D620N) cells analysed. ***P0.0001 by 2-tailed Student's t-test.


( d ) GFP - VPS35 WT and D620N - expressing cells were transfected with HA - Q74 and immunostained for HA . The percentage of transfected cells with aggregates was counted by a blinded experimenter . The quantification shows the mean of three experiments in triplicate with minimum 200 cells per replicate . * * * P = 0 . 00086 by 1 - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP-VPS35 WT and D620N-expressing cells were transfected with HA-Q74 and immunostained for HA. The percentage of transfected cells with aggregates was counted by a blinded experimenter. The quantification shows the mean of three experiments in triplicate with minimum 200 cells per replicate. ***P=0.00086 by 1-tailed Student's t-test.


( e ) Confocal images representative of the experiment described in d . Scale bar , 20 μm .

(e) Confocal images representative of the experiment described in d. Scale bar, 20 μm.


( f ) Cells stably expressing WT and D620N GFP - VPS35 were transfected with GFP - α - synuclein A53T and GFP for 48 h and analyzed by western blotting .

(f) Cells stably expressing WT and D620N GFP-VPS35 were transfected with GFP-α-synuclein A53T and GFP for 48 h and analyzed by western blotting.


g ) Quantification of the representative experiment in triplicate in f , in which the level of α - synuclein was expressed as a ratio to GFP . A representative experiment of two independent experiments is shown . * * P = 0 . 0047 by 2 - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the representative experiment in triplicate in f, in which the level of α-synuclein was expressed as a ratio to GFP. A representative experiment of two independent experiments is shown. **P=0.0047 by 2-tailed Student's t-test.


( h ) VPS35 was knocked down with two individual siRNA nucleotides in HeLa cells , and cells were treated with bafilomycin A1 and lysed as in a . A representative experiment is shown in

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

(h) VPS35 was knocked down with two individual siRNA nucleotides in HeLa cells, and cells were treated with bafilomycin A1 and lysed as in a. A representative experiment is shown in triplicate


( i ) Quantification of three independent experiments in triplicate . * P = 0 . 026 ; other results non - significant by 2 - tailed Student ' s t - test .

O O O O O O O O O O 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) Quantification of three independent experiments in triplicate. *P=0.026; other results non-significant by 2-tailed Student's t-test.


( j ) Protein levels of CSC , including VPS35 , VPS26 and VPS29 ) , were assessed upon VPS35 knockdown , confirming previous results that knockdown of one component destabilizes the CSC . All error bars indicate s . e . m .

O O O O O O O O O O O O O O O O O O O O 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) Protein levels of CSC, including VPS35, VPS26 and VPS29), were assessed upon VPS35 knockdown, confirming previous results that knockdown of one component destabilizes the CSC. All error bars indicate s.e.m.


( a ) HeLa cells were transfected with pEGFP vector or GFP - FAM21 tail for 48 h and subsequently treated with bafilomycin A1 as in Fig . 4 . Endogenous LC3 - II and tubulin levels were assessed by western blot . ( b ) Quantification of the representative experiment in triplicate shown in a , of two independent experiments . * P = 0 . 02 ( DMSO ) and 0 . 08 ( Baf ) by 2 - tailed Student ' s t - test .

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

(a) HeLa cells were transfected with pEGFP vector or GFP-FAM21 tail for 48 h and subsequently treated with bafilomycin A1 as in Fig. 4. Endogenous LC3-II and tubulin levels were assessed by western blot. (b) Quantification of the representative experiment in triplicate shown in a, of two independent experiments. *P=0.02 (DMSO) and 0.08 (Baf) by 2-tailed Student's t-test.


( c ) HeLa cells stably expressing GFP - VPS35 WT and D620N were transfected with pEGFP vector or GFP - FAM21 , and subsequently treated with bafilomycin A1 , lysed and subjected to western blot as in a . ( d ) Quantification of the representative experiment in triplicate shown in c , of two independent experiments . * * P = 0 . 008 by 2 - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 stably expressing GFP-VPS35 WT and D620N were transfected with pEGFP vector or GFP-FAM21, and subsequently treated with bafilomycin A1, lysed and subjected to western blot as in a. (d) Quantification of the representative experiment in triplicate shown in c, of two independent experiments. **P=0.008 by 2-tailed Student's t-test.


( e ) Cells were depleted of WASH1 , treated with bafilomycin A1 and examined for endogenous LC3 - II , tubulin , WASH1 and GAPDH levels . A representative experiment of six independent experiments is shown . ( f ) Quantification of the representative experiment shown in e . * * P = 0 . 002 ( DMSO ) and P = 0 . 0028 ( Baf ) by 2 - tailed Student ' s t - test .

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

(e) Cells were depleted of WASH1, treated with bafilomycin A1 and examined for endogenous LC3-II, tubulin, WASH1 and GAPDH levels. A representative experiment of six independent experiments is shown. (f) Quantification of the representative experiment shown in e. **P=0.002 (DMSO) and P=0.0028 (Baf) by 2-tailed Student's t-test.


g ) HeLa cells depleted of WASH1 by siRNA were transfected with GFP - LC3 for 24 h . The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy . A representative experiment of two independent experiments is shown , with 757 ( control ) and 695 ( knockdown ) cells analysed . * * * P0 . 0001 by 2 - tailed Student ' s t - test .

g) HeLa cells depleted of WASH1 by siRNA were transfected with GFP-LC3 for 24 h. The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy. A representative experiment of two independent experiments is shown, with 757 (control) and 695 (knockdown) cells analysed. ***P0.0001 by 2-tailed Student's t-test.


( h ) HeLa cells depleted of FKBP15 were transfected with GFP - LC3 and vesicles were quantified as in g . A representative experiment of two independent experiments in triplicate is shown , with 579 ( control ) and 815 ( knockdown ) cells analysed in total .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells depleted of FKBP15 were transfected with GFP-LC3 and vesicles were quantified as in g. A representative experiment of two independent experiments in triplicate is shown, with 579 (control) and 815 (knockdown) cells analysed in total.


( i ) HeLa cells depleted of FKBP15 were subsequently transfected with a GFP - Q74 construct for 24 h and fixed in PFA . The percentage of transfected cells with aggregates was counted by a blinded experimenter . The quantification shows the mean of two experiments , each in triplicate with min 220 cells counted per

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells depleted of FKBP15 were subsequently transfected with a GFP-Q74 construct for 24 h and fixed in PFA. The percentage of transfected cells with aggregates was counted by a blinded experimenter. The quantification shows the mean of two experiments, each in triplicate with min 220 cells counted per replicate


( j ) FKBP15 was depleted in HeLa cells as in e . A representative blot from three independent experiments in triplicate is shown . Error bars indicate s . e . m .

(j) FKBP15 was depleted in HeLa cells as in e. A representative blot from three independent experiments in triplicate is shown. Error bars indicate s.e.m.


( a ) HeLa cells were immunostained for endogenous ATG9A and VPS35 and subjected to confocal microscopy . Magnified areas are shown on the right of the pictures . ( b ) HeLa cells were transfected with ATG9A - GFP for 24 h , and subsequently fixed , immunostained for endogenous WASH1 and subjected to confocal microscopy . ( c ) HeLa cells were transfected with ATG9A - GFP as in b , but immunostained instead for endogenous FAM21 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were immunostained for endogenous ATG9A and VPS35 and subjected to confocal microscopy. Magnified areas are shown on the right of the pictures. (b) HeLa cells were transfected with ATG9A-GFP for 24 h, and subsequently fixed, immunostained for endogenous WASH1 and subjected to confocal microscopy. (c) HeLa cells were transfected with ATG9A-GFP as in b, but immunostained instead for endogenous FAM21.


( d ) HeLa cells stably expressing GFP - VPS35 WT and D620N were depleted of endogenous VPS35 using 40 nM of siRNA , and subsequently immunostained for TGN46 and endogenous ATG9A and subjected to confocal microscopy . ( e ) Colocalization between TGN and ATG9A is expressed in terms of the Pearson ' s Coefficient . n = 25 cells ( WT ) and 33 cells ( D620N ) . Error bars represent s . e . m . and * * P = 0 . 01 by 2 - tailed Student ' s t - test . Scale bars in ( a - d ) , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 stably expressing GFP-VPS35 WT and D620N were depleted of endogenous VPS35 using 40 nM of siRNA, and subsequently immunostained for TGN46 and endogenous ATG9A and subjected to confocal microscopy. (e) Colocalization between TGN and ATG9A is expressed in terms of the Pearson's Coefficient. n=25 cells (WT) and 33 cells (D620N). Error bars represent s.e.m. and **P=0.01 by 2-tailed Student's t-test. Scale bars in (a-d), 10 μm.


( a ) HeLa cells depleted of WASH1 with two successive siRNA treatments were either starved in Hank ' s balanced salt solution for 1 h or kept in full medium . Following fixation , cells were immunostained for TGN46 , ATG9A and VPS35 and subjected to confocal microscopy . Representative pictures with magnified areas are shown . Scale bar , 10 μm . ( b ) Colocalization between TGN and ATG9A is expressed in terms of the Pearson ' s coefficient . Number of cells analysed : 38 ( control basal ) , 46 ( knockdown basal ) , 36 ( control starved ) , 53 ( knockdown starved ) . Error bars represent s . e . m . and * * * P = 0 . 0005 ( basal ) and P = 0 . 0004 ( starved ) by 2 - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells depleted of WASH1 with two successive siRNA treatments were either starved in Hank's balanced salt solution for 1 h or kept in full medium. Following fixation, cells were immunostained for TGN46, ATG9A and VPS35 and subjected to confocal microscopy. Representative pictures with magnified areas are shown. Scale bar, 10 μm. (b) Colocalization between TGN and ATG9A is expressed in terms of the Pearson's coefficient. Number of cells analysed: 38 (control basal), 46 (knockdown basal), 36 (control starved), 53 (knockdown starved). Error bars represent s.e.m. and ***P=0.0005 (basal) and P=0.0004 (starved) by 2-tailed Student's t-test.


( a ) HeLa cells stably expressing GFP - VPS35 WT and D620N were transfected with mRFP - LC3 for 24 h , immunostained for endogenous ATG9A , and imaged by confocal microscopy .

O O O O O O O O O O O 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) HeLa cells stably expressing GFP-VPS35 WT and D620N were transfected with mRFP-LC3 for 24 h, immunostained for endogenous ATG9A, and imaged by confocal microscopy.


( b ) Colocalization is expressed in terms of Mander ' s coefficient M1 to indicate the proportion of LC3 intensities that also contain ATG9A intensities . A representative experiment of three independent experiments is shown , in which at least 33 cells were analysed per condition . Error bars indicate s . e . m . , and * * * P0 . 001 by 2 - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colocalization is expressed in terms of Mander's coefficient M1 to indicate the proportion of LC3 intensities that also contain ATG9A intensities. A representative experiment of three independent experiments is shown, in which at least 33 cells were analysed per condition. Error bars indicate s.e.m., and ***P0.001 by 2-tailed Student's t-test.


( c ) HeLa cells depleted of WASH1 were transfected with GFP - LC3 for 24 h , immunostained for endogenous ATG9A , and imaged by confocal microscopy .

O O O O O O 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 depleted of WASH1 were transfected with GFP-LC3 for 24 h, immunostained for endogenous ATG9A, and imaged by confocal microscopy.


( d ) Colocalization expressed as M1 , as in b . A representative experiment of two independent experiments is shown , in which at least 24 cells were analysed per condition . Error bars indicate s . e . m . , and * * * P0 . 001 by 2 - tailed Student ' s t - test . Scale bars in ( a , c ) , 10 μm . In b , d , colocalization was measured for the entire area of the cell slice in each image . In order to ensure that results were not confounded by diffuse nuclear staining often observed in the LC3 channel , the analyses were repeated with the nuclei excluded , and all of the results remained statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colocalization expressed as M1, as in b. A representative experiment of two independent experiments is shown, in which at least 24 cells were analysed per condition. Error bars indicate s.e.m., and ***P0.001 by 2-tailed Student's t-test. Scale bars in (a,c), 10 μm. In b,d, colocalization was measured for the entire area of the cell slice in each image. In order to ensure that results were not confounded by diffuse nuclear staining often observed in the LC3 channel, the analyses were repeated with the nuclei excluded, and all of the results remained statistically significant.


( a ) SH - SY5Y cells were depleted of WASH1 , treated with bafilomycin A1 and examined for LC3 - II , tubulin , WASH1 and GAPDH levels . Blots shown are representative of two independent experiments in triplicate . ( b ) Quantification of the representative experiment in triplicate shown in ( a ) , of two independent experiments . Error bars indicate s . e . m . * * P = 0 . 0058 by 1 - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SH-SY5Y cells were depleted of WASH1, treated with bafilomycin A1 and examined for LC3-II, tubulin, WASH1 and GAPDH levels. Blots shown are representative of two independent experiments in triplicate. (b) Quantification of the representative experiment in triplicate shown in (a), of two independent experiments. Error bars indicate s.e.m. **P=0.0058 by 1-tailed Student's t-test.


( c ) WASH1 - depleted SH - SY5Y cells were trypsinized , stained with propidium iodide and analysed by flow cytometry . Living cells , not stained with propidium iodide , are shown as a percentage of total cells . The graph depicts a representative experiment in triplicate out of two independent experiments , in which at least 10 , 000 cells were analysed in each replicate . Error bars indicate s . e . m . * P = 0 . 045 by 1 - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WASH1-depleted SH-SY5Y cells were trypsinized, stained with propidium iodide and analysed by flow cytometry. Living cells, not stained with propidium iodide, are shown as a percentage of total cells. The graph depicts a representative experiment in triplicate out of two independent experiments, in which at least 10,000 cells were analysed in each replicate. Error bars indicate s.e.m. *P=0.045 by 1-tailed Student's t-test.


( B ) Bar graph representing the normalized average value of the neuronal ATP / ATP + ADP ratio on the basal condition , on glutamate stimulation for 5 minutes , on glutamate stimulation with anisomycin ( 25μM ) and on glutamate stimulation with cycloheximide ( 350μM ) . Data presented as mean ± SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n = 5 independent platings . One - way ANOVA followed by Bonferroni ' s multiple comparison test .

O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 O O O O O 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) Bar graph representing the normalized average value of the neuronal ATP/ATP+ADP ratio on the basal condition, on glutamate stimulation for 5 minutes, on glutamate stimulation with anisomycin (25μM) and on glutamate stimulation with cycloheximide (350μM). Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. **p<0.01, ***p<0.001, ****p<0.0001, n=5 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.


( C ) Bar graph representing the normalized average value of neuronal ATP / ATP + ADP ratio on the basal condition , on glutamate stimulation for 5 minutes , on glutamate stimulation with MPEP , on glutamate stimulation with CNQX + D - AP5 and on glutamate stimulation with CNQX + D - AP5 + anisomycin . Data presented as mean ± SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . . * p < 0 . 05 , * * * p < 0 . 001 , n = 4 - 5 independent platings . One - way ANOVA followed by Bonferroni ' s multiple comparison test .

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

(C) Bar graph representing the normalized average value of neuronal ATP/ATP+ADP ratio on the basal condition, on glutamate stimulation for 5 minutes, on glutamate stimulation with MPEP, on glutamate stimulation with CNQX + D-AP5 and on glutamate stimulation with CNQX + D-AP5 + anisomycin. Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment.. *p<0.05, ***p<0.001, n=4-5 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.


( D ) Representative current - clamp and voltage - clamp traces showing a single neuron firing spontaneous bursts of action potentials . The traces on the right shows a single representative burst at a higher temporal resolution .

O O O O O O O O O O O O O O O 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) Representative current-clamp and voltage-clamp traces showing a single neuron firing spontaneous bursts of action potentials. The traces on the right shows a single representative burst at a higher temporal resolution.


( E ) Bar graph representing the burst frequency and burst duration both in the voltage and current - clamp mode . n = 16 neurons from ≥ 3 independent platings . Data presented as mean ± SEM with scattered data points .

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

(E) Bar graph representing the burst frequency and burst duration both in the voltage and current-clamp mode. n=16 neurons from ≥3 independent platings. Data presented as mean ± SEM with scattered data points.


( F ) Representative voltage - clamp traces of miniature EPSCs recorded ( Vhold = - 70mV ) from a neuron in presence of TTX .

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

(F) Representative voltage-clamp traces of miniature EPSCs recorded (Vhold= -70mV) from a neuron in presence of TTX.


( G ) Bar graph representing the baseline mEPSC frequency and amplitude in the current - clamp mode . N = 11 neurons from ≥ 3 independent platings . Data presented as mean ± SEM with scattered data points .

O O O O O O O O O O O O O O O O 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) Bar graph representing the baseline mEPSC frequency and amplitude in the current-clamp mode. N=11 neurons from ≥3 independent platings. Data presented as mean ± SEM with scattered data points.