figf1 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(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.(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.


figf2 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(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.(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.(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.


figf3 ( 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 . ( 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 B-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(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.(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.


figf4 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(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.(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.


figf5 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(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.(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.


Figure 1 ( 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 .

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Figure 1(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.


Figure 2 ( 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 .

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Figure 2(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.


Figure 3 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 3. (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)


Figure 4 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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).


Figure 5 ( 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 .

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Figure 5(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.


f1 ( 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 . ( 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 .

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f1(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.(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.


f2a ) 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 . ( c ) Cells stably expressing WT or D620N GFP - VPS35 were treated with indicated siRNAs , lysed and immunoprecipitated as in ( a ) .

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f2a) 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.(c) Cells stably expressing WT or D620N GFP-VPS35 were treated with indicated siRNAs, lysed and immunoprecipitated as in (a).


f3 ( 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 . ( 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 .

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f3(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.(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.


f4a ) 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 . ( 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 . ( 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 . ( 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 . ( 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 .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f4a) 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.(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.(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.(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.(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.


f5 ( 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 . ( 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 . ( 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f5(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.(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.(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 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.


f6 ( 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 .

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f6(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.


f7 ( 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 .

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f7(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.


f8 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f8(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.(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.


f9 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f9(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.


Figure 1 ( 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 . ( 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 . ( 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 . ( I ) Immunoblots depicting levels of PSD 95 , GFAP , Synapsin 1 and tubulin in the whole cortical lysate and the synaptoneurosome preparations . Quantification representing the average fold enrichments of PSD 95 , Synapsin 1 and GFAP in the synaptoneurosome preparations compared to the whole cortical lysate . Data presented as mean ± SEM with scattered data points . Data points for all the proteins were normalized to their respective whole lysate values . * * p < 0 . 05 , * * p < 0 . 01 , n ≥ 7 animals for each group . One - sample t - test . ( J ) Bar graph representing the normalized average value of synaptic ATP level on the basal condition , on DHPG treatment for 5 minutes , on DHPG treatment with MPEP , on DHPG treatment with anisomycin and on DHPG treatment 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 . 05 , n = 8 animals . ( K ) Bar graph representing the normalized average value of the synaptic ATP level on the basal condition , on NMDA treatment for 5 minutes and on NMDA treatment with D - AP5 . Data presented as mean + / - SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . n = 8 independent platings .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.(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.(I) Immunoblots depicting levels of PSD 95, GFAP, Synapsin 1 and tubulin in the whole cortical lysate and the synaptoneurosome preparations. Quantification representing the average fold enrichments of PSD 95, Synapsin 1 and GFAP in the synaptoneurosome preparations compared to the whole cortical lysate. Data presented as mean ± SEM with scattered data points. Data points for all the proteins were normalized to their respective whole lysate values. **p<0.05, **p<0.01, n ≥ 7 animals for each group. One-sample t-test.(J) Bar graph representing the normalized average value of synaptic ATP level on the basal condition, on DHPG treatment for 5 minutes, on DHPG treatment with MPEP, on DHPG treatment with anisomycin and on DHPG treatment 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<0.05, n=8 animals.(K) Bar graph representing the normalized average value of the synaptic ATP level on the basal condition, on NMDA treatment for 5 minutes and on NMDA treatment with D-AP5. Data presented as mean +/- SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. n=8 independent platings.


Figure 2 ( B ) Representative neurons showing changes in the PercevalHR fluorescence ratio ( ~ ATP / ADP ratio ) in somatic compartments on the bath application of DHPG and NMDA . Scale bar 10μm . ( C ) Representative neurons showing changes in the PercevalHR fluorescence ratio ( ~ ATP / ADP ratio ) in dendritic compartments on the bath application of DHPG and NMDA . Scale bar 10μm . ( D ) Average traces depicting the time course of the normalized somatic ATP / ADP ratio on the basal conditions , on DHPG treatment , and on NMDA treatment . The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n = 5 - 6 cells from ≥ 3 independent platings . ( E ) Average traces depicting the time course of normalized dendritic ATP / ADP ratio on the basal condition , on the basal condition in the presence of anisomycin , on DHPG treatment , on NMDA treatment , on DHPG treatment in the presence of anisomycin and on NMDA treatment in the presence of anisomycin . The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n = 6 - 9 cells from ≥ 3 independent platings . ( G ) Line graph showing the average normalized synaptic ATP level at various time points after DHPG treatment until 5 minutes of recovery in the presence or absence of anisomycin . Data presented as mean + / - SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 for comparison with 0 minute . # # p < 0 . 01 and # # # # p < 0 . 0001 for comparison between DHPG + anisomycin . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n ≥ 5 animals per group . ( H ) Line graph showing the average normalized synaptic ATP level at various time points after NMDA treatment until 5 minutes after recovery in the presence or absence of anisomycin . Data presented as mean + / - SEM , * * p < 0 . 01 for comparison with 0 minutes . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n ≥ 4 animals per group .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Representative neurons showing changes in the PercevalHR fluorescence ratio (~ATP/ADP ratio) in somatic compartments on the bath application of DHPG and NMDA. Scale bar 10μm. (C) Representative neurons showing changes in the PercevalHR fluorescence ratio (~ATP/ADP ratio) in dendritic compartments on the bath application of DHPG and NMDA. Scale bar 10μm.(D) Average traces depicting the time course of the normalized somatic ATP/ADP ratio on the basal conditions, on DHPG treatment, and on NMDA treatment. The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n= 5-6 cells from ≥3 independent platings.(E) Average traces depicting the time course of normalized dendritic ATP/ADP ratio on the basal condition, on the basal condition in the presence of anisomycin, on DHPG treatment, on NMDA treatment, on DHPG treatment in the presence of anisomycin and on NMDA treatment in the presence of anisomycin. The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n= 6-9 cells from ≥3 independent platings.(G) Line graph showing the average normalized synaptic ATP level at various time points after DHPG treatment until 5 minutes of recovery in the presence or absence of anisomycin. Data presented as mean +/- SEM, *p<0.05, **p<0.01, ****p<0.0001 for comparison with 0 minute. ##p<0.01 and ####p<0.0001 for comparison between DHPG + anisomycin. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n≥ 5 animals per group.(H) Line graph showing the average normalized synaptic ATP level at various time points after NMDA treatment until 5 minutes after recovery in the presence or absence of anisomycin. Data presented as mean +/- SEM, **p<0.01 for comparison with 0 minutes. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n≥ 4 animals per group.


Figure 3 ( B ) Representative images showing newly synthesized proteins on the basal condition visualized through FUNCAT metabolic labeling ( pseudo - colored ) in cortical neurons . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm . ( C ) Representative images showing newly synthesized proteins visualized through FUNCAT metabolic labeling ( pseudo - colored ) in cortical neurons at various time points following DHPG and NMDA treatment . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm . ( D ) Line graph showing the change in the average normalized FUNCAT intensity representing the quantity of newly synthesized proteins at various time points following DHPG and NMDA treatments . Data presented as mean + / - SEM . Data points in all groups were normalized to the average of the basal group . * * * p < 0 . 001 n = 21 - 54 neurons per group from 3 independent platings . ( E ) Representative images showing phospho - eEF2 immunolabeling ( Pseudo - colored ) in low - density cortical neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm . ( F ) Box plot showing phospho - eEF2 normalized intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , n = 31 - 49 cells per group from 3 independent platings . ( G ) Representative immunoblots describing changes in the phospho - eEF2 and total - eEF2 levels at various time points after DHPG and NMDA treatment in cortical synaptoneurosomes . Note in each case phospho and total eEF2 levels were normalized individually to their respective tubulin levels for calculating the phospho / total undefined ratio . ( H ) Line graph showing the average value of the synaptic phospho / total ratio of eEF2 at various time points after DHPG and NMDA treatment until 5 minutes of recovery . Data presented as mean + / - SEM , * * p < 0 . 01 . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * * * p < 0 . 001 , * * * * p < 0 . 0001 , n ≥ 5 animals per group .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Representative images showing newly synthesized proteins on the basal condition visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.(C) Representative images showing newly synthesized proteins visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons at various time points following DHPG and NMDA treatment. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.(D) Line graph showing the change in the average normalized FUNCAT intensity representing the quantity of newly synthesized proteins at various time points following DHPG and NMDA treatments. Data presented as mean +/- SEM. Data points in all groups were normalized to the average of the basal group. ***p<0.001 n= 21-54 neurons per group from 3 independent platings.(E) Representative images showing phospho-eEF2 immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.(F) Box plot showing phospho-eEF2 normalized intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=31-49 cells per group from 3 independent platings.(G) Representative immunoblots describing changes in the phospho-eEF2 and total-eEF2 levels at various time points after DHPG and NMDA treatment in cortical synaptoneurosomes. Note in each case phospho and total eEF2 levels were normalized individually to their respective tubulin levels for calculating the phospho/total undefined ratio.(H) Line graph showing the average value of the synaptic phospho/total ratio of eEF2 at various time points after DHPG and NMDA treatment until 5 minutes of recovery. Data presented as mean +/- SEM, **p<0.01. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. ***p<0.001, ****p<0.0001, n≥ 5 animals per group.


Figure 4 ( A ) Representative images showing phospho - AMPK immunolabeling ( Pseudo - colored ) in low - density cortical neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm . ( B ) Box plot showing the normalized phospho - AMPK intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , n = 30 - 46 cells per group from 3 independent platings . ( C ) Representative immunoblots describing changes in the phospho - AMPK and total - AMPK levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin . Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho / total ratio of AMPK . ( D ) Line graph showing the normalized average value of the synaptic phospho / total ratio of AMPK at various time points after DHPG treatment in the presence or absence of anisomycin . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 5 animals per group . ( E ) Representative immunoblots depicting the changes in the α - SNAP protein levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin . ( F ) Line graph showing the normalized average value of the synaptic α - SNAP protein levels at various time points after DHPG treatment in the presence or absence of anisomycin . Data presented as mean + / - SEM . * p < 0 . 05 . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * * p < 0 . 01 , n ≥ 4 animals per group . ( G ) Representative images showing the α - SNAP ( cyan ) and AMPK ( magenta ) immunolabeling in low - density cortical neurons . Merge shows the colocalization of the two channels both on the basal condition and on DHPG treatment for 5 minutes . Scale bar 10μm . Zoomed in representative images of dendrites showing the merge of both channels on the basal and DHPG treated conditions . Scale bar 5μm . ( H ) Box plot depicting the quantification of co - localization through Pearson ' s correlation coefficient between α - SNAP and AMPK in cortical neurons on the basal condition and on DHPG treatment for 5 minutes . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 01 , n ≥ 35 cells per group from 4 independent platings . Unpaired - sample t - test . ( I ) Representative images showing the phospho - AMPK immunolabeling ( Pseudo - colored ) in low - density cortical neurons on the basal condition and on DHPG treatment for 5 minutes in the presence of scrambled siRNA and in the presence of α - SNAP siRNA . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm . ( J ) Box plot showing the normalized phospho - AMPK intensity distribution across multiple neurons on the basal condition in the presence of scrambled siRNA , on DHPG treatment for 5 minutes in the presence of scrambled siRNA , on the basal condition in the presence of α - SNAP siRNA , on DHPG treatment for 5 minutes in the presence of α - SNAP siRNA . Data points in all groups were normalized to the average of the scrambled siRNA basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , n = 29 - 35 cells per group from 3 independent platings .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Representative images showing phospho-AMPK immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.(B) Box plot showing the normalized phospho-AMPK intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=30-46 cells per group from 3 independent platings.(C) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total ratio of AMPK.(D) Line graph showing the normalized average value of the synaptic phospho/total ratio of AMPK at various time points after DHPG treatment in the presence or absence of anisomycin. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. *p<0.05, **p<0.01, ****p<0.0001, n= 5 animals per group.(E) Representative immunoblots depicting the changes in the α-SNAP protein levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin.(F) Line graph showing the normalized average value of the synaptic α-SNAP protein levels at various time points after DHPG treatment in the presence or absence of anisomycin. Data presented as mean +/- SEM. *p<0.05. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. **p<0.01, n≥ 4 animals per group.(G) Representative images showing the α-SNAP (cyan) and AMPK (magenta) immunolabeling in low-density cortical neurons. Merge shows the colocalization of the two channels both on the basal condition and on DHPG treatment for 5 minutes. Scale bar 10μm. Zoomed in representative images of dendrites showing the merge of both channels on the basal and DHPG treated conditions. Scale bar 5μm.(H) Box plot depicting the quantification of co-localization through Pearson's correlation coefficient between α-SNAP and AMPK in cortical neurons on the basal condition and on DHPG treatment for 5 minutes. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.01, n≥35 cells per group from 4 independent platings. Unpaired-sample t-test.(I) Representative images showing the phospho-AMPK immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition and on DHPG treatment for 5 minutes in the presence of scrambled siRNA and in the presence of α-SNAP siRNA. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.(J) Box plot showing the normalized phospho-AMPK intensity distribution across multiple neurons on the basal condition in the presence of scrambled siRNA, on DHPG treatment for 5 minutes in the presence of scrambled siRNA, on the basal condition in the presence of α-SNAP siRNA, on DHPG treatment for 5 minutes in the presence of α-SNAP siRNA. Data points in all groups were normalized to the average of the scrambled siRNA basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, n=29-35 cells per group from 3 independent platings.


Figure 5 ( A ) Representative immunoblots describing changes in the phospho - AMPK and total - AMPK levels at various time points after NMDA treatment in cortical synaptoneurosomes in the presence or absence of extracellular Ca2 + . Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho / total AMPK ratio . ( B ) Line graph showing the normalized average value for the synaptic phospho / total ratio of AMPK at various time points after NMDA treatment until 5 minutes of recovery in the presence or absence of extracellular Ca2 + . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 6 animals per group . ( C ) Representative images depicting intracellular Ca2 + levels through Fluo8 fluorescence ( Pseudo - colored ) of a cortical neuron plated at low - density before stimulation , 15 sec ( immediate ) after NMDA treatment , 300 sec ( delayed ) after NMDA treatment and after ionomycin treatment in the presence of 10mM Ca2 + . Scale bar 20μm . ( D ) Representative time trace showing the normalized change in Fluo8 fluorescence on NMDA treatment in the presence or absence of extracellular Ca2 + and D - AP5 . Values in all other groups were normalized to the unsimulated condition for the corresponding experiment and all the data points are represented as a fraction of the initial time point . Ionomycin treatment in the presence of 10mM Ca2 + was used to calculate fluorescence maximum for a particular cell . ( E ) Box plot showing the distribution of ΔF / F0 across multiple neurons on the basal condition , on NMDA treatment in the presence Ca2 + , on NMDA treatment in the absence of Ca2 + , on NMDA treatment with D - AP5 and on NMDA treatment along with MK - 801 . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * * * * p < 0 . 0001 , n ≥ 30 neurons per group from 3 independent platings .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after NMDA treatment in cortical synaptoneurosomes in the presence or absence of extracellular Ca2+. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total AMPK ratio.(B) Line graph showing the normalized average value for the synaptic phospho/total ratio of AMPK at various time points after NMDA treatment until 5 minutes of recovery in the presence or absence of extracellular Ca2+. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. **p<0.01, ****p<0.0001, n= 6 animals per group.(C) Representative images depicting intracellular Ca2+ levels through Fluo8 fluorescence (Pseudo-colored) of a cortical neuron plated at low-density before stimulation, 15 sec (immediate) after NMDA treatment, 300 sec (delayed) after NMDA treatment and after ionomycin treatment in the presence of 10mM Ca2+. Scale bar 20μm.(D) Representative time trace showing the normalized change in Fluo8 fluorescence on NMDA treatment in the presence or absence of extracellular Ca2+ and D-AP5. Values in all other groups were normalized to the unsimulated condition for the corresponding experiment and all the data points are represented as a fraction of the initial time point. Ionomycin treatment in the presence of 10mM Ca2+ was used to calculate fluorescence maximum for a particular cell.(E) Box plot showing the distribution of ΔF/F0 across multiple neurons on the basal condition, on NMDA treatment in the presence Ca2+, on NMDA treatment in the absence of Ca2+, on NMDA treatment with D-AP5 and on NMDA treatment along with MK-801. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. ****p<0.0001, n≥30 neurons per group from 3 independent platings.