( C ) HCT116 XIAP WT XIAP KO were treated with 1 μM API ‐ 2 for 6 h . lysates were analysed by western blotting . data are representative of three biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S6C .

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(C) HCT116 XIAP WT and XIAP KO cells were treated with 1 μM API‐2 for 6 h. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6C.


( D ) HCT116 were treated with or without EBSS for 4 h . lysates were subjected to immunoprecipitation with anti ‐ XIAP antibody followed by western blot . data are representative of two biological replicates .

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(D) HCT116 cells were treated with or without EBSS for 4 h. Cell lysates were subjected to immunoprecipitation with anti‐XIAP antibody followed by western blot analysis. The data are representative of two biological replicates.


( E ) Lysates from HCT116 were immunoprecipitated with anti ‐ Mdm2 antibody . Immunoprecipitates lysates were analysed by western blotting with anti ‐ p ‐ XIAP antibody . data are representative of two biological replicates .

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(E) Lysates from HCT116 cells were immunoprecipitated with anti‐Mdm2 antibody. Immunoprecipitates and cell lysates were analysed by western blotting with anti‐p‐XIAP antibody. The data are representative of two biological replicates.


( F ) HCT116 XIAP KO were transfected with Flag ‐ XIAP , Flag ‐ XIAP S87A , Flag ‐ XIAP S87D or control vector . Twenty ‐ four hours after transfection , were harvested . LC3 conversion was evaluated by western blot . data are representative of three biological replicates . of LCII / LC3I to actin is presented in Supplementary Figure S6D .

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

(F) HCT116 XIAP KO cells were transfected with Flag‐XIAP, Flag‐XIAP S87A, Flag‐XIAP S87D or control vector. Twenty‐four hours after transfection, cells were harvested. LC3 conversion was evaluated by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6D.


( G ) p53 − / − Mdm2 − / − MEF were transfected with HA ‐ Mdm2 plus Flag ‐ XIAP , Flag ‐ XIAP S87A or Flag ‐ XIAP S87D . Twenty ‐ four hours later , were treated with 20 μM MG - 132 for another 4h . lysates were subjected to immunoprecipitation with anti ‐ HA antibody . Immunoprecipitates were analysed by western blotting . data are representative of two biological replicates .

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(G) p53−/−Mdm2−/− MEF cells were transfected with HA‐Mdm2 plus Flag‐XIAP, Flag‐XIAP S87A or Flag‐XIAP S87D. Twenty‐four hours later, cells were treated with 20 μM MG-132 for another 4h. Cell lysates were subjected to immunoprecipitation with anti‐HA antibody. Immunoprecipitates were analysed by western blotting. The data are representative of two biological replicates.


( A - C ) 1 × 106HCT116XIAP WT 1 × 106XIAP KO ( group 1 ) , 1 × 106HCT116XIAP KO ( KO ‐ Ctrl ) 1 × 106HCT116XIAP KO stably expressing wild ‐ type XIAP ( KO ‐ XIAP ) ( group 2 ) , 1 × 106HCT116XIAP KO ( KO ‐ Ctrl ) 1 × 106HCT116XIAP KO stably expressing XIAP S87A mutant ( KO ‐ XIAP SA ) ( group 3 ) , or 1 × 106HCT116XIAP KO ( KO ‐ Ctrl ) 1 × 106HCT116XIAP KO stably expressing XIAP S87D mutant ( KO ‐ XIAP SD ) ( group 4 ) were individually injected to left flank ( Left ) right flank ( Right ) of nude mice as indicated . Five after injection , mice were sacrificed photographed ( A ) . volumes of flank tumours excised from six mice in each group were compared ( n = 6 ) ( B ) . Tumour weights were represented as means ± s . d . from six mice in each group ( C ) .

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(A-C) 1 × 106HCT116XIAP WT and 1 × 106XIAP KO cells (group 1), 1 × 106HCT116XIAP KO cells (KO‐Ctrl) and 1 × 106HCT116XIAP KO cells stably expressing wild‐type XIAP (KO‐XIAP) (group 2), 1 × 106HCT116XIAP KO cells (KO‐Ctrl) and 1 × 106HCT116XIAP KO cells stably expressing XIAP S87A mutant (KO‐XIAP SA) (group 3), or 1 × 106HCT116XIAP KO cells (KO‐Ctrl) and 1 × 106HCT116XIAP KO cells stably expressing XIAP S87D mutant (KO‐XIAP SD) (group 4) were individually injected to the left flank (Left) and right flank (Right) of nude mice as indicated. Five weeks after injection, the mice were sacrificed and photographed (A). The volumes of all flank tumours excised from six mice in each group were compared (n=6) (B). Tumour weights were represented as means±s.d. from six mice in each group (C).


( D - F ) 1 × 106HCT116XIAP KO or 1 × 106HCT116XIAP KO stably expressing XIAP D148A / 310A were injected to right flank of nude mice as indicated ( n = 3 ) . Five after injection , mice were sacrificed photographed ( D ) . volumes of excised tumours were shown ( E ) . Tumour weights were represented as means ± s . d . from three mice in each group ( F ) .

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(D-F) 1 × 106HCT116XIAP KO cells or 1 × 106HCT116XIAP KO cells stably expressing XIAP D148A/310A were injected to the right flank of nude mice as indicated (n=3). Five weeks after injection, the mice were sacrificed and photographed (D). The volumes of excised tumours were shown (E). Tumour weights were represented as means±s.d. from three mice in each group (F).


( G , H ) human tumour tissues ( T ) from Oesophagus ( G ) Colon ( H ) their adjacent normal tissues ( N ) were homogenized for extraction . extracts were analysed by western blotting with indicated antibodies . data are representative of three biological replicates . blots in ( G , H ) were qualified of LC3II / LC3I to actin was then calculated shown in Supplementary Figure 9A B .

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(G, H) The human tumour tissues (T) from Oesophagus (G) and Colon (H) and their adjacent normal tissues (N) were homogenized for protein extraction. Protein extracts were analysed by western blotting with the indicated antibodies. The data are representative of three biological replicates. The blots in (G, H) were qualified and the ratio of LC3II/LC3I to actin was then calculated and shown in Supplementary Figure 9A and B.


A . Confocal microscopic image showing HeLa immunostained for Nup358 ( green , upper panel ) or Nup214 ( green , lower panel ) RanGAP1 ( red ) using specific antibodies . was stained with Hoechst 33342 ( blue ) . Scale bars , 10 μm .

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A. Confocal microscopic image showing HeLa cells immunostained for Nup358 (green, upper panel) or Nup214 (green, lower panel) and RanGAP1 (red) using specific antibodies. DNA was stained with Hoechst 33342 (blue). Scale bars, 10 μm.


B . COS - 7 transfected with GFP - Nup358 one of was subjected for live imaging using confocal microscopy frames at indicated time points have been provided . Arrows denoted by different colors indicate AL structures . Note that one of AL structures ( marked by yellow arrow ) originated from NE ( dotted ) fused with another AL structure ( marked by red arrow ) , which later fused with a different AL structure ( marked by blue arrow ) . N , nucleus ; C , cytoplasm . Scale bar , 5 μm .

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B. COS-7 cells transfected with GFP-Nup358 and one of the cells was subjected for live imaging using confocal microscopy and the frames at indicated time points have been provided. Arrows denoted by different colors indicate individual AL structures. Note that one of the AL structures (marked by yellow arrow) originated from the NE (dotted line) and fused with another AL structure (marked by red arrow), which later fused with a different AL structure (marked by blue arrow). N, nucleus; C, cytoplasm. Scale bar, 5 μm.


C . Maximum projection confocal image of a sodium arsenite treated HeLa immunostained for endogenous Nup358 ( green ) , P bodies ( red , Dcp1a as a marker ) SGs ( blue , eIF3η as a marker ) using specific antibodies . Scale bar , 10 μm . histograms represent profile along dotted arrows . Adjacent graph represents quantitative data showing of P bodies ( top ) or SGs ( bottom ) associated with envelope ( NE ) , Nup358 positive AL , with each other or unassociated with any of other mentioned structures ( others ) . Data are presented as mean SD ( n = 3 ) .

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C. Maximum intensity projection confocal image of a sodium arsenite treated HeLa cell immunostained for endogenous Nup358 (green), P bodies (red, Dcp1a as a marker) and SGs (blue, eIF3η as a marker) using specific antibodies. Scale bar, 10 μm. The histograms represent fluorescence intensity profile along the dotted arrows. Adjacent graph represents quantitative data showing percentage of P bodies (top) or SGs (bottom) associated with nuclear envelope (NE), Nup358 positive AL, with each other or unassociated with any of the other mentioned structures (others). Data are presented as mean SD (n = 3).


D . HeLa were transfected with Nup358 siRNA ( siNup358 ) or Nup214 siRNA ( siNup214 ) . Western blot ( WB ) indicates extent of Nup358 depletion level of Dcp1a .

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D. HeLa cells were transfected with Nup358 siRNA (siNup358) or Nup214 siRNA (siNup214). Western blot (WB) indicates the extent of Nup358 depletion and the level of Dcp1a.


D . HeLa were transfected with Nup358 siRNA ( siNup358 ) or Nup214 siRNA ( siNup214 ) . were fixed stained for endogenous Nup358 ( green , upper panel ) or Nup214 ( green , lower panel ) endogenous P marker ( Dcp1a , red ) . Graph represents quantitative data as mean ± SD . data was obtained from three independent experiments , in each experiment , 100 were counted from different fields for presence of intact microscopically distinct P bodies expressed as .

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D. HeLa cells were transfected with Nup358 siRNA (siNup358) or Nup214 siRNA (siNup214). Cells were fixed and stained for endogenous Nup358 (green, upper panel) or Nup214 (green, lower panel) and endogenous P body marker (Dcp1a, red). Graph represents the quantitative data as mean ± SD. The data was obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact microscopically distinct P bodies and expressed as percentage.


A . Western of HeLa , treated with control ( siControl ) , Dicer ( siDicer ) , Nup214 ( siNup214 ) or Nup358 ( siNup358 ) siRNA , for assessing extent of depletion using indicated antibodies . Vinculin was used as loading control .

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A. Western analysis of HeLa cells, treated with control (siControl), Dicer (siDicer), Nup214 (siNup214) or Nup358 (siNup358) siRNA, for assessing the extent of protein depletion using indicated antibodies. Vinculin was used as loading control.


B . HeLa were initially transfected with indicated siRNAs , followed by renilla luciferase ( RL ) reporter constructs ; RL - control ( no let - 7a binding site in 3 ' UTR ) or RL - 3xBulge ( 3 imperfect let - 7a binding sites in 3 ' - UTR ) . Firefly luciferase ( FL ) was co - transfected to serve as internal control . RL / FL luminescence was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ .

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

B. HeLa cells were initially transfected with indicated siRNAs, followed by renilla luciferase (RL) reporter constructs; RL-control (no let-7a binding site in the 3'UTR) or RL-3xBulge (3 imperfect let-7a binding sites in the 3'-UTR). Firefly luciferase (FL) was co-transfected to serve as internal control. RL/FL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


C . HEK293T were transfected with indicated siRNAs , followed by pCMV - FL - miR30 ( P ) reporter with either pSUPER - control or pSUPER - miR30 constructs . RL was co - transfected as internal control . FL / RL luminescence was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ .

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C. HEK293T cells were transfected with indicated siRNAs, followed by pCMV-FL-miR30 (P) reporter with either pSUPER-control or pSUPER-miR30 constructs. RL was co-transfected as internal control. FL/RL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


D . HeLa were initially transfected with control ( siControl ) , Dicer ( siDicer ) or Nup358 ( siNup358 ) specific siRNAs , followed by FL constructs containing wild type ( HMGA2 - wt ) or mutant ( HMGA2 - mut ) HMGA2 3 ' - UTR , along with RL as internal control . Graph was plotted using data from three independent experiments . FL / RL luminescence was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ .

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D. HeLa cells were initially transfected with control (siControl), Dicer (siDicer) or Nup358 (siNup358) specific siRNAs, followed by FL constructs containing wild type (HMGA2-wt) or mutant (HMGA2-mut) HMGA2 3'-UTR, along with RL as internal control. Graph was plotted using data from three independent experiments. FL/RL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


A . HeLa were transfected with control , Dicer or Nup358 specific siRNAs , as indicated . Upper panel , total was isolated analyzed by northern blotting for let - 7a using radio - labeled probe . Ethidium bromide ( EtBr ) stained gel indicates equal loading of samples .

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A. HeLa cells were transfected with control, Dicer or Nup358 specific siRNAs, as indicated. Upper panel, total RNA was isolated and analyzed by northern blotting for let-7a using radio-labeled probe. Ethidium bromide (EtBr) stained gel indicates equal loading of RNA samples.


A . HeLa were transfected with control , Dicer or Nup358 specific siRNAs , as indicated . Bottom panel , total was isolated from control , Dicer or Nup358 siRNA treated was reverse - transcribed using TaqMan microRNA reverse transcription kit . of let - 7a were quantified by qPCR following manufacturer ' s instructions . Graph represents relative of miRNAs as compared to U6 control . values were further normalized to let - 7a in control siRNA knockdown condition . Data are presented as mean SD ( n = 3 ) .

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A. HeLa cells were transfected with control, Dicer or Nup358 specific siRNAs, as indicated. Bottom panel, total RNA was isolated from control, Dicer or Nup358 siRNA treated cells and was reverse-transcribed using TaqMan microRNA reverse transcription kit. The levels of let-7a were quantified by qPCR following manufacturer's instructions. Graph represents the relative levels of miRNAs as compared to U6 RNA control. The values were further normalized to let-7a levels in control siRNA knockdown condition. Data are presented as mean SD (n = 3).


B . HeLa were transfected with indicated siRNAs , followed by RL - 3xBulge construct . Immunoprecipitation ( IP ) was performed with control mouse IgG ( IgG IP ) or anti - AGO2 antibody ( AGO2 IP ) using lysates prepared from these . Bottom left panel ; isolated from IgG control or AGO2 immunoprecipitate samples was reverse - transcribed using oligo ( dT ) primer . RL - 3xBulge mRNA association with AGO2 was quantified by q - PCR normalized to level of GAPDH mRNA associated with AGO2 .

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B. HeLa cells were transfected with indicated siRNAs, followed by RL-3xBulge construct. Immunoprecipitation (IP) was performed with control mouse IgG (IgG IP) or anti-AGO2 antibody (AGO2 IP) using lysates prepared from these cells. Bottom left panel; RNA isolated from IgG control or AGO2 immunoprecipitate samples was reverse-transcribed using oligo(dT) primer. RL-3xBulge mRNA association with AGO2 was quantified by q-PCR and normalized to the level of GAPDH mRNA associated with AGO2.


B . HeLa were transfected with indicated siRNAs , followed by RL - 3xBulge construct . Immunoprecipitation ( IP ) was performed with control mouse IgG ( IgG IP ) or anti - AGO2 antibody ( AGO2 IP ) using lysates prepared from these . Total was isolated from control AGO2 immunoprecipitates of AGO2 associated let - 7a ( top left panel ) miR - 17 ( top right panel ) were quantified by q - PCR . Graph represents extent of miRNAs associated with AGO2 as compared to U6 ( as a negative control ) with AGO2 .

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B. HeLa cells were transfected with indicated siRNAs, followed by RL-3xBulge construct. Immunoprecipitation (IP) was performed with control mouse IgG (IgG IP) or anti-AGO2 antibody (AGO2 IP) using lysates prepared from these cells. Total RNA was isolated from control and AGO2 immunoprecipitates and the levels of AGO2 associated let-7a (top left panel) and miR-17 (top right panel) were quantified by q-PCR. Graph represents the extent of miRNAs associated with AGO2 as compared to U6 RNA (as a negative control) with AGO2.


B . HeLa were transfected with indicated siRNAs , followed by RL - 3xBulge construct . Immunoprecipitation ( IP ) was performed with control mouse IgG ( IgG IP ) or anti - AGO2 antibody ( AGO2 IP ) using lysates prepared from these . Bottom right panel ; AGO2 immunoprecipitates were analyzed by western blotting using AGO2 specific antibody . Vinculin was used as loading control . Data are presented as mean SD ( n = 3 ) .

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B. HeLa cells were transfected with indicated siRNAs, followed by RL-3xBulge construct. Immunoprecipitation (IP) was performed with control mouse IgG (IgG IP) or anti-AGO2 antibody (AGO2 IP) using lysates prepared from these cells. Bottom right panel; AGO2 immunoprecipitates were analyzed by western blotting using AGO2 specific antibody. Vinculin was used as loading control. Data are presented as mean SD (n = 3).


C . HeLa were transfected with control ( siControl ) or Nup358 specific siRNA ( siNup358 ) for 96 h . were lysed subjected to immunoprecipitation with control IgG ( IgG IP ) or anti - AGO2 ( AGO2 IP ) . Total was extracted from initial lysate IP samples analyzed by q - PCR for validated miRNA targets , Serbp1 Dnajb11 . GAPDH was considered as negative control . Data expressed as relative amount of target mRNA associated with AGO2 as compared to GAPDH mRNA .

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C. HeLa cells were transfected with control (siControl) or Nup358 specific siRNA (siNup358) for 96 h. Cells were lysed and subjected to immunoprecipitation with control IgG (IgG IP) or anti-AGO2 (AGO2 IP). Total RNA was extracted from initial lysate and IP samples and analyzed by q-PCR for validated miRNA targets, Serbp1 and Dnajb11. GAPDH was considered as negative control. Data expressed as the relative amount of target mRNA associated with AGO2 as compared to GAPDH mRNA.


C . HeLa were transfected with control ( siControl ) or Nup358 specific siRNA ( siNup358 ) for 96 h . Western blots indicate extent of Nup358 depletion AGO2 immunoprecipitation in siControl siNup358 samples . Vinculin was used as loading control . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ .

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C. HeLa cells were transfected with control (siControl) or Nup358 specific siRNA (siNup358) for 96 h. Western blots indicate the extent of Nup358 depletion and AGO2 immunoprecipitation in siControl and siNup358 samples. Vinculin was used as loading control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


D . HeLa were treated with indicated siRNAs for 96 h . were lysed analyzed for extent of Nup358 depletion for of validated miRNA targets , Ras c - Myc by western . Vinculin was used as loading control .

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D. HeLa cells were treated with indicated siRNAs for 96 h. Cells were lysed and analyzed for extent of Nup358 depletion and for the levels of validated miRNA targets, Ras and c-Myc by western analysis. Vinculin was used as loading control.


E . HeLa were lysed immunoprecipitation was performed with control ( IgG - IP ) or Nup358 ( Nup358 - IP , left panel ) or AGO2 ( AGO2 - IP , right panel ) using specific antibodies . immunoprecipitates were washed with a buffer containing ( + ) or not containing ( - ) RNAse A probed for presence of indicated by western blotting .

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E. HeLa cells were lysed and immunoprecipitation was performed with control (IgG-IP) or Nup358 (Nup358-IP, left panel) or AGO2 (AGO2-IP, right panel) using specific antibodies. The immunoprecipitates were washed with a buffer containing (+) or not containing (-) RNAse A and probed for the presence of indicated proteins by western blotting.


F . HeLa were transfected with RL - 3xBulge construct immunoprecipitation was performed using control ( IgG - IP ) or Nup358 ( Nup358 - IP ) antibodies . Total was isolated from immunoprecipitates analyzed for let - 7a miRNA ( left panel ) , RL - 3xBulge mRNA endogenous miRNA target Dnajb11 using q - PCR ( right panel ) . Fold enrichment in Nup358 - IP as compared to IgG - control was calculated data are presented as mean SD ( n = 3 ) , P ‐ value was obtained using Student ' s t ‐ .

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F. HeLa cells were transfected with RL-3xBulge construct and immunoprecipitation was performed using control (IgG-IP) or Nup358 (Nup358-IP) antibodies. Total RNA was isolated from the immunoprecipitates and analyzed for let-7a miRNA (left panel), RL-3xBulge mRNA and the endogenous miRNA target Dnajb11 using q-PCR (right panel). Fold enrichment in Nup358-IP as compared to IgG-control was calculated and the data are presented as mean SD (n = 3), P‐value was obtained using Student's t‐test.


B . HEK293T were co - transfected with GFP - maltose binding ( MBP ) - control or GFP - tagged version of indicated Nup358 fragments along with HA - AGO2 . Immunoprecipitation ( IP ) was performed using GFP antibodies presence of AGO2 was detected by western blotting ( WB ) using HA antibodies .

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B. HEK293T cells were co-transfected with GFP-maltose binding protein (MBP)-control or GFP-tagged version of indicated Nup358 fragments along with HA-AGO2. Immunoprecipitation (IP) was performed using GFP antibodies and the presence of AGO2 was detected by western blotting (WB) using HA antibodies.


C . were transfected with indicated constructs IP WB were performed as described for B .

C. Cells were transfected with indicated constructs and IP and WB were performed as described for B.


D . HEK293T transfected with GFP - MBP ( control ) , GFP - Nup358 - C or GFP - Nup358 - CΔIR ( a mutant devoid of IR region ) HA - AGO2 . IP WB analyses were performed as indicated .

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D. HEK293T cells transfected with GFP-MBP (control), GFP-Nup358-C or GFP-Nup358-CΔIR (a mutant devoid of IR region) and HA-AGO2. IP and WB analyses were performed as indicated.


E . were transfected with HA - tagged version of indicated AGO subfamily member GFP - Nup358 - IR . IP WB analyses were performed using indicated antibodies .

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E. Cells were transfected with HA-tagged version of indicated AGO subfamily member and GFP-Nup358-IR. IP and WB analyses were performed using indicated antibodies.


F . Lysates from expressing GFP - control , GFP - human ( h ) Nup358 - IR or GFP - zebrafish ( z ) Nup358 - IR HA - AGO2 were immunoprecipitated using GFP antibodies immunoprecipitates were analyzed for presence of HA - AGO2 by western blotting .

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F. Lysates from cells expressing GFP-control, GFP-human (h) Nup358-IR or GFP-zebrafish (z) Nup358-IR and HA-AGO2 were immunoprecipitated using GFP antibodies and the immunoprecipitates were analyzed for the presence of HA-AGO2 by western blotting.


B . HEK293T were transfected with indicated constructs immunoprecipitation ( IP ) was performed using GFP specific antibodies probed for HA - AGO2 by western blotting ( WB ) using HA antibodies . Endogenous RanGAP Ubc9 were probed with specific antibodies .

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B. HEK293T cells were transfected with indicated constructs and immunoprecipitation (IP) was performed using GFP specific antibodies and probed for HA-AGO2 by western blotting (WB) using HA antibodies. Endogenous RanGAP and Ubc9 were probed with specific antibodies.


C . Lysates prepared from expressing indicated constructs were subjected to IP WB using indicated antibodies . Presence of endogenous RanGAP Ubc9 was determined by WB .

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C. Lysates prepared from cells expressing the indicated constructs were subjected to IP and WB using indicated antibodies. Presence of endogenous RanGAP and Ubc9 was determined by WB.


D . GFP - control , GFP - IR1 + 2 wild type or mutants were co - expressed with HA - AGO IP WB were performed to detect interaction using indicated antibodies .

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D. GFP-control, GFP-IR1+2 wild type or mutants were co-expressed with HA-AGO and IP and WB analysis were performed to detect the interaction using indicated antibodies.


E . Lysates prepared from expressing indicated were immunoprecipitated with GFP specific antibodies presence of specific in immunoprecipitates was detected by WB with indicated antibodies .

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E. Lysates prepared from cells expressing the indicated proteins were immunoprecipitated with GFP specific antibodies and the presence of specific proteins in the immunoprecipitates was detected by WB with indicated antibodies.


A . Top panel ; depicts acid corresponding to Nup358 - SIM1 region substitutions introduced in SIM1 mutant . Bottom panel : HEK293T were co - transfected with GFP - control , GFP - SIM1 or GFP - SIM1 - mut along with HA - AGO2 immunoprecipitation ( IP ) was performed with GFP specific antibodies western ( WB ) of input lysate immunoprecipitates were carried out using indicated antibodies .

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A. Top panel; depicts the amino acid sequence corresponding to the Nup358-SIM1 region and substitutions introduced in the SIM1 mutant. Bottom panel: HEK293T cells were co-transfected with GFP-control, GFP-SIM1 or GFP-SIM1-mut along with HA-AGO2 and immunoprecipitation (IP) was performed with GFP specific antibodies and western analysis (WB) of the input lysate and immunoprecipitates were carried out using indicated antibodies.


B . Top panel ; depiction of acid corresponding to SIMs in indicated used in this study . Hydrophobic residues involved in SUMO binding are indicated in red . Bottom panel ; constructs expressing GFP - control or GFP - fused SIMs of indicated were co - transfected with HA - AGO2 in HEK293T IP WB were performed to determine extent of interaction .

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B. Top panel; depiction of amino acid sequence corresponding to the SIMs in the indicated proteins used in this study. Hydrophobic residues involved in SUMO binding are indicated in red. Bottom panel; constructs expressing GFP-control or GFP-fused SIMs of indicated proteins were co-transfected with HA-AGO2 in HEK293T cells and IP and WB were performed to determine the extent of interaction.


C . HA - MBP - control , HA - AGO1 or HA - AGO2 was co - expressed with GFP - SIM1 lysates were subjected to IP with HA antibodies WB with indicated antibodies to detect presence of in immunoprecipitates .

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C. HA-MBP-control, HA-AGO1 or HA-AGO2 was co-expressed with GFP-SIM1 and the cell lysates were subjected to IP with HA antibodies and WB analysis with indicated antibodies to detect the presence of proteins in the immunoprecipitates.


D . SIM1 directly interacts with AGO2 . lysates expressing MBP control or MBP - AGO2 GST or GST - SIM1 were mixed GST pull down was performed . presence of in pull down samples was analyzed by WB using specific antibodies . extent of GST pull down was monitored by coomassie of ( coomassie ) .

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D. SIM1 directly interacts with AGO2. Bacterial lysates expressing MBP control or MBP-AGO2 and GST or GST-SIM1 were mixed and GST pull down assay was performed. The presence of proteins in the pull down samples was analyzed by WB using specific antibodies. The extent of GST pull down was monitored by coomassie staining of the membrane (coomassie).


E . SIM1 interacts with PIWI subfamily of . HEK293T were co - transfected with indicated constructs expressing HA - tagged version of PIWI GFP - SIM1 . IP was performed using HA antibodies . immunoprecipitates were probed for presence of GFP - SIM1 using GFP specific antibodies by WB .

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E. SIM1 interacts with PIWI subfamily of proteins. HEK293T cells were co-transfected with indicated constructs expressing HA-tagged version of PIWI proteins and GFP-SIM1. IP was performed using HA antibodies. The immunoprecipitates were probed for the presence of GFP-SIM1 using GFP specific antibodies by WB.


F . Putative SIM - binding regions in AGO2 . Three distinct superimpositions of AGO2 ( grey ) SUMO ( blue ) , in with Nup358 - SIM1 ( red ) , are shown in cartoon representation rendered with Chimera [ 90 ] . residue numbers on AGO2 that could be putative SIM - binding regions are labelled . To identify possible Nup358 binding sites on AGO2 , 3D structure of AGO2 ( PDB ID : 4W5O ) [ 89 ] was compared with that of SUMO ( PDB ID : 1Z5S ) [ 47 ] .

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F. Putative SIM-binding regions in AGO2. Three distinct superimpositions of AGO2 (grey) and SUMO (blue), in complex with Nup358-SIM1(red), are shown in cartoon representation rendered with Chimera [90]. The residue numbers on the AGO2 that could be the putative SIM-binding regions are labelled. To identify possible Nup358 binding sites on AGO2, the 3D structure of AGO2 (PDB ID: 4W5O) [89] was compared with that of SUMO (PDB ID: 1Z5S) [47].


A . Right panel ; Western blots showing expression of transfected constructs using indicated antibodies . a - tubulin was used as loading control .

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A. Right panel; Western blots showing the expression of transfected constructs using indicated antibodies. a-tubulin was used as loading control.


A . Left panel ; HeLa were co - transfected with GFP - MBP ( control ) , GFP - SIM1 or GFP - SIM1 - mut ( AGO interaction defective ) along with RL - control or RL - 3xbulge . Firefly luciferase was used as transfection control . Dual luciferase was performed . RL / FL luminescence ration was plotted after normalization with GFP - control . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ .

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A. Left panel; HeLa cells were co-transfected with GFP-MBP (control), GFP-SIM1 or GFP-SIM1-mut (AGO interaction defective) along with RL-control or RL-3xbulge. Firefly luciferase was used as transfection control. Dual luciferase assay was performed. RL/FL luminescence ration was plotted after normalization with GFP-control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


B . Bottom right panel ; Western blot for monitoring expression of indicated using antibodies . a - tubulin was used as loading control .

B. Bottom right panel; Western blot for monitoring the expression levels of indicated proteins using antibodies. a-tubulin was used as loading control.


B . Bottom left panel ; HEK293T were co - transfected with indicated expression constructs RL reporter . Firefly luciferase ( FL ) was used as transfection control . Dual luciferase was performed . of RL / FL luminescence was plotted after normalization with N - HA - MBP control . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ .

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B. Bottom left panel; HEK293T cells were co-transfected with indicated expression constructs and the RL reporter. Firefly luciferase (FL) was used as transfection control. Dual luciferase assay was performed. The ratio of RL/FL luminescence was plotted after normalization with N-HA-MBP control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


C . Ectopic expression of AGO2 restores P formation , but fails to rescue impairment in miRNA function in Nup358 depleted . HeLa were transfected with control ( siContriol ) or Nup358 ( siNup358 ) specific siRNA , followed by indicated constructs . Middle panel : HeLa were transfected with indicated siRNAs , followed by expression constructs miRNA RL reporters . FL was used as transfection control . of RL / FL luminescence was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was obtained using Student ' s t ‐ .

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C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Middle panel: HeLa cells were transfected with indicated siRNAs, followed by expression constructs and miRNA RL reporters. FL was used as transfection control. The ratio of RL/FL luminescence was calculated. Data are presented as mean SD (n = 3), P‐value was obtained using Student's t‐test.


C . Ectopic expression of AGO2 restores P formation , but fails to rescue impairment in miRNA function in Nup358 depleted . HeLa were transfected with control ( siContriol ) or Nup358 ( siNup358 ) specific siRNA , followed by indicated constructs . Right panel : Western blot for relative expression of indicated constructs in HEK293T . a - tubulin is used as loading control .

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C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Right panel: Western blot analysis for the relative expression of indicated constructs in HEK293T cells. a-tubulin is used as loading control.


C . Ectopic expression of AGO2 restores P formation , but fails to rescue impairment in miRNA function in Nup358 depleted . HeLa were transfected with control ( siContriol ) or Nup358 ( siNup358 ) specific siRNA , followed by indicated constructs . Left panel : Quantitative representation showing number of showing microscopically distinct P bodies in different indicated conditions . data was obtained from three independent experiments , in each experiment , 100 were counted from different fields for presence of intact P bodies expressed as .

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C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Left panel: Quantitative representation showing the number of cells showing microscopically distinct P bodies in different indicated conditions. The data was obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact P bodies and expressed as percentage.


D . HeLa were transfected with siControl or siNup358 subjected to flotation . Fractions were collected analyzed for presence of indicated by western blotting .

D. HeLa cells were transfected with siControl or siNup358 and subjected to membrane flotation assay. Fractions were collected and analyzed for the presence of indicated proteins by western blotting.


LC - MS / MS measurements of m6A in total ( B ) or in poly ( A ) + ( C ) upon KD of predicted methyltransferases in Drosophila S2R + . m6A abundance in total is significantly reduced when Mettl5 is depleted , while its depletion has no effect on m6A level in mRNA . As expected , KD of Mettl3 Mettl14 reduce m6A in mRNA . Bar chart represents mean ± standard deviation of three technical measurements from three biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( two - t - ) .

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LC-MS/MS measurements of m6A levels in total RNA (B) or in poly(A)+ RNA (C) upon KD of predicted methyltransferases in Drosophila S2R+ cells. m6A abundance in total RNA is significantly reduced when Mettl5 is depleted, while its depletion has no effect on m6A level in mRNA. As expected, the KD of Mettl3 and Mettl14 reduce m6A levels in mRNA. Bar chart represents the mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test).


Subcellular localization of Mettl5 - FLAG ( purple ) Trmt112 - eGFP ( green ) expressed under control of their own promoter in S2R + . 63X magnified merge of immunofluorescently - labelled S2R + . Scale bar : 4 . 47 μm .

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Subcellular localization of Mettl5-FLAG (purple) and Trmt112-eGFP (green) expressed under the control of their own promoter in S2R+ cells. 63X magnified merge of immunofluorescently-labelled S2R+ cells. Scale bar: 4.47 μm.


Developmental expression of Mettl5 transcript assayed by RT - qPCR . figure shows mean ± standard deviation of three technical measurements from three biological replicates .

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Developmental expression of Mettl5 transcript assayed by RT-qPCR analysis. The figure shows mean ± standard deviation of three technical measurements from three biological replicates.


In situ hybridization of Mettl5 transcript at different embryonic stages . central nervous system ( CNS ) is highlighted in schematics . Data retrieved from FlyExpress 7 . ( http : / / www . flyexpress . net / search . php ? type = image & search = FBgn0036856 ) .

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In situ hybridization of Mettl5 transcript at different embryonic stages. The central nervous system (CNS) is highlighted in the schematics. Data retrieved from FlyExpress 7. (http://www.flyexpress.net/search.php?type=image&search=FBgn0036856).


LC - MS / MS measurements of m6A in total of WT Mettl5 mutant flies . Bars represent mean ± standard deviation of measurements of three biological replicates . * * * P < 0 . 001 ( two - t - ) .

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LC-MS/MS measurements of m6A levels in total RNA of WT and Mettl5 mutant flies. Bars represent mean ± standard deviation of measurements of three biological replicates. ***P<0.001 (two-tailed t-test).


Purified 18S rRNA analyzed for its m6A content by quantitative HPLC . 18S rRNA was extracted from 40S subunits isolated on sucrose gradients . calibration control is a commercial source of m6A ( in grey ) . m6A elutes at 48 min .

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Purified 18S rRNA analyzed for its m6A content by quantitative HPLC. The 18S rRNA was extracted from 40S subunits isolated on sucrose gradients. The calibration control is a commercial source of m6A (in grey). m6A elutes at 48 min.


Mature rRNA on ethidium - stained denaturing agarose gels . same amounts of total extracted from indicated flies , from S2R + depleted or not of Mettl5 , were loaded . 28S / 18S was established by densitometry .

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Mature rRNA analysis on ethidium-stained denaturing agarose gels. The same amounts of total RNA extracted from the indicated flies, and from S2R+ cells depleted or not of Mettl5, were loaded. The 28S/18S ratio was established by densitometry.


of 18S rRNA maturation in WT mutant flies ( Mettl5fs ) . Total extracted from indicated was resolved on denaturing agarose gels processed for northern blotting with specific probes ( complementary to ITS1 or ITS2 sequences ) . pre - rRNAs detected accumulate to normal indicating that processing is unaffected .

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Analysis of 18S rRNA maturation in WT and mutant flies (Mettl5fs). Total RNA extracted from the indicated animals was resolved on denaturing agarose gels and processed for northern blotting with specific probes (complementary to ITS1 or ITS2 sequences). The pre-rRNAs detected accumulate to normal levels indicating that processing is unaffected.


Western blot validation of co - immunoprecipitated Mettl5 - FLAG - Myc Trmt112 , bearing either a N - terminal HA tag ( top ) or a C - terminal GFP tag ( bottom ) , in S2R + . arrows point at indicated . HA - Trmt12 migrates below antibody chains ( top ) . Trmt112 - eGFP migrates above heavy chains indicated by an asterisk ( bottom ) .

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Western blot validation of the co-immunoprecipitated proteins Mettl5-FLAG-Myc and Trmt112, bearing either a N-terminal HA tag (top) or a C-terminal GFP tag (bottom), in S2R+ cells. The arrows point at the indicated proteins. HA-Trmt12 migrates below the antibody light chains (top). Trmt112-eGFP migrates above the heavy chains indicated by an asterisk (bottom).


Trmt112 depletion reduces m6A in total from Drosophila S2R + . Mean ± standard deviation of three technical measurements from three biological replicates . * P < 0 . 05 ( two - t - ) .

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Trmt112 depletion reduces m6A levels in total RNA from Drosophila S2R+ cells. Mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05 (two-tailed t-test).


Representative trajectories of WT Mettl5 mutant flies analysed by Buridan ' s Paradigm . blue lines indicate when fly stops changes direction . Orientation evaluated with help of Buridan ' s paradigm for Mettl5 mutant flies . Orientation was measured as angular deviation from straight path needed to move from one landmark to another in arena . Number of flies tested per genotype : 30 . Mean ± standard deviation . Shapiro - Wilk was used to for normal distribution in each group . Normally distributed groups were tested by t - . Due to multiple comparison Bonferroni correction was applied . ( p < 0 . 05 = * ; p < 0 . 0001 = * * * * ) .

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Representative trajectories of WT and Mettl5 mutant flies analysed by the Buridan's Paradigm. The blue lines indicate when the fly stops and changes direction. Orientation evaluated with the help of the Buridan's paradigm for Mettl5 mutant flies. Orientation was measured as the angular deviation from the straight path needed to move from one landmark to another in the arena. Number of flies tested per genotype: 30. Mean ± standard deviation. Shapiro-Wilk test was used to test for normal distribution in each group. Normally distributed groups were tested by t-test. Due to multiple comparison Bonferroni correction was applied. (p<0.05 = *; p<0.0001=****).


( A ) smFISH targeting Bmal1 , Cry1 Nr1d1 in wild - type NIH 3T3 at 17h , 29h 25h after synchronisation with Dex , respectively . Nuclei are stained with DAPI ( blue ) . Each dot ( ) corresponds to a single transcript . Segmented boundaries are delineated in grey . blue channels represent maximum z - projections . Scale bar : 20μm .

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(A) smFISH targeting Bmal1, Cry1 and Nr1d1 in wild-type NIH 3T3 cells at 17h, 29h and 25h after synchronisation with Dex, respectively. Nuclei are stained with DAPI (blue). Each fluorescent dot (white) corresponds to a single transcript. Segmented cell boundaries are delineated in grey. The blue and white channels represent maximum z-projections. Scale bar: 20μm.


( B ) Number of Bmal1 , Cry1 , Nr1d1 transcripts per as a function of time after treatment with Dex . These data combine smFISH hybridizations from 4h sampled Nr1d1 / Cry1 Bmal1 / Cry1 experiment . Each dot shows average over one replicate from an independent slide ( Methods ) . solid lines represent fits using a two - harmonic cosinor model ( Equation ( 1 ) , Methods ) for each gene individually .

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(B) Number of Bmal1, Cry1, and Nr1d1 transcripts per cell as a function of time after treatment with Dex. These data combine all smFISH hybridizations from the 4h sampled Nr1d1/Cry1 and Bmal1/Cry1 experiment. Each dot shows the average over one replicate from an independent slide (Methods). The solid lines represent fits using a two-harmonic cosinor model (Equation (1), Methods) for each gene individually.


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(D) Distributions of Nr1d1, Cry1 and Bmal1 transcripts at 21h, 29h and 37h after synchronisation with Dex. μ represents the mean of the distribution, CV represent the coefficient of variation (standard deviation / mean), and N is the number of cells at the given time point. Total number of cells analysed for the Nr1d1/Cry1 pair: 21h - 449; 25h - 414; 29h - 521; 33h - 463; 37h - 477; 41h - 429. Total number of cells analysed for the Bmal1/Cry1 pair: 17h - 465; 21h - 490; 25h - 504; 29h - 436; 33h - 407; 37h - 454; 41h - 404.


( A ) Log conditional mean mRNA for a given as a function of log . Each is sorted by by placing it in one of 20 bins that range from 5th to 95th percentile . conditional mean given E [ m | A ] is then mean mRNA of each bin . is normalised such that average is equal to one . Solid lines represent a least - squares linear fit to data .

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(A) Log conditional mean mRNA count for a given area as a function of the log area. Each cell is sorted by area by placing it in one of 20 bins that range from the 5th to the 95th percentile. The conditional mean given the area E[m|A] is then the mean mRNA count of each bin. The area is normalised such that the average area is equal to one. Solid lines represent a least-squares linear fit to the data.


( B ) Top image : Dual - channel smFISH targeting Bmal1 Cry1 simultaneously , taken at 17h after Dex synchronisation . Blue , nuclei stained with DAPI ; green dots , Bmal1 transcripts ; red , Cry1 transcripts . Bottom image : Dual - channel smFISH targeting Nr1d1 Cry1 simultaneously , taken at 25h after Dex synchronisation . Blue , nuclei stained with DAPI ; green dots , Nr1d1 transcripts ; red , Cry1 transcripts . Scale bar : 20μm .

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(B) Top image: Dual-channel smFISH targeting Bmal1 and Cry1 simultaneously, taken at 17h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Bmal1 transcripts; red, Cry1 transcripts. Bottom image: Dual-channel smFISH targeting Nr1d1 and Cry1 simultaneously, taken at 25h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Nr1d1 transcripts; red, Cry1 transcripts. Scale bar: 20μm.


( C ) Bivariate distributions of mRNA per for dual - channel smFISH targeting either Bmal1 Cry1 or Nr1d1 Cry1 . Each dot corresponds to a single , contours represent KDE estimates of density . Time represents number of hours after Dex synchronisation , R represents Pearson correlation coefficient .

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(C) Bivariate distributions of mRNA counts per cell for dual-channel smFISH targeting either Bmal1 and Cry1 or Nr1d1 and Cry1. Each dot corresponds to a single cell, and the contours represent KDE estimates of the density. Time represents the number of hours after Dex synchronisation, and R represents the Pearson correlation coefficient.


( C ) Posterior parameter estimates . Burst frequency is plotted as a function of time , where solid represents posterior mean shaded represents 90 % confidence interval . Posterior probability densities are shown for average burst , β ( which controls dependence between burst ) ρ ( representing correlation in burst between genes ( in log space ) ) , where β is inferred from model M2 average burst ρ are calculated from model M4 .

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(C) Posterior parameter estimates. Burst frequency is plotted as a function of time, where the solid line represents the posterior mean and the shaded area represents the 90% confidence interval. Posterior probability densities are shown for the average burst size, β (which controls the dependence between burst size and cell area) and ρ (representing the correlation in burst size between genes (in log space)), where β is inferred from model M2 and the average burst size and ρ are calculated from model M4.


( D ) Comparison of probability density of data ( kernel density estimates ) with model ( using model M4 ) . To estimate probability from model , data set was simulated 15 times using posterior mean parameter values combined with measured areas .

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(D) Comparison of the probability density of the data (kernel density estimates) with the model (using model M4). To estimate the probability from the model, the data set was simulated 15 times using the posterior mean parameter values combined with the measured cell areas.


( a ) FLAG - AMBRA1 was transfected into HEK293 together with ULK1WT or ULK1K46I Myc - tagged . extracts were immunoprecipitated ( IP ) using anti - FLAG antibody .

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(a) FLAG-AMBRA1 was transfected into HEK293 cells together with ULK1WT or ULK1K46I Myc-tagged proteins. Protein extracts were immunoprecipitated (IP) using anti-FLAG antibody.


( b ) HEK293 were grown either in normal or in EBS medium for 1 h ; extracts were immunoprecipitated using anti - ULK1 antibody or rabbit immunoglobulin as a negative control ( IgG ) . Autophagy induction is shown by p62 degradation .

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(b) HEK293 cells were grown either in normal or in EBS medium for 1 h; protein extracts were immunoprecipitated using anti-ULK1 antibody or rabbit immunoglobulin as a negative control (IgG). Autophagy induction is shown by p62 degradation.


( c ) HEK293 were treated as in b , in presence of leupeptin stained by anti - AMBRA1 ( red ) anti - ULK1 ( green ) antibodies . merge of two signals DAPI ( blue ) for is shown in right panels . Scale bar , 6 μm . outlined areas are shown at a higher magnification in lower panels . arrows point to co - localization . Measurements by Metamorph indicate a co - localization of 46 % between ULK1 AMBRA1 on autophagy induction .

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(c) HEK293 cells were treated as in b, in the presence of leupeptin and stained by anti-AMBRA1 (red) and anti-ULK1 (green) antibodies. The merge of the two signals and DAPI (blue) for nuclear staining is shown in the right panels. Scale bar, 6 μm. The outlined areas are shown at a higher magnification in the lower panels. White arrows point to the co-localization puncta. Measurements by Metamorph indicate a co-localization ratio of 46% between ULK1 and AMBRA1 on autophagy induction.


( d , e ) ULK1 AMBRA1 constructs are illustrated . Asterisks mark nonspecific bands corresponding to immunoglobulins . ( d ) HEK293 were co - transfected with vectors encoding AMBRA1 together with ULK1 - Myc FL ( full ) or its deletion mutants encoding kinase proline - rich domains ( Δ829 - 1 , 051 ) , ULK1 carboxy - terminal domain ( Δ1 - 828 ) . extracts were immunoprecipitated using anti - Myc antibody .

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(d,e) ULK1 and AMBRA1 constructs are illustrated. Asterisks mark nonspecific bands corresponding to immunoglobulins. (d) HEK293 cells were co-transfected with vectors encoding AMBRA1 together with ULK1-Myc FL (full length) or its deletion mutants encoding the kinase and the proline-rich domains (Δ829-1,051), and the ULK1 carboxy-terminal domain (Δ1-828). Protein extracts were immunoprecipitated using anti-Myc antibody.


( d , e ) ULK1 AMBRA1 constructs are illustrated . Asterisks mark nonspecific bands corresponding to immunoglobulins . ( e ) HEK293 were co - transfected with vectors encoding ULK1 - HA together with β - galactosidase - Myc ( βgal ) , AMBRA1 full - ( FL ) , F1 , F2 or F3 Myc - tagged . extracts were immunoprecipitated using anti - HA antibody . relative amounts of co - immunoprecipitatedULK1 F1 or F3 , respectively , are same ( see upper lower left panel ) .

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(d,e) ULK1 and AMBRA1 constructs are illustrated. Asterisks mark nonspecific bands corresponding to immunoglobulins. (e) HEK293 cells were co-transfected with vectors encoding ULK1-HA together with β-galactosidase-Myc (βgal), AMBRA1 full-length (FL), and F1, F2 or F3 Myc-tagged proteins. Protein extracts were immunoprecipitated using anti-HA antibody. The relative amounts of co-immunoprecipitatedULK1 and F1 or F3, respectively, are the same (see upper and lower left panel).


( f ) HEK293 were co - transfected with vectors encoding AMBRA1 , ULK1 - Myc ATG13 - FLAG . extracts were immunoprecipitated using anti - FLAG antibody .

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(f) HEK293 cells were co-transfected with vectors encoding AMBRA1, ULK1-Myc and ATG13-FLAG. Protein extracts were immunoprecipitated using anti-FLAG antibody.


( g ) HEK293 were transfected with scramble siRNA or ATG13 siRNA . extracts were immunoprecipitated using ULK1 antibody or IgG . band density of immunoprecipitated AMBRA1 relative to immunoprecipitated ULK1 is analysed , with control arbitrarily defined as 1 . 00 . n = 3 extracts prepared from independent experiments ; data are presented as means ± s . d . significance is P = 0 . 04 . Uncropped of blots / gels are shown in Supplementary Fig . S6 . Source data of statistical are shown in Supplementary Table S1 .

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(g) HEK293 cells were transfected with scramble siRNA or ATG13 siRNA. Protein extracts were immunoprecipitated using ULK1 antibody or IgG. The band density ratio of immunoprecipitated AMBRA1 relative to immunoprecipitated ULK1 is analysed, with the control ratio arbitrarily defined as 1.00. n = 3 extracts prepared from independent experiments; data are presented as means±s.d. and significance is P = 0.04. Uncropped images of blots/gels are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.


( a ) HEK293 were transfected with AMBRA1 or empty vector , as a control . ULK1 , AMBRA1 actin were analysed by western blotting .

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(a) HEK293 cells were transfected with AMBRA1 or the empty vector, as a control. ULK1, AMBRA1 and actin were analysed by western blotting.


( b ) extracts from wild - type ( Ambra1 + / + ) homozygous ( Ambra1gt / gt ) for Ambra1 gene - trap mutation at developmental day 14 . 5 were analysed by western blotting using anti - Ulk1 or anti - Hsp90 ( loading control ) antibodies . is shown on right . n = 6 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 037 .

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(b) Protein extracts from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos for the Ambra1 gene-trap mutation at developmental day 14.5 were analysed by western blotting using anti-Ulk1 or anti-Hsp90 (loading control) antibodies. Quantification is shown on the right. n = 6 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.037.


( c ) AMBRA1 , ULK1 actin ( as a loading control ) were analysed by western blotting in HeLa transiently transfected with scramble siRNA or AMBRA1 siRNA . is shown on right . n = 5 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 007 .

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(c) AMBRA1, ULK1 and actin (as a protein loading control) were analysed by western blotting in HeLa cells transiently transfected with scramble siRNA or AMBRA1 siRNA. Quantification is shown on the right. n = 5 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.007.


( d ) HeLa stably interfered for AMBRA1 were transiently transfected with empty vector or AMBRA1 vector ; AMBRA1 , ULK1 , ATG13 , p - ATG13 actin were analysed by western blotting . of ULK1 pATG13 bands is shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 006 for ULK1 P = 0 . 044 P = 0 . 011 for pATG13 .

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(d) HeLa cells stably interfered for AMBRA1 were transiently transfected with empty vector or AMBRA1 vector; AMBRA1, ULK1, ATG13, p-ATG13 and actin levels were analysed by western blotting. Quantification of ULK1 and pATG13 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.006 for ULK1 and P = 0.044 and P = 0.011 for pATG13.


( e ) HeLa were transfected or not with vector encoding AMBRA1 treated with 35 μM cycloheximide ( CHX ) for 6 8 h before extraction . ULK1 , actin AMBRA1 were analysed by western blotting . is shown below . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 049 P = 0 . 02 .

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(e) HeLa cells were transfected or not with vector encoding AMBRA1 and treated with 35 μM cycloheximide (CHX) for 6 and 8 h before extraction. ULK1, actin and AMBRA1 were analysed by western blotting. Quantification is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.049 and P = 0.02.


( f ) HEK293 were transfected as in a ; ATG13 , pATG13 , ULK1 , AMBRA1 actin were analysed by western blotting .

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(f) HEK293 cells were transfected as in a; ATG13, pATG13, ULK1, AMBRA1 and actin were analysed by western blotting.


( g ) Endogenous ULK1 is immunoprecipitated from HeLa transfected with β - galactosidase or AMBRA1 plasmid respectively using an anti - ULK1 antibody . As a control , rabbit IgG was used . resulting precipitates were subjected to ULK1kinase using MBP as substrate . Purified complexes were analysed by autoradiography by western blotting using an anti - ULK1 antibody . Total extracts were analysed using an anti - AMBRA1 antibody .

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(g) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected with β-galactosidase or AMBRA1 plasmid respectively using an anti-ULK1 antibody. As a control, rabbit IgG was used. The resulting precipitates were subjected to ULK1kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody. Total extracts were analysed using an anti-AMBRA1 antibody.


( a ) extracts from HeLa were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control analysed by western blotting using anti - Lys63 specific ubiquitin anti - ULK1 antibodies .

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(a) Protein extracts from HeLa cells were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control and analysed by western blotting using anti-Lys63 specific ubiquitin and anti-ULK1 antibodies.


( b ) HeLa were co - transfected with ULK1 - Myc alone or in combination with wild - type ubiquitin or its mutant ( K48R or K63R ) HA - tagged constructs . extracts were immunoprecipitated using an anti - Myc antibody . Purified complexes total extracts were analysed by western blotting using anti - Myc anti - HA antibodies .

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(b) HeLa cells were co-transfected with ULK1-Myc alone or in combination with wild-type ubiquitin or its mutant (K48R or K63R) HA-tagged constructs. Protein extracts were immunoprecipitated using an anti-Myc antibody. Purified complexes and total extracts were analysed by western blotting using anti-Myc and anti-HA antibodies.


( c ) In HeLa , AMBRA1 was downregulated by RNAi . extracts were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control . Purified complexes total extracts were analysed by western blotting using anti - ULK1 , anti - Lys - 63 ubiquitin anti - AMBRA1 antibodies . Of note , ULK1 in co - immunoprecipitation experiment do not change in this case in AMBRA1 siRNA lane , owing to saturation of anti - ULK1 antibody ( see also Fig . 4h ) .

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(c) In HeLa cells, AMBRA1 was downregulated by RNAi. Protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting using anti-ULK1, anti-Lys-63 ubiquitin and anti-AMBRA1 antibodies. Of note, ULK1 levels in the co-immunoprecipitation experiment do not change in this case in the AMBRA1 siRNA lane, owing to saturation of the anti-ULK1 antibody (see also Fig. 4h).


( d ) HeLa were co - transfected with vectors encoding for ubiquitin Lys - 63 - HA alone or in combination with AMBRA1 . extracts were immunoprecipitated using an anti - ULK1 antibody or with IgG as a negative control Ub - Lys - 63 - HA , ULK1 AMBRA1 were analysed by western blotting .

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(d) HeLa cells were co-transfected with vectors encoding for ubiquitin Lys-63-HA alone or in combination with AMBRA1. Protein extracts were immunoprecipitated using an anti-ULK1 antibody or with IgG as a negative control and Ub-Lys-63-HA, ULK1 and AMBRA1 were analysed by western blotting.


( e , f ) HeLa were cultured in complete or starvation medium for 30 min or in presence of 100 nM of rapamycin for 3 h then extracts were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control . Purified complexes total extracts were analysed by western blotting as in a . Quantifications are shown on right . In both panels , n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 003 P = 0 . 0015 , respectively .

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(e,f) HeLa cells were cultured in complete or starvation medium for 30 min or in the presence of 100 nM of rapamycin for 3 h and then protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting as in a. Quantifications are shown on the right. In both panels, n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.003 and P = 0.0015, respectively.


( a ) HeLa were transfected with FLAG - TRAF6 or grown in starvation medium for 1 h . Some of them were treated with chloroquine . LC3 , p62 actin were analysed by western blotting . of LC3II p62 bands is shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 017 for LC3 P = 0 . 01 P = 0 . 04 for p62 .

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(a) HeLa cells were transfected with FLAG-TRAF6 or grown in starvation medium for 1 h. Some of them were treated with chloroquine. LC3, p62 and actin were analysed by western blotting. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.017 for LC3 and P = 0.01 and P = 0.04 for p62.


( b ) HeLa were transduced with AMBRA1 lentiviral shRNA transfected or not with FLAG - TRAF6 . of LC3II p62 bands is shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 001 P = 0 . 0018 for LC3 P = 0 . 004 for p62 .

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(b) HeLa cells were transduced with AMBRA1 lentiviral shRNA and transfected or not with FLAG-TRAF6. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001 and P = 0.0018 for LC3 and P = 0.004 for p62.


( c ) of LC3 in HeLa transduced as in b . Five different confocal microscopy for each experiment were randomly chosen GFP - positive dots were examined on with identical brightness contrast settings . results are represented as average number of LC3 dots with 100 analysed per sample . Confocal are shown in Supplementary Fig . S3b .

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(c) Count of LC3 puncta in HeLa cells transduced as in b. Five different confocal microscopy images for each experiment were randomly chosen and GFP-positive dots were examined on the images with identical brightness and contrast settings. The results are represented as the average number of LC3 dots with 100 cells analysed per sample. Confocal images are shown in Supplementary Fig. S3b.


( d ) HEK293 were transfected with AMBRA1 FLAG - TRAF6 as indicated . extracts were immunoprecipitated using an anti - FLAG antibody .

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(d) HEK293 cells were transfected with AMBRA1 and FLAG-TRAF6 as indicated. Protein extracts were immunoprecipitated using an anti-FLAG antibody.


( e ) HeLa were transfected as in b FLAG - TRAF6 was purified as in d . of immunoprecipitated TRAF6 is shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 0003 .

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(e) HeLa cells were transfected as in b and FLAG-TRAF6 was purified as in d. Quantification of immunoprecipitated TRAF6 is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.0003.


( f ) In HeLa , TRAF6 was downregulated by RNAi . AMBRA1 , ULK1 , TRAF6 , ATG13 actin were analysed by western blotting .

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(f) In HeLa cells, TRAF6 was downregulated by RNAi. AMBRA1, ULK1, TRAF6, ATG13 and actin were analysed by western blotting.


( g ) HeLa were co - transfected with vectors encoding ULK1 - Myc , FLAG - TRAF6 Ub - Lys - 63 - HA , as indicated . extracts were immunoprecipitated using an anti - Myc antibody .

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(g) HeLa cells were co-transfected with vectors encoding ULK1-Myc, FLAG-TRAF6 and Ub-Lys-63-HA, as indicated. Protein extracts were immunoprecipitated using an anti-Myc antibody.


( h ) HeLa stably interfered for AMBRA1 were transfected with FLAG - TRAF6 Ub - Lys - 63 - HA as indicated . extracts were immunoprecipitated using anti - ULK1 antibody .

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(h) HeLa cells stably interfered for AMBRA1 were transfected with FLAG-TRAF6 and Ub-Lys-63-HA as indicated. Protein extracts were immunoprecipitated using anti-ULK1 antibody.


( i ) Endogenous ULK1 is immunoprecipitated from HeLa transfected as in b . resulting precipitates were subjected to a ULK1 kinase using MBP as substrate . Purified complexes were analysed by autoradiography by western blotting using an anti - ULK1 antibody .

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(i) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected as in b. The resulting precipitates were subjected to a ULK1 kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody.


( a - c ) Two canonical TRAF6 - binding sites in AMBRA1 are essential for its interaction with TRAF6 . HEK293 were co - transfected with vectors encoding FLAG - TRAF6 together with AMBRA1WT mutant constructs . extracts were immunoprecipitated using an anti - FLAG antibody . AMBRA1 FLAG - TRAF6 were analysed by western blotting .

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(a-c) Two canonical TRAF6-binding sites in AMBRA1 are essential for its interaction with TRAF6. HEK293 cells were co-transfected with vectors encoding FLAG-TRAF6 together with AMBRA1WT and mutant constructs. Protein extracts were immunoprecipitated using an anti-FLAG antibody. AMBRA1 and FLAG-TRAF6 were analysed by western blotting.


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( d ) HeLa were transfected with AMBRA1 RNAi oligonucleotide ( siRNA AMBRA1 ) . Then , were co - transfected with vectors encoding ubiquitin - HA AMBRA1WT or AMBRA1AA , respectively . extracts were immunoprecipitated using anti - ULK1 antibody ; ubiquitin - HA , ULK1 AMBRA1 were analysed by western blotting .

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(d) HeLa cells were transfected with AMBRA1 RNAi oligonucleotide (siRNA AMBRA1). Then, cells were co-transfected with vectors encoding ubiquitin-HA and AMBRA1WT or AMBRA1AA, respectively. Protein extracts were immunoprecipitated using anti-ULK1 antibody; ubiquitin-HA, ULK1 and AMBRA1 were analysed by western blotting.


( e ) HeLa stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β - galactosidase ( negative control ) AMBRA1WT or AMBRA1AA respectively ; extracts were analysed by western blotting using anti - ULK1 , anti - AMBRA1 anti - ATG13 , anti - pATG13 anti - actin antibodies .

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(e) HeLa cells stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β-galactosidase (negative control) AMBRA1WT or AMBRA1AA respectively; protein extracts were analysed by western blotting using anti-ULK1, anti-AMBRA1 anti-ATG13, anti-pATG13 and anti-actin antibodies.


( f ) HeLa were transfected as in e some of them were treated with chloroquine for 1 h . LC3 , p62 , AMBRA1 actin were analysed by western blotting . of LC3II p62 are shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 03 for LC3 P = 0 . 045 for p62 .

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(f) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h. LC3, p62, AMBRA1 and actin were analysed by western blotting. Quantification of LC3II and p62 are shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.03 for LC3 and P = 0.045 for p62.


( g ) GFP - LC3 MEF from wild - type ( Ambra1 + / + ) homozygous ( Ambra1gt / gt ) were transduced with indicated lentiviral vectors of LC3 per is shown on right . Bars represent mean ± s . e . m . of triplicate samples with 50 analysed per sample ; n = 3 independent experiments . Significance is P = 0 . 001 P = 0 . 003 ( n = 3 ) . Scale bar , 10 μm .

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(g) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with the indicated lentiviral vectors and quantification of LC3 puncta per cells is shown on the right. Bars represent mean±s.e.m. of triplicate samples with 50 cells analysed per sample; n = 3 independent experiments. Significance is P = 0.001 and P = 0.003 (n = 3). Scale bar, 10 μm.


( a ) HEK293 were co - transfected with vectors encoding ULK1 - HA ULK1 - Myc then grown either in normal ( Ctrl ) or in starvation ( EBS ) medium for 1 h . extracts were immunoprecipitated using an anti - Myc antibody , analysed by western blotting using anti - HA , anti - Myc antibodies . is shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 001 .

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(a) HEK293 cells were co-transfected with vectors encoding ULK1-HA and ULK1-Myc and then grown either in normal (Ctrl) or in starvation (EBS) medium for 1 h. Protein extracts were immunoprecipitated using an anti-Myc antibody, and analysed by western blotting using anti-HA, and anti-Myc antibodies. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001.


( b ) HEK293 were transfected with TRAF6 RNAi oligonucleotide ( TRAF6 siRNA ) or unrelated oligonucleotides as a control ( Ctrl siRNA ) . Then , were co - transfected with vectors encoding ULK1 - HA ULK1 - Myc complexes were immunoprecipitated using anti - Myc antibody . extracts were analysed by western blotting using anti - HA , anti - Myc anti - TRAF6 antibodies . of ULK1 - HA relative to ULK1 - Myc is reported on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 037 .

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(b) HEK293 cells were transfected with TRAF6 RNAi oligonucleotide (TRAF6 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected with vectors encoding ULK1-HA and ULK1-Myc and protein complexes were immunoprecipitated using anti-Myc antibody. Protein extracts were analysed by western blotting using anti-HA, anti-Myc and anti-TRAF6 antibodies. Quantification of ULK1-HA relative to ULK1-Myc is reported on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.037.


( c ) HEK293 were transfected with AMBRA1 RNAi oligonucleotide ( AMBRA1 siRNA ) or unrelated oligonucleotides as a control ( Ctrl siRNA ) . Then , were co - transfected analysed as in a . is shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 044 .

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(c) HEK293 cells were transfected with AMBRA1 RNAi oligonucleotide (AMBRA1 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.044.


( d ) HEK293 were co - transfected with vectors encoding for ULK1 - HA , ULK1 - Myc AMBRA1WT or AMBRA1AA respectively . complexes were immunoprecipitated analysed as in a . is shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 014 .

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(d) HEK293 cells were co-transfected with vectors encoding for ULK1-HA, ULK1-Myc and AMBRA1WT or AMBRA1AA respectively. Protein complexes were immunoprecipitated and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.014.


( a ) Highly purified mTOR was incubated with radiolabelled ATP without substrate , with purified FLAG - AMBRA1 purified FLAG - AMBRA1S52A analysed by autoradiography . lower panels show a FLAG mTOR western blot ( WB ) .

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(a) Highly purified mTOR was incubated with radiolabelled ATP without substrate, with purified FLAG-AMBRA1 and purified FLAG-AMBRA1S52A and analysed by autoradiography. The lower panels show a FLAG and mTOR western blot (WB).


( b ) HEK293 expressing FLAG - RAPTOR were subjected to FLAG immunoprecipitation . complexes were analysed by western blotting using anti - FLAG anti - AMBRA1 antibodies .

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(b) HEK293 cells expressing FLAG-RAPTOR were subjected to FLAG immunoprecipitation. Protein complexes were analysed by western blotting using anti-FLAG and anti-AMBRA1 antibodies.


( c ) HEK293 were treated with Torin1 for 3 h or cultured in starvation medium for 2 h extracts were analysed by western blotting using anti - pSer - 52 anti - AMBRA1 antibodies .

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(c) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti-pSer-52 and anti-AMBRA1 antibodies.


( d ) HeLa stably interfered for AMBRA1 were transduced with indicated lentiviral vectors encoding for β - galactosidase , AMBRA1WT or AMBRA1S52A respectively some of them were treated with chloroquine for 1 h . extracts were analysed by western blotting using anti - LC3 , anti - p62 anti - actin antibodies . of LC3II p62 bands is shown below . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 038 .

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(d) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β-galactosidase, AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h. Protein extracts were analysed by western blotting using anti-LC3, anti-p62 and anti-actin antibodies. Quantification of LC3II and p62 bands is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.038.


( e ) GFP - LC3 MEF from wild - type ( Ambra1 + / + ) homozygous ( Ambra1gt / gt ) were transduced with indicated lentiviral vectors of LC3 per is shown on right . Bars represent mean ± s . e . m . of triplicate samples 50 analysed per sample . n = 3 independent experiments . Significance is P = 0 . 001 P = 0 . 003 . Scale bar , 10

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(e) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with indicated lentiviral vectors and quantification of LC3 puncta per cell is shown on the right. Bars represent mean±s.e.m. of triplicate samples and 50 cells analysed per sample. n = 3 independent experiments. Significance is P = 0.001 and P = 0.003. Scale bar, 10 μm


( f ) HeLa stably interfered for AMBRA1 were co - transfected with vectors encoding Ub - Lys - 63 - HA AMBRA1WT or AMBRA1S52A , respectively . extracts were immunoprecipitated using an anti - ULK1 antibody ubiquitin Lys - 63 - HA , ULK1 , AMBRA1 actin were analysed by western blotting .

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(f) HeLa cells stably interfered for AMBRA1 were co-transfected with vectors encoding Ub-Lys-63-HA and AMBRA1WT or AMBRA1S52A, respectively. Protein extracts were immunoprecipitated using an anti-ULK1 antibody and ubiquitin Lys-63-HA, ULK1, AMBRA1 and actin were analysed by western blotting.


( g ) HeLa stably interfered for AMBRA1 were transduced as in d ; extracts were analysed by western blotting using anti - AMBRA1 , anti - ATG13 , anti - pATG13 anti - actin antibodies . of p - ATG13 band is shown below .

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(g) HeLa cells stably interfered for AMBRA1 were transduced as in d; protein extracts were analysed by western blotting using anti-AMBRA1, anti-ATG13, anti-pATG13 and anti-actin antibodies. Quantification of the p-ATG13 band is shown below.


( h ) HeLa transfected with ULK1 - HA , ULK1 - Myc different AMBRA1 constructs , respectively . complexes were immunoprecipitated using anti - HA antibody analysed by western blotting using anti - HA , anti - Myc anti - AMBRA1 antibodies .

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(h) HeLa cells transfected with ULK1-HA, ULK1-Myc and different AMBRA1 constructs, respectively. Protein complexes were immunoprecipitated using anti-HA antibody and analysed by western blotting using anti-HA, anti-Myc and anti-AMBRA1 antibodies.


( B ) Phase - contrast of wild - type or PABPC4KO HeLa depleted of PABPC1 by siRNA - mediated knockdown . Scale bar , 100 μm . ( C ) Total numbers of viable in ( B ) were quantified with acridine orange propidium iodide graphed as a of wild - type HeLa . Error bars represent of three biological replicates .

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(B) Phase-contrast images of wild-type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA-mediated knockdown. Scale bar, 100 μm. (C) Total numbers of viable cells in (B) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild-type HeLa cells. Error bars represent the SEM of three biological replicates.


( C ) Phase - contrast of PABPDHFR , or PABPDHFR transfected with a plasmid coding for wild - type PABPC1 , 48 hours post - TMP removal . Scale bar , 100 μm .

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(C) Phase-contrast images of PABPDHFR cells, or PABPDHFR cells transfected with a plasmid coding for wild-type PABPC1, 48 hours post-TMP removal. Scale bar, 100 μm.


( D ) Western blot of lysates derived from imaged in ( C ) .

(D) Western blot analysis of lysates derived from cells imaged in (C).


( B ) SUnSET of PABPDHFR grown in presence or absence of TMP for 12 hours to maintain or deplete PABP . were subsequently pulsed with either puromycin or puromycin cycloheximide ( control ) , lysed equal amounts were resolved by SDS - PAGE . Western blot was performed using a monoclonal antibody against puromycin or actin ( loading control ) .

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(B) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP. Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide (control), lysed and equal protein amounts were resolved by SDS-PAGE. Western blot analysis was performed using a monoclonal antibody against puromycin or actin (loading control).


( D ) Scatter plot comparing log2 fold - changes [ ( - ) PABP vs ( + ) PABP ] in polysome - associated mRNA ( y - axis ) to corresponding changes in total mRNA . Transcripts identified as regulated via altered translation efficiency ( orange red ) or abundance ( green ) according to anota2seq are visualized together with non - regulated transcripts ( grey ) .

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(D) Scatter plot comparing log2 fold-changes [(-) PABP vs (+) PABP] in polysome-associated mRNA (y-axis) to corresponding changes in total cytoplasmic mRNA. Transcripts identified as regulated via altered translation efficiency (orange and red) or abundance (light and dark green) according to anota2seq are visualized together with non-regulated transcripts (grey).