Immunoblotting of TNF - induced cleavage of PARP and caspase 3 in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF ( 100ng / ml ) with or without CHX ( 1µg / ml ) for indicated times . Representative of three independent experiments . H

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Immunoblotting of TNF-induced cleavage of PARP and caspase 3 in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF (100ng/ml) with or without CHX (1µg/ml) for indicated times. Representative of three independent experiments. H


TNF - dependent induction of Caspase 3 activation in immortalized MEFs of the indicated genotype measured by DEVD - AFC . MEFs treated with hTNF ( 100ng / ml ) alone , or with CHX ( 1µg / ml ) for 4 hours ( H ) or 2 hours ( I ) subjected to the Caspase 3 activity assays . A representative data of two independent experiments , n = 4 .

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TNF-dependent induction of Caspase 3 activation in immortalized MEFs of the indicated genotype measured by DEVD-AFC. MEFs treated with hTNF (100ng/ml) alone, or with CHX (1µg/ml) for 4 hours (H) or 2 hours (I) subjected to the Caspase 3 activity assays. A representative data of two independent experiments, n=4.


TNF - induced Caspase 8 activity in immortalized MEFs of the indicated genotype . Luminol - dependent activity of Caspase 8 in immortalized MEFs treated with hTNF ( 100ng / ml ) with or without CHX ( 1µg / ml ) . A representative data of three independent experiments , n = 4 .

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TNF-induced Caspase 8 activity in immortalized MEFs of the indicated genotype. Luminol-dependent activity of Caspase 8 in immortalized MEFs treated with hTNF (100ng/ml) with or without CHX (1µg/ml) . A representative data of three independent experiments, n=4.


( A ) Representative images of GFP ‐ LC3 staining in HCT116 XIAP WT and XIAP KO cells with stable expression of GFP ‐ LC3 . Quantification of LC3 punctate cells was shown on the right . The data are represented as means ± s . d . of three independent experiments .

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(A) Representative images of GFP‐LC3 staining in HCT116 XIAP WT and XIAP KO cells with stable expression of GFP‐LC3. Quantification of LC3 punctate cells was shown on the right. The data are represented as means±s.d. of three independent experiments.


( B ) Endogenous LC3 expression in HCT116XIAP WT and XIAP KO cells was analysed by western blotting with anti ‐ LC3 antibody . The data are representative of three biological replicates . The ratio of LC3II / LC3I to actin is presented in Supplementary Figure S1A .

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(B) Endogenous LC3 expression in HCT116XIAP WT and XIAP KO cells was analysed by western blotting with anti‐LC3 antibody. The data are representative of three biological replicates. The ratio of LC3II/LC3I to actin is presented in Supplementary Figure S1A.


( C ) Lysates from XIAP + / + and XIAP − / − mouse embryonic fibroblasts were analysed by western blotting with the indicated antibodies . The data are representative of two biological replicates .

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(C) Lysates from XIAP+/+ and XIAP−/− mouse embryonic fibroblasts were analysed by western blotting with the indicated antibodies. The data are representative of two biological replicates.


( D ) Autophagic vesicles in HCT116 XIAP WT and XIAP KO cells were evaluated by electron microscopy . The images are representative of three biological replicates .

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(D) Autophagic vesicles in HCT116 XIAP WT and XIAP KO cells were evaluated by electron microscopy. The images are representative of three biological replicates.


( E ) HCT116 cells were treated with Embelin as indicated . LC3 ‐ II accumulation was determined by western blot analysis with anti ‐ LC3 antibody . The data are representative of three biological replicates . The ratio of LCII / LC3I to actin is presented in Supplementary Figure S1B .

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(E) HCT116 cells were treated with Embelin as indicated. LC3‐II accumulation was determined by western blot analysis with anti‐LC3 antibody. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1B.


( F ) HCT116 XIAP KO cells were treated with Embelin as indicated . LC3 ‐ II accumulation was determined by western blot analysis with anti ‐ LC3 antibody . The data are representative of three biological replicates .

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(F) HCT116 XIAP KO cells were treated with Embelin as indicated. LC3‐II accumulation was determined by western blot analysis with anti‐LC3 antibody. The data are representative of three biological replicates.


( G ) HCT116 cells were treated with Leupeptin and Embelin in the indicated combinations . 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 S1D .

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(G) HCT116 cells were treated with Leupeptin and Embelin in the indicated combinations. 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 S1D.


( H ) MEF ATG5 WT and ATG5 KO cells were individually transfected with XIAP ‐ specific or control siRNAs . Forty ‐ eight hours after transfection , cell lysates were analysed by western blotting with the indicated antibodies . The data are representative of three biological replicates . The ratio of LCII / LC3I to actin is presented in Supplementary Figure S1E .

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(H) MEF ATG5 WT and ATG5 KO cells were individually transfected with XIAP‐specific or control siRNAs. Forty‐eight hours after transfection, cell lysates were analysed by western blotting with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1E.


( I ) HCT116XIAP WT and XIAP KO cells were treated with 20 ‐ μM Z ‐ VAD ‐ FMK or DMSO for 2 h . Cell lysates were then analysed by western blotting . The data are representative of three biological replicates . The ratio of LCII / LC3I to actin is presented in Supplementary Figure S1F .

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(I) HCT116XIAP WT and XIAP KO cells were treated with 20 ‐μM Z‐VAD‐FMK or DMSO for 2 h. Cell lysates were then analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1F.


( J ) HCT116 XIAP KO cells were transfected with either Flag ‐ XIAP ( D148A / W310A ) or control plasmid . Twenty ‐ four hours after transfection , cell lysates were subjected to western blot analysis with the indicated antibodies . The data are representative of three biological replicates . The ratio of LCII / LC3I to actin is presented in Supplementary Figure S1G .

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(J) HCT116 XIAP KO cells were transfected with either Flag‐XIAP (D148A/W310A) or control plasmid. Twenty‐four hours after transfection, cell lysates were subjected to western blot analysis with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1G.


( A ) A549 and IMR90 cells were transfected with XIAP ‐ specific or control siRNAs . Cell lysates were subjected to western blot analysis with the indicated antibodies . The data are representative of two biological replicates . The ratio of LCII / LC3I to actin is presented in Supplementary Figure S2A .

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(A) A549 and IMR90 cells were transfected with XIAP‐specific or control siRNAs. Cell lysates were subjected to western blot analysis with the indicated antibodies. The data are representative of two biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2A.


( B ) HCT116 cells expressing XIAP ‐ specific or control siRNAs , HCT116 XIAP WT and XIAP KO cells were individually transfected with Flag ‐ XIAP or control vector . 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 S2D .

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(B) HCT116 cells expressing XIAP‐specific or control siRNAs, HCT116 XIAP WT and XIAP KO cells were individually transfected with Flag‐XIAP or control vector. 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 S2D.


( C ) HCT116 XIAP WT and XIAP KO cells were individually transfected with Flag ‐ XIAP or control vector . Cytoplasmic / nuclear fractionation was performed to analyse cellular localization of Mdm2 and p53 . PARP and GAPDH were used as nuclear ( N ) and cytoplasmic ( C ) fraction markers , respectively . The data are representative of three biological replicates .

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(C) HCT116 XIAP WT and XIAP KO cells were individually transfected with Flag‐XIAP or control vector. Cytoplasmic/nuclear fractionation was performed to analyse cellular localization of Mdm2 and p53. PARP and GAPDH were used as nuclear (N) and cytoplasmic (C) fraction markers, respectively. The data are representative of three biological replicates.


( D ) HCT116 p53 + / + and p53 − / − cells were individually transfected with XIAP ‐ specific or control siRNAs . LC3 conversion was detected by western blot analysis . The data are representative of three biological replicates . The ratio of LCII / LC3I to actin is presented in Supplementary Figure S2E .

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

(D) HCT116 p53+/+ and p53−/− cells were individually transfected with XIAP‐specific or control siRNAs. LC3 conversion was detected by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2E.


( E ) HCT116 p53 + / + and p53 − / − cells were individually transfected with Flag ‐ XIAP or control vector . LC3 conversion was detected by western blot analysis . The data are representative of three biological replicates . The ratio of LCII / LC3I to actin is presented in Supplementary Figure S2F .

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(E) HCT116 p53+/+ and p53−/− cells were individually transfected with Flag‐XIAP or control vector. LC3 conversion was detected by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2F.


( F ) HCT116 XIAP WT and XIAP KO cells treated with 10 μM Nutlin3 for 6 h . 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 S2H .

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(F) HCT116 XIAP WT and XIAP KO cells treated with 10 μM Nutlin3 for 6 h. 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 S2H.


( G ) HCT116 XIAP KO cells were treated with the indicated amounts of Nutlin for 8 h . Cell lysates were analysed by western blotting with anti ‐ LC3 antibody . The data are representative of three biological replicates .

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(G) HCT116 XIAP KO cells were treated with the indicated amounts of Nutlin for 8 h. Cell lysates were analysed by western blotting with anti‐LC3 antibody. The data are representative of three biological replicates.


( A ) Lysates from HCT116 cells were immunoprecipitated separately with anti ‐ XIAP antibody and an isotype ‐ matched control IgG . Immunoprecipitates and input were analysed by western blotting . The data are representative of two biological replicates .

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(A) Lysates from HCT116 cells were immunoprecipitated separately with anti‐XIAP antibody and an isotype‐matched control IgG. Immunoprecipitates and input were analysed by western blotting. The data are representative of two biological replicates.


( B ) Recombinant His ‐ XIAP protein was incubated separately with purified GST and GST ‐ Mdm2 fusion proteins on glutathione beads for 4 h followed by western blot analysis using anti ‐ His antibody . GST and GST ‐ Mdm2 were analysed by Coomassie blue staining . The data are representative of two biological replicates .

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(B) Recombinant His‐XIAP protein was incubated separately with purified GST and GST‐Mdm2 fusion proteins on glutathione beads for 4 h followed by western blot analysis using anti‐His antibody. GST and GST‐Mdm2 were analysed by Coomassie blue staining. The data are representative of two biological replicates.


( C ) HCT116 cells were treated with XIAP ‐ specific or control siRNAs . Twenty ‐ four hours later , cells were cultured in the presence of 50 μg ml − 1 cycloheximide for the indicated periods of time , and subsequently analysed by western blotting . We should mention that amounts of cell lysates were adjusted to achieve similar expression levels of Mdm2 at time 0 , while the same amounts of cell lysates were used to examine levels of XIAP and actin . The data are representative of three biological replicates . The ratio of Mdm2 to actin is presented in Supplementary Figure S3B .

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(C) HCT116 cells were treated with XIAP‐specific or control siRNAs. Twenty‐four hours later, cells were cultured in the presence of 50 μg ml−1 cycloheximide for the indicated periods of time, and subsequently analysed by western blotting. We should mention that amounts of cell lysates were adjusted to achieve similar expression levels of Mdm2 at time 0, while the same amounts of cell lysates were used to examine levels of XIAP and actin. The data are representative of three biological replicates. The ratio of Mdm2 to actin is presented in Supplementary Figure S3B.


( D ) p53 − / − Mdm2 − / − MEF cells were co ‐ transfected with the indicated Mdm2 , XIAP , and p53 constructs . Cell lysates were analysed by western blotting with the indicated antibodies . We should mention that XIAP H467A expressing plasmid was always used less than XIAP expressing plasmid to ensure expression levels of XIAP and XIAP H467A were similar . The data are representative of three biological replicates .

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(D) p53−/−Mdm2−/− MEF cells were co‐transfected with the indicated Mdm2, XIAP, and p53 constructs. Cell lysates were analysed by western blotting with the indicated antibodies. We should mention that XIAP H467A expressing plasmid was always used less than XIAP expressing plasmid to ensure expression levels of XIAP and XIAP H467A were similar. The data are representative of three biological replicates.


( E ) p53 − / − Mdm2 − / − MEF cells were transfected with the indicated plasmids . Twenty ‐ four hours after transfection , cells were treated with 20 μM MG ‐ 132 for additional 4 h . Cell lysates were denatured before proteins conjugated to His ‐ ubiquitin were pulled down by Ni2 + ‐ NTA beads . The bead ‐ bound proteins and total cell lysates ( TCLs ) were analysed by western blot with anti ‐ Mdm2 antibody . The data are representative of three biological replicates .

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(E) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Twenty‐four hours after transfection, cells were treated with 20 μM MG‐132 for additional 4 h. Cell lysates were denatured before proteins conjugated to His‐ubiquitin were pulled down by Ni2+‐NTA beads. The bead‐bound proteins and total cell lysates (TCLs) were analysed by western blot with anti‐Mdm2 antibody. The data are representative of three biological replicates.


( F ) In all , 2 μM Mdm2 or its C464A mutant and 1 μM XIAP or its H467A mutant proteins were incubated with 100 nM E1 , 2 μM E2 and 200 μM Ub in a total 20 μlin vitro ubiquitination reaction buffer at 37 ° C for 1 h . The reaction mixtures were analysed by western blotting with anti ‐ Mdm2 antibody . The data are representative of three biological replicates .

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(F) In all, 2 μM Mdm2 or its C464A mutant and 1 μM XIAP or its H467A mutant proteins were incubated with 100 nM E1, 2 μM E2 and 200 μM Ub in a total 20 μlin vitro ubiquitination reaction buffer at 37°C for 1 h. The reaction mixtures were analysed by western blotting with anti‐Mdm2 antibody. The data are representative of three biological replicates.


( G ) p53 − / − Mdm2 − / − MEF cells were transfected with the indicated plasmids . Cell lysates were analysed by western blotting . The data are representative of three biological replicates .

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(G) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Cell lysates were analysed by western blotting. The data are representative of three biological replicates.


( H ) p53 − / − Mdm2 − / − MEF cells were transfected with the indicated plasmids . Twenty ‐ four hours after transfection , cells were treated with 20 μM MG ‐ 132 for additional 4 h . Cell lysates were denatured before proteins conjugated to His ‐ ubiquitin were pulled down by Ni2 + ‐ NTA beads . The bead ‐ bound proteins were analysed by western blotting with anti ‐ p53 antibody . The data are representative of three biological replicates .

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(H) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Twenty‐four hours after transfection, cells were treated with 20 μM MG‐132 for additional 4 h. Cell lysates were denatured before proteins conjugated to His‐ubiquitin were pulled down by Ni2+‐NTA beads. The bead‐bound proteins were analysed by western blotting with anti‐p53 antibody. The data are representative of three biological replicates.


( A ) HCT116 XIAP WT and XIAP KO cells were treated with EBSS for the indicated periods of time . GFP ‐ LC3 puncta formation was observed by a microscope . Quantification of LC3 punctate cells was shown on the right . The data are represented as means ± s . d . of three independent experiments .

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(A) HCT116 XIAP WT and XIAP KO cells were treated with EBSS for the indicated periods of time. GFP‐LC3 puncta formation was observed by a microscope. Quantification of LC3 punctate cells was shown on the right. The data are represented as means±s.d. of three independent experiments.


( B ) HCT116 XIAP WT and XIAP KO cells were treated with EBSS for 4 h followed by western blot analysis with the indicated antibodies . The data are representative of three biological replicates . The ratio of LCII / LC3I to actin is presented in Supplementary Figure S6B .

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(B) HCT116 XIAP WT and XIAP KO cells were treated with EBSS for 4 h followed by western blot analysis with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6B.


( 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 .

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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 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 .

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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 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 .

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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 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 .

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(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 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 .

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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 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 ) .

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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 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 ) .

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

(D-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 ) 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 .

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

(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 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 .

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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 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 .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 ) .

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

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 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 .

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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 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 .

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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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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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 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 ) .

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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 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 .

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

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 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 .

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

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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

B. 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 . Cells were transfected with indicated constructs and IP and 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 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 cells expressing the indicated constructs were subjected to IP and WB using indicated antibodies . Presence of endogenous RanGAP and 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 and IP and WB analysis were performed to detect the 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 . 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 ) .

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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 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 .

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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 ) 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 ] .

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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 the 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 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 .

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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 the expression levels of indicated proteins using antibodies . a - tubulin was used as loading control .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 ) 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 .

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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 analysis . The 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 . 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 ) .

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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 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 ) .

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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 . 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 .

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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 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 .

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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.


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 .

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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 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 ) .

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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 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 ) .

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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 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 = * * * * ) .

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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 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 .

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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 , 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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(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.


( 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells together with ULK1WT or ULK1K46I Myc - tagged proteins . Protein 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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 cells were co - transfected with vectors encoding AMBRA1 , ULK1 - Myc and ATG13 - FLAG . Protein 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 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 .

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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 cells were transfected with AMBRA1 or the empty vector , as a control . ULK1 , AMBRA1 and 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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells were transfected as in a ; ATG13 , pATG13 , ULK1 , AMBRA1 and 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 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 .

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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 ) 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 .

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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 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 .

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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.