Immunofluorescence of c - Jun ( green ) , CD1a or CD1c or CD14 ( red ) and DAPI in psoriatic lesions . Shown is a representative image ( Magnification 25x , Scale Bar : 100µm ) with an Inset ( Magnification 63x , Scale Bar : 20 µm , Deconvoluted ) . Arrows indicate c - Jun + DCs . Asterisk highlights a DC with prominent c - Jun expression as shown enlarged in 2 white - framed windows ( DC Marker ( red ) + c - Jun ( green , left ) or DAPI ( blue , right ) ) .

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Immunofluorescence of c-Jun (green), CD1a or CD1c or CD14 (red) and DAPI in psoriatic lesions. Shown is a representative image (Magnification 25x, Scale Bar: 100µm) with an Inset (Magnification 63x, Scale Bar: 20 µm, Deconvoluted). Arrows indicate c-Jun+ DCs. Asterisk highlights a DC with prominent c-Jun expression as shown enlarged in 2 white-framed windows (DC Marker (red) + c-Jun (green, left) or DAPI (blue, right)).


D - E . Representative immunofluorescence of c - Jun ( green ) , CD1a ( red ) , CCL2 ( D ) or Il23p19 ( E ) ( white ) and DAPI in psoriatic lesions . Arrows indicate triple - positive cells and an asterisk highlights a representative one that is shown enlarged in a white - framed inlet . Magnification : 40x . Scale bar : 50 µm . ( n = 2 patient samples ) .

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D-E. Representative immunofluorescence of c-Jun (green), CD1a (red), CCL2 (D) or Il23p19 (E) (white) and DAPI in psoriatic lesions. Arrows indicate triple-positive cells and an asterisk highlights a representative one that is shown enlarged in a white-framed inlet. Magnification: 40x. Scale bar: 50 µm. (n = 2 patient samples).


B - C Human mo - DCs pre - treated with JNKi ( SP600125 , 25µM ) ( B ) or AP - 1 Inhibitor ( T - 5224 , 20 µM ) ( C ) for 1h were stimulated with R848 ( 10 µg / ml ) . CCL2 and IL - 23 were analyzed by ELISA . ( n = 5 - 11 ; 2 - 3 independent experiments ) . Data information : Data are shown as mean ± SEM . P ‐ values were calculated by paired , two - tailed t - test ( B , C , Statistical significance : ns > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . See Appendix Table S3 for exact P ‐ values .

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B-C Human mo-DCs pre-treated with JNKi (SP600125, 25µM) (B) or AP-1 Inhibitor (T-5224, 20 µM) (C) for 1h were stimulated with R848 (10 µg/ml). CCL2 and IL-23 were analyzed by ELISA. (n = 5-11; 2-3 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by paired, two-tailed t-test (B, C, Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S3 for exact P‐values.


E - F Human moDCs described in ( D ) were analyzed for mean fluorescence intensity of CD80 or CD86 by flow cytometry ( E ) and CCL2 or IL - 23 protein by ELISA ( F ) . ( n = 7 ; 2 independent experiments ) . Data information : Data are shown as mean ± SEM . P ‐ values were calculated by paired , two - tailed t - test E and F ) . Statistical significance : ns > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . See Appendix Table S3 for exact P ‐ values .

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E-F Human moDCs described in (D) were analyzed for mean fluorescence intensity of CD80 or CD86 by flow cytometry (E) and CCL2 or IL-23 protein by ELISA (F). (n = 7; 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by paired, two-tailed t-test E and F). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S3 for exact P‐values.


B . Western blot analysis of hnRNP R in three independent HNRNPR wildtype ( + / + ) , heterozygous ( + / - ) and homozygous ( - / - ) HeLa cell lines .

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B. Western blot analysis of hnRNP R in three independent HNRNPR wildtype (+/+), heterozygous (+/-) and homozygous (-/-) HeLa cell lines.


C . Confocal microscopy images of nuclei stained with DAPI . Scale bar : 10 µm . D . Quantification of the DAPI area in ( C ) . Data are mean with standard deviation ( SD ) ; * * * P ≤ 0 . 001 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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C. Confocal microscopy images of nuclei stained with DAPI. Scale bar: 10 µm. D. Quantification of the DAPI area in (C). Data are mean with standard deviation (SD); ***P ≤ 0.001; unpaired two-tailed t-test (n = 3 biological replicates).


E . RNA FISH with a Cy3 - labeled oligo - dT probe . DAPI was used for nuclear staining . Scale bar : 10 µm . F . Quantification of mean fluorescence intensity of the Cy3 oligo - dT signal in ( E ) . Data are mean with SD ; * * * * P ≤ 0 . 0001 ; Mann - Whitney test ( n = 129 cells for HNRNPR + / + and n = 176 cells for HNRNPR - / - ) .

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E. RNA FISH with a Cy3-labeled oligo-dT probe. DAPI was used for nuclear staining. Scale bar: 10 µm. F. Quantification of mean fluorescence intensity of the Cy3 oligo-dT signal in (E). Data are mean with SD; ****P ≤ 0.0001; Mann-Whitney test (n = 129 cells for HNRNPR+/+ and n = 176 cells for HNRNPR-/-).


A . Western blot analysis of Ser2 - phosphorylated and total RNA pol II , Cyclin T1 , CDK9 and GAPDH protein levels .

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A. Western blot analysis of Ser2-phosphorylated and total RNA pol II, Cyclin T1, CDK9 and GAPDH protein levels.


B . Quantification of relative expression of Ser2 - phosphorylated and total RNA pol II , Cyclin T1 and CDK9 in ( A ) . Data are mean with SD ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . not significant ; unpaired two - tailed t - tests ( n = 3 biological replicates ) .

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B. Quantification of relative expression of Ser2-phosphorylated and total RNA pol II, Cyclin T1 and CDK9 in (A). Data are mean with SD; **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; unpaired two-tailed t-tests (n = 3 biological replicates).


F . Western blot analysis of subcellular fractions for Calnexin , GAPDH , histone H3 , Ser2 - phosphorylated and total RNA pol II , Cyclin T1 and CDK9 . Cyt , cytosol ; nuc + org , nuclear soluble proteins and organelles ; chr , chromatin - associated proteins . G . Quantification of relative expression of Ser2 - phosphorylated and total RNA pol II , Cyclin T1 and CDK9 in subcellular fractions in ( F ) . Data are mean with SD ; * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . not significant ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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F. Western blot analysis of subcellular fractions for Calnexin, GAPDH, histone H3, Ser2-phosphorylated and total RNA pol II, Cyclin T1 and CDK9. Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins. G. Quantification of relative expression of Ser2-phosphorylated and total RNA pol II, Cyclin T1 and CDK9 in subcellular fractions in (F). Data are mean with SD; *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


A . Western blot analysis of the indicated proteins in the input ( In ) lysates used for co - immunoprecipitation . B . Western blot analysis of Cyclin T1 , HEXIM1 , CDK9 and hnRNP A1 co - immunoprecipitated by an anti - LARP7 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control . C . Western blot analysis of Cyclin T1 , HEXIM1 , CDK9 , hnRNP A1 and LARP7 co - immunoprecipitated by an anti - MePCE antibody . Immunoprecipitation with rabbit - IgG antibody was used as control . D . Quantification of CDK9 , Cyclin T1 and HEXIM1 co - immunoprecipitating with LARP7 in ( B ) . Data are mean with SD ; * * P ≤ 0 . 01 , n . s . not significant ; unpaired two - tailed t - tests ( n = 3 biological replicates ) . E . Quantification of CDK9 , Cyclin T1 and HEXIM1 co - immunoprecipitating with MePCE in ( C ) . Data are mean with SD ; * P ≤ 0 . 05 , n . s . not significant ; unpaired two - tailed t - tests ( n = 3 biological replicates ) . F . Quantification of hnRNP A1 co - immunoprecipitating with LARP7 and MePCE in ( B ) and ( C ) . Data are mean with SD ; * P ≤ 0 . 05 ; unpaired two - tailed t - tests ( n = 3 biological replicates ) .

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A. Western blot analysis of the indicated proteins in the input (In) lysates used for co-immunoprecipitation. B. Western blot analysis of Cyclin T1, HEXIM1, CDK9 and hnRNP A1 co-immunoprecipitated by an anti-LARP7 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control. C. Western blot analysis of Cyclin T1, HEXIM1, CDK9, hnRNP A1 and LARP7 co-immunoprecipitated by an anti-MePCE antibody. Immunoprecipitation with rabbit-IgG antibody was used as control. D. Quantification of CDK9, Cyclin T1 and HEXIM1 co-immunoprecipitating with LARP7 in (B). Data are mean with SD; **P ≤ 0.01, n.s. not significant; unpaired two-tailed t-tests (n = 3 biological replicates). E. Quantification of CDK9, Cyclin T1 and HEXIM1 co-immunoprecipitating with MePCE in (C). Data are mean with SD; *P ≤ 0.05, n.s. not significant; unpaired two-tailed t-tests (n = 3 biological replicates). F. Quantification of hnRNP A1 co-immunoprecipitating with LARP7 and MePCE in (B) and (C). Data are mean with SD; *P ≤ 0.05; unpaired two-tailed t-tests (n = 3 biological replicates).


G . Western blot analysis of subcellular fractions Calnexin , GAPDH , histone H3 , hnRNP R and hnRNP A1 . Cyt , cytosol ; nuc + org , nuclear soluble proteins and organelles ; chr , chromatin - associated proteins .

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G. Western blot analysis of subcellular fractions Calnexin, GAPDH, histone H3, hnRNP R and hnRNP A1. Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins.


H . Western blot analysis of hnRNP R co - immunoprecipitated by an anti - hnRNP A1 antibody from subcellular fractions . Immunoprecipitation with mouse - IgG antibody was used as control .

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H. Western blot analysis of hnRNP R co-immunoprecipitated by an anti-hnRNP A1 antibody from subcellular fractions. Immunoprecipitation with mouse-IgG antibody was used as control.


I . Quantification of 7SK RNA in subcellular fractions by qPCR . Data are mean with SD ; * * P ≤ 0 . 01 , n . s . not significant ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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I. Quantification of 7SK RNA in subcellular fractions by qPCR. Data are mean with SD; **P ≤ 0.01, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


J . Quantification of 7SK RNA co - immunoprecipitating with hnRNP A1 from subcellular fractions . Data are mean with SD ; * P ≤ 0 . 05 , n . s . not significant ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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J. Quantification of 7SK RNA co-immunoprecipitating with hnRNP A1 from subcellular fractions. Data are mean with SD; *P ≤ 0.05, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


A . Western blot analysis of BRD4 , hnRNP R , Cyclin T1 , CDK9 , α - Tubulin and histone H3 in the input lysates used for co - immunoprecipitation .

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A. Western blot analysis of BRD4, hnRNP R, Cyclin T1, CDK9, α-Tubulin and histone H3 in the input lysates used for co-immunoprecipitation.


B . Western blot analysis of hnRNP R , Cyclin T1 , CDK9 and histone H3 co - immunoprecipitated by an anti - BRD4 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control .

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B. Western blot analysis of hnRNP R, Cyclin T1, CDK9 and histone H3 co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.


C . Quantification of CDK9 co - immunoprecipitating with BRD4 in ( B ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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C. Quantification of CDK9 co-immunoprecipitating with BRD4 in (B). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).


D . Western blot analysis of BRD4 ( long and short exposure ) co - immunoprecipitated by an anti - CDK9 antibody . Immunoprecipitation with mouse - IgG antibody was used as control .

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D. Western blot analysis of BRD4 (long and short exposure) co-immunoprecipitated by an anti-CDK9 antibody. Immunoprecipitation with mouse-IgG antibody was used as control.


E . Quantification of BRD4 co - immunoprecipitating with CDK9 in ( D ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

E. Quantification of BRD4 co-immunoprecipitating with CDK9 in (D). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).


F . Quantification of histone H3 co - immunoprecipitating with BRD4 in ( B ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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F. Quantification of histone H3 co-immunoprecipitating with BRD4 in (B). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).


G . Western blot analysis of BRD4 , hnRNP R , Cyclin T1 , CDK9 , α - Tubulin and histone H3 in the input lysates used for co - immunoprecipitation . Lysates were pre - treated with RNase as indicated .

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G. Western blot analysis of BRD4, hnRNP R, Cyclin T1, CDK9, α-Tubulin and histone H3 in the input lysates used for co-immunoprecipitation. Lysates were pre-treated with RNase as indicated.


H . Western blot analysis of hnRNP R , Cyclin T1 , CDK9 and histone H3 co - immunoprecipitated by an anti - BRD4 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control .

H. Western blot analysis of hnRNP R, Cyclin T1, CDK9 and histone H3 co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.


I . Western blot analysis of BRD4 , hnRNP R , CDK9 and α - Tubulin in the input lysates used for co - immunoprecipitation .

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I. Western blot analysis of BRD4, hnRNP R, CDK9 and α-Tubulin in the input lysates used for co-immunoprecipitation.


J . Western blot analysis of hnRNP R and CDK9 co - immunoprecipitated by an anti - BRD4 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control . K . Quantification of CDK9 co - immunoprecipitating with BRD4 in ( J ) . Data are mean with SD ; * * * * P ≤ 0 . 0001 ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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J. Western blot analysis of hnRNP R and CDK9 co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control. K. Quantification of CDK9 co-immunoprecipitating with BRD4 in (J). Data are mean with SD; ****P ≤ 0.0001; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


L . Western blot analysis of phospho - CDK9 ( Thr186 ) co - immunoprecipitated by an anti - BRD4 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control . M . Quantification of phospho - CDK9 ( Thr186 ) co - immunoprecipitating with BRD4 in ( L ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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L. Western blot analysis of phospho-CDK9(Thr186) co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control. M. Quantification of phospho-CDK9(Thr186) co-immunoprecipitating with BRD4 in (L). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).


A , B . Western blot analysis of Ser2 - phosphorylated and total RNA pol II levels in HNRNPR + / + ( A ) and - / - ( B ) cells treated with DMSO or CHX for the indicated durations . GAPDH and α - Tubulin served as loading control .

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A, B. Western blot analysis of Ser2-phosphorylated and total RNA pol II levels in HNRNPR+/+ (A) and -/- (B) cells treated with DMSO or CHX for the indicated durations. GAPDH and α-Tubulin served as loading control.


C . Quantification of relative expression levels of Ser2 - phosphorylated and total RNA pol II in ( A , B ) . Data are mean with SD ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ; unpaired two - tailed t - tests ( n = 4 biological replicates ) .

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C. Quantification of relative expression levels of Ser2-phosphorylated and total RNA pol II in (A, B). Data are mean with SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; unpaired two-tailed t-tests (n = 4 biological replicates).


D , Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Calnexin , GAPDH and histone H3 ( D ) Cyt , cytosol ; nuc + org , nuclear soluble proteins and organelles ; chr , chromatin - associated proteins .

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D, Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Calnexin, GAPDH and histone H3 (D) Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins.


E . Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Cyclin T1 and CDK9 ( E ) . Cyt , cytosol ; nuc + org , nuclear soluble proteins and organelles ; chr , chromatin - associated proteins . F . Quantification of relative expression of Cyclin T1 and CDK9 protein levels in subcellular fractions in ( E ) . Data are mean with SD ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . not significant ; one - way ANOVA with Tukey ' s multiple comparisons test ( n = 3 biological replicates ) .

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E. Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Cyclin T1 and CDK9 (E). Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins. F. Quantification of relative expression of Cyclin T1 and CDK9 protein levels in subcellular fractions in (E). Data are mean with SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; one-way ANOVA with Tukey's multiple comparisons test (n = 3 biological replicates).


G . Western blot analysis of Cyclin T1 levels in HNRNPR + / + and - / - cells treated with DMSO or MG132 . α - Tubulin served as loading control . H . Quantification of relative expression levels of Cyclin T1 in ( G ) . Data are mean with SD ; * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . not significant ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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G. Western blot analysis of Cyclin T1 levels in HNRNPR+/+ and -/- cells treated with DMSO or MG132. α-Tubulin served as loading control. H. Quantification of relative expression levels of Cyclin T1 in (G). Data are mean with SD; ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


B . Western blot analysis of Cyclin K , CDK9 and α - Tubulin in the input lysates used for co - immunoprecipitation .

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B. Western blot analysis of Cyclin K, CDK9 and α-Tubulin in the input lysates used for co-immunoprecipitation.


C . Western blot analysis of Cyclin K co - immunoprecipitated by an anti - CDK9 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control .

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C. Western blot analysis of Cyclin K co-immunoprecipitated by an anti-CDK9 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.


D . Quantification of Cyclin K co - immunoprecipitating with CDK9 in ( C ) . Data are mean with SD ; * P ≤ 0 . 05 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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D. Quantification of Cyclin K co-immunoprecipitating with CDK9 in (C). Data are mean with SD; *P ≤ 0.05; unpaired two-tailed t-test (n = 3 biological replicates).


Effects of ubiquitin surface mutants based on the UbcH7 - Ub : R0RBR : pUb model for the ubiquitin loading and off - loading potential of pParkinCH ( pParkinC431S , H433A ) . Coomassie stained gels ( top ) depict the formation of a pParkinCH ~ Ub oxyester intermediate after 60 minutes in the absence and presence of pUb using the indicated ubiquitin species . Anti - HA blots ( bottom ) depict the ubiquitination of HA - tagged Miro1181 - 579 by pParkin after 10 minutes in the absence and presence of pUb using the indicated ubiquitin species . The E24A substitution in Ub is a negative control as this residue faces away from the interface with parkin .

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Effects of ubiquitin surface mutants based on the UbcH7-Ub:R0RBR:pUb model for the ubiquitin loading and off-loading potential of pParkinCH (pParkinC431S,H433A). Coomassie stained gels (top) depict the formation of a pParkinCH ~ Ub oxyester intermediate after 60 minutes in the absence and presence of pUb using the indicated ubiquitin species. Anti-HA blots (bottom) depict the ubiquitination of HA-tagged Miro1181-579 by pParkin after 10 minutes in the absence and presence of pUb using the indicated ubiquitin species. The E24A substitution in Ub is a negative control as this residue faces away from the interface with parkin.


Auto - ubiquitination assay for parkin using ARJP and non - ARJP substitutions in parkin observed near the interface with UbcH7 - Ub . Assays were done in the absence and presence of pUb . Experiments with PINK1 were done by treating parkin : pUb with PINK1 for 30 minutes prior to adding other reagents needed for ubiquitination . Assays were monitored using a DyLight - labelled Ub protein and measuring fluorescence at 800 nm .

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Auto-ubiquitination assay for parkin using ARJP and non-ARJP substitutions in parkin observed near the interface with UbcH7-Ub. Assays were done in the absence and presence of pUb. Experiments with PINK1 were done by treating parkin:pUb with PINK1 for 30 minutes prior to adding other reagents needed for ubiquitination. Assays were monitored using a DyLight-labelled Ub protein and measuring fluorescence at 800 nm.


Exposure of the catalytic Cys431 is parkin as assessed by reaction with a UbVS probe . The different stages of activation using parkin , parkin : pUb , pParkin : pUb and pParkin : pUb in combination with an isopeptide linked UbcH7 - Ub conjugate are indicated above each gel . Following addition of UbVS , samples were taken at the times indicated ( 0 - 10 minutes ) and visualized by SDS - PAGE . Relative percentages of pParkin and the pParkin - Ub adduct as a function of time . Intensity percentages were calculated as a function of total intensity of pParkin - Ub , parkin and UbVS / pUb bands . Error bars represent standard deviation from the average for duplicate measurements . Exposure of the catalytic Cys431 is R0RBR parkin as assessed by reaction with a UbVS probe . The different stages of activation using R0RBR , R0RBR : pUb , R0RBR : pUb : pUbl and R0RBR : pUb : pUbl in combination with an isopeptide linked UbcH7 - Ub conjugate are indicated above each gel . Following addition of UbVS , samples were taken at the times indicated ( 0 - 60 minutes ) and visualized by SDS - PAGE Relative percentages of R0RBR and the R0RBR - Ub adduct as a function of time . Intensity percentages were calculated as a function of total intensity of the R0RBR - Ub , R0RBR and UbVS / pUb / pUbl bands .

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Exposure of the catalytic Cys431 is parkin as assessed by reaction with a UbVS probe. The different stages of activation using parkin, parkin:pUb, pParkin:pUb and pParkin:pUb in combination with an isopeptide linked UbcH7-Ub conjugate are indicated above each gel. Following addition of UbVS, samples were taken at the times indicated (0-10 minutes) and visualized by SDS-PAGE.   Relative percentages of pParkin and the pParkin-Ub adduct as a function of time. Intensity percentages were calculated as a function of total intensity of pParkin -Ub, parkin and UbVS/pUb bands. Error bars represent standard deviation from the average for duplicate measurements.  Exposure of the catalytic Cys431 is R0RBR parkin as assessed by reaction with a UbVS probe. The different stages of activation using R0RBR, R0RBR:pUb, R0RBR:pUb:pUbl and R0RBR:pUb:pUbl in combination with an isopeptide linked UbcH7-Ub conjugate are indicated above each gel. Following addition of UbVS, samples were taken at the times indicated (0-60 minutes) and visualized by SDS-PAGE   Relative percentages of R0RBR and the R0RBR-Ub adduct as a function of time. Intensity percentages were calculated as a function of total intensity of the R0RBR-Ub, R0RBR and UbVS/pUb/pUbl bands.


. Co - immunoprecipitation analysis of the HOIP K784R mutant with SHARPIN and HOIL - 1L using total cell extracts of HEK293T cells transiently expressing Myc - HOIP wildtype ( WT ) , or Myc - HOIP - K784R with HOIL - 1L - HA and Flag - SHARPIN . Anti - Vinculin antibody used to monitor protein loading . Representative data shown from three independent experiments .

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. Co-immunoprecipitation analysis of the HOIP K784R mutant with SHARPIN and HOIL-1L using total cell extracts of HEK293T cells transiently expressing Myc-HOIP wildtype (WT), or Myc-HOIP-K784R with HOIL-1L-HA and Flag-SHARPIN. Anti-Vinculin antibody used to monitor protein loading. Representative data shown from three independent experiments.


. Immunoblotting to detect ubiquitination of NEMO in HEK293T cells transiently expressing Flag - NEMO , GFP - SHARPIN , and HOIL - 1L - HA with Myc - HOIP wildtype ( WT ) , Myc - HOIP K784R or Myc - HOIP C885A . Total cell lysates in denaturing conditions subjected to SDS - PAGE followed up by immunoblotting using antibodies as indicated . Anti - Vinculin antibody to monitor loading . Representative data from three independent experiments .

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. Immunoblotting to detect ubiquitination of NEMO in HEK293T cells transiently expressing Flag-NEMO, GFP-SHARPIN, and HOIL-1L-HA with Myc-HOIP wildtype (WT), Myc-HOIP K784R or Myc-HOIP C885A. Total cell lysates in denaturing conditions subjected to SDS-PAGE followed up by immunoblotting using antibodies as indicated. Anti-Vinculin antibody to monitor loading. Representative data from three independent experiments.


Immunoblotting to detect TNF - induced degradation and phosphorylation of IκB - α , or phosphorylation of IKK in immortalized Hoip + / + and HoipK778R / K778R MEFs treated with human TNF ( 20ng / ml ) for the indicated times . Immunoblots of anti - Vinculin antibody and anti - α - Tubulin antibody shown for monitoring loading amount . Representative data from three independent

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Immunoblotting to detect TNF-induced degradation and phosphorylation of IκB-α, or phosphorylation of IKK in immortalized Hoip+/+ and HoipK778R/K778R MEFs treated with human TNF (20ng/ml) for the indicated times. Immunoblots of anti-Vinculin antibody and anti-α-Tubulin antibody shown for monitoring loading amount. Representative data from three independent experiments


TNF - dependent induction of Caspase 3 activation in immortalized Hoip + / + or HoipK778R / K778R MEFs measured by using DEVD - AFC . MEFs treated with hTNF ( 100ng / ml ) and CHX ( 1µg / ml ) for 4 hours subjected to the Caspase 3 activity assays .

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TNF-dependent induction of Caspase 3 activation in immortalized Hoip+/+ or HoipK778R/K778R MEFs measured by using DEVD-AFC. MEFs treated with hTNF (100ng/ml) and CHX (1µg/ml) for 4 hours subjected to the Caspase 3 activity assays.


TNF - induced Caspase 8 activity in Hoip + / + or HoipK778R / K778R immortalized MEFs treated with hTNF ( 100ng / ml ) with or without Cycloheximide ( CHX ) ( 1µg / ml ) or z - VAD ( 20µM ) . Representative data from three independent experiments , n = 4 .

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TNF-induced Caspase 8 activity in Hoip+/+ or HoipK778R/K778R immortalized MEFs treated with hTNF (100ng/ml) with or without Cycloheximide (CHX) (1µg/ml) or z-VAD (20µM). Representative data from three independent experiments, n=4.


H & E staining , Keratin 14 ( KRT14 ) and Cleaved Caspase 3 immunostaining of dorsal skin sections from mice of the indicated genotypes at 4 or 8 weeks of age . Scale bars : 50 µm .

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H&E staining, Keratin 14 (KRT14) and Cleaved Caspase 3 immunostaining of dorsal skin sections from mice of the indicated genotypes at 4 or 8 weeks of age. Scale bars: 50 µm.


Immunostaining ( H & E , Keratin 14 ( KRT14 ) , Ly6G , F4 / 80 and Cleaved Caspase 3 ) of mouse dorsal skin sections of the indicated genotypes . Scale bars : 50 µm . C

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Immunostaining (H&E, Keratin 14 (KRT14), Ly6G, F4/80 and Cleaved Caspase 3) of mouse dorsal skin sections of the indicated genotypes. Scale bars: 50 µm. C


Immunoblotting to examine TNF - induced degradation and phosphorylation of IκB - α in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF ( 20ng / ml ) for the indicated times . Representative of three independent experiments . G

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Immunoblotting to examine TNF-induced degradation and phosphorylation of IκB-α in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF (20ng/ml) for the indicated times. Representative of three independent experiments. G


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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

O O O B-MEASURED_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 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

(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 .

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

(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 .

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

(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 .

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

(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 .

O 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

(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 .

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-MEASURED_VAR O O O O O O O O O O O

(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 .

O O O O B-CONTROLLED_VAR O 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 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 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 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 .

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

(G) 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 ) .

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

(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 .

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

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 .

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

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 .

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

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 .

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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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 . 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.