Figure 6A , B . Tables showing top 20 down - regulated ( A ) genes in Ahr - / - B cells as compared to Ahr + / + cells , after analysis by RNA sequencing . B cells were activated with 10 µg / ml α - IgM + 20 ng / ml IL - 4 for 8h ; 250 nM FICZ was added to the culture during the last 4h . The average read counts are directly proportional to the extent of expression of a given gene in Ahr + / + or Ahr - / - cells . Data from n = 3 mice per group . Average read counts for the housekeeping gene Hprt1 in both Ahr + / + and Ahr - / - B cells are indicated below the table in figure 6A . A , B . Tables showing top up - regulated ( B ) genes in Ahr - / - B cells as compared to Ahr + / + cells , after analysis by RNA sequencing . B cells were activated with 10 µg / ml α - IgM + 20 ng / ml IL - 4 for 8h ; 250 nM FICZ was added to the culture during the last 4h . The average read counts are directly proportional to the extent of expression of a given gene in Ahr + / + or Ahr - / - cells . Data from n = 3 mice per group . Average read counts for the housekeeping gene Hprt1 in both Ahr + / + and Ahr - / - B cells are indicated below the table in figure 6A . C . qPCR analysis of Ccno expression in splenic CD19 + cells isolated from non - immune Ahrfl / + mb1Cre + ( Ahr + / - B cells , black ) and Ahrfl / - mb1Cre + ( Ahr - / - B cells , white ) mice , stimulated with 10 µg / ml anti - IgM for the indicated time points . Ccno expression was normalized to Hprt1 . n = 3 independent experiments ; mean ± SEM ; two - way ANOVA , Sidak ' s multiple comparison test . D . qPCR analysis of Ccno expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 8h with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM . Ccno expression was normalized to Hprt1 . n = 3 independent experiments ; mean ± SEM ; one - way ANOVA , Tukey ' s multiple comparison test . E . qPCR analysis of Ccno expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for the indicated time points with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM in presence or absence of 250 nM FICZ or 1 μM 3 - MC . Ccno expression was normalized to Hprt1 . n = 3 independent experiments ; mean ± SEM ; one - way ANOVA , Tukey ' s multiple comparison test . F . Chromatin Immunoprecipitation ( ChIP ) analysis of AhR interaction with the Ccno promoter and an irrelevant region ( - 3 . 3 Kb from Ccno transcription starting site ) in Ahr + / + ( black ) and Ahr - / - ( white ) B cells 5h after activation with 10 µg / ml α - IgM + 20 ng / ml IL - 4 . 250 nM FICZ was added in the last hour of culture . Representative data of n = 2 independent experiments ; mean ± SEM ; two - way ANOVA , Sidak ' s multiple comparison test .

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

Figure 6A, B. Tables showing top 20 down-regulated (A) genes in Ahr-/- B cells as compared to Ahr+/+ cells, after analysis by RNA sequencing. B cells were activated with 10 µg/ml α-IgM + 20 ng/ml IL-4 for 8h; 250 nM FICZ was added to the culture during the last 4h. The average read counts are directly proportional to the extent of expression of a given gene in Ahr+/+ or Ahr-/- cells. Data from n = 3 mice per group. Average read counts for the housekeeping gene Hprt1 in both Ahr+/+ and Ahr-/- B cells are indicated below the table in figure 6A.A, B. Tables showing top up-regulated (B) genes in Ahr-/- B cells as compared to Ahr+/+ cells, after analysis by RNA sequencing. B cells were activated with 10 µg/ml α-IgM + 20 ng/ml IL-4 for 8h; 250 nM FICZ was added to the culture during the last 4h. The average read counts are directly proportional to the extent of expression of a given gene in Ahr+/+ or Ahr-/- cells. Data from n = 3 mice per group. Average read counts for the housekeeping gene Hprt1 in both Ahr+/+ and Ahr-/- B cells are indicated below the table in figure 6A.C. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from non-immune Ahrfl/+mb1Cre+ (Ahr+/- B cells, black) and Ahrfl/-mb1Cre+ (Ahr-/- B cells, white) mice, stimulated with 10 µg/ml anti-IgM for the indicated time points. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; two-way ANOVA, Sidak's multiple comparison test.D. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 8h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey's multiple comparison test.E. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for the indicated time points with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM in presence or absence of 250 nM FICZ or 1 μM 3-MC. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey's multiple comparison test.F. Chromatin Immunoprecipitation (ChIP) analysis of AhR interaction with the Ccno promoter and an irrelevant region (-3.3 Kb from Ccno transcription starting site) in Ahr+/+ (black) and Ahr-/- (white) B cells 5h after activation with 10 µg/ml α-IgM + 20 ng/ml IL-4. 250 nM FICZ was added in the last hour of culture. Representative data of n = 2 independent experiments; mean ± SEM; two-way ANOVA, Sidak's multiple comparison test.


figf1 ( a - c ) HeLa cells were either left untreated , amino - acid ( aa ) / FBS starved for 5 h , or starved and then recovered in amino - acid / FBS - containing medium , then immunostained , or immunoblotted using antibodies as shown . Co - localization panels show an overlap between mTOR and LAMP1 signals . Note that changes in the positioning of lysosomal mTOR ( quantified as the percentage of cells with predominantly peripheral localization of LAMP1 - positive vesicles ; a , c ) correlate with mTORC1 activity ( levels of phosphorylated S6K relative to the total S6K ; b ) . ( d ) Visualization of Akt activated in response to recovery after serum starvation . After nutrient recovery , LAMP1 - positive vesicles localize to peripheral regions with higher concentrations of phospho - Akt . DAPI , 4 , 6 − diamidino − 2 − phenylindole . For all panels , values are means ± s . e . m . of three independent experiments carried out in triplicate . * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - test ; other comparisons are not significant ( n . s . ) . Representative maximum - intensity projections of serial confocal optical sections are shown . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

figf1(a-c) HeLa cells were either left untreated, amino-acid (aa)/FBS starved for 5 h, or starved and then recovered in amino-acid/FBS-containing medium, then immunostained, or immunoblotted using antibodies as shown. Co-localization panels show an overlap between mTOR and LAMP1 signals. Note that changes in the positioning of lysosomal mTOR (quantified as the percentage of cells with predominantly peripheral localization of LAMP1-positive vesicles; a,c) correlate with mTORC1 activity (levels of phosphorylated S6K relative to the total S6K; b).(d) Visualization of Akt activated in response to recovery after serum starvation. After nutrient recovery, LAMP1-positive vesicles localize to peripheral regions with higher concentrations of phospho-Akt. DAPI, 4,6−diamidino−2−phenylindole. For all panels, values are means ± s.e.m. of three independent experiments carried out in triplicate. *P0.05, **P0.01, ***P0.005 Student's t-test; other comparisons are not significant (n.s.). Representative maximum-intensity projections of serial confocal optical sections are shown. Uncropped images of blots are shown in Supplementary Fig. S7.


figf2 ( a , b ) Nocodazole flattens the differences in lysosomal mTOR localization and dampens mTORC1 signalling in response to changes in nutrient availability . Cells were treated as in Fig . 1a followed by incubation with dimethylsulphoxide ( vehicle ) or with nocodazole during the last 2 h before fixation / lysis . Samples were analysed by immunofluorescence ( a ) or immunoblotting ( b ) . Quantification of phopho - S6K levels is shown in ( b ) . ( c - e ) Changes in lysosomal positioning induced by kinesin - or small GTPase - family members ( c , d ) correlate with changes in mTORC1 activity ( e ) . HeLa cells were transfected with overexpression constructs ( OE ) or with siRNA as shown , followed by immunofluorescence ( c , d ) or by immunoblotting ( e ) analyses . Values are means ± s . e . m . of three independent experiments carried out in triplicate . All comparisons are with the control within each treatment condition , * P0 . 05 , * * * P0 . 005 Student ' s t - test ; n . s . not significant . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

figf2(a,b) Nocodazole flattens the differences in lysosomal mTOR localization and dampens mTORC1 signalling in response to changes in nutrient availability. Cells were treated as in Fig. 1a followed by incubation with dimethylsulphoxide (vehicle) or with nocodazole during the last 2 h before fixation/lysis. Samples were analysed by immunofluorescence (a) or immunoblotting (b). Quantification of phopho-S6K levels is shown in (b).(c-e) Changes in lysosomal positioning induced by kinesin- or small GTPase-family members (c,d) correlate with changes in mTORC1 activity (e). HeLa cells were transfected with overexpression constructs (OE) or with siRNA as shown, followed by immunofluorescence (c,d) or by immunoblotting (e) analyses. Values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.


figf3 ( a - c ) HeLa cells transfected with ARL8B or KIF2 siRNA , or with ARL8B overexpression construct ( non - targeting siRNA and empty pcDNA vector used as transfection controls ) , were either left untreated , serum / amino - acid starved for 5 h , or starved and then recovered in amino - acid - and FBS - containing medium for 30 min . Cells were immunostained using LAMP1 antibody ( a ) and the percentage of cells with predominantly peripheral localization of LAMP1 - positive vesicles was quantified ( b ) or subjected to immunoblotting ( c ) using antibodies as shown . Quantification of phospho - S6K levels relative to the total S6K is shown in ( c ) . Values are means ± s . e . m . of three independent experiments carried out in triplicate . All comparisons are with the control within each treatment condition , * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - test ; n . s . not significant . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

figf3(a-c) HeLa cells transfected with ARL8B or KIF2 siRNA, or with ARL8B overexpression construct (non-targeting siRNA and empty pcDNA vector used as transfection controls), were either left untreated, serum/amino-acid starved for 5 h, or starved and then recovered in amino-acid- and FBS-containing medium for 30 min. Cells were immunostained using LAMP1 antibody (a) and the percentage of cells with predominantly peripheral localization of LAMP1-positive vesicles was quantified (b) or subjected to immunoblotting (c) using antibodies as shown. Quantification of phospho-S6K levels relative to the total S6K is shown in (c). Values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, **P0.01, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.


figf4 ( a ) Starvation increases pHi in HeLa cells allowed to starve using three different protocols ( see Methods ) with or without subsequent recovery . ( b - d ) Changing pHi from 7 . 1 to 7 . 7 is sufficient to affect localization of lysosomes and mTORC1 activity . pHi was titrated in full tissue - culture medium containing nigericin , which enables changes in pHi to be forced by altering pH in the medium , followed by immunostaining ( b , c ) or western blotting ( d ) using antibodies as shown . ( e ) Changes in lysosomal localization have no effect on pHi . ARL8B and KIF2 were overexpressed or knocked down in HeLa cells followed by pHi measurement . ( f , g ) Nutrients and pHi affect levels of ARL8 and KIF2 in lysosomal fractions . Protein levels and their quantification in total cellular lysates or in isolated lysosomal fractions from HeLa cells subjected to 1 h nutrient deprivation - recovery ( f ) or to 1 h changes of pHi in full tissue - culture medium containing nigericin ( g ) are shown . ( h , i ) Effect of nutrients and pHi on binding of ARL8 and KIF2 to polymerized microtubules . HeLa cells treated as in ( f ) and ( g ) , followed by isolation of polymerized microtubule fraction and western blotting . The asterisk indicates a nonspecific band . For all panels values are means ± s . e . m . of three independent experiments carried out in triplicate . All comparisons are with the control within each treatment condition , * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - test ; n . s . not significant . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

figf4(a) Starvation increases pHi in HeLa cells allowed to starve using three different protocols (see Methods) with or without subsequent recovery.(b-d) Changing pHi from 7.1 to 7.7 is sufficient to affect localization of lysosomes and mTORC1 activity. pHi was titrated in full tissue-culture medium containing nigericin, which enables changes in pHi to be forced by altering pH in the medium, followed by immunostaining (b,c) or western blotting (d) using antibodies as shown.(e) Changes in lysosomal localization have no effect on pHi. ARL8B and KIF2 were overexpressed or knocked down in HeLa cells followed by pHi measurement.(f,g) Nutrients and pHi affect levels of ARL8 and KIF2 in lysosomal fractions. Protein levels and their quantification in total cellular lysates or in isolated lysosomal fractions from HeLa cells subjected to 1 h nutrient deprivation-recovery (f) or to 1 h changes of pHi in full tissue-culture medium containing nigericin (g) are shown.(h,i) Effect of nutrients and pHi on binding of ARL8 and KIF2 to polymerized microtubules. HeLa cells treated as in (f) and (g), followed by isolation of polymerized microtubule fraction and western blotting. The asterisk indicates a nonspecific band. For all panels values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, **P0.01, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.


figf5 ( b ) Serum and amino - acid starvation of HeLa cells for 5 h reduces LC3 - II levels ( above ) , but increases autolysosome numbers detected using tfLC3 ( below ) . With tfLC3 , GFP - and RFP - positive puncta represent autophagosomes before lysosomal fusion , whereas RFP - positive / GFP - negative puncta represent autolysosomes - GFP is more rapidly quenched by low lysosomal pH ( see Methods ) . Increased autolysosomes indicate enhanced starvation - induced flux of LC3 to lysosomes . ( c ) ARL8B knockdown increases autophagosomal synthesis . siRNA - transfected HeLa cells were incubated for 48 h , then left untreated or incubated with bafilomycin A1 . LC3 - II levels versus actin were quantified ( bottom graphs ) . Asterisk : nonspecific band . ( d ) ARL8B overexpression inhibits autophagosome synthesis and degradation . HeLa cells overexpressing ARL8B or empty vector were analysed as in ( c ) . ( e ) HeLa cells were co - transfected with either ARL8B overexpression construct or siRNA ( non - targeting siRNA and empty peGFP vector were transfection controls ) together with mCherry - LC3 for 48 h . After fixation , cells were stained for endogenous LAMP1 and DNA ( DAPI ) . Representative maximum - intensity projections of serial confocal optical sections are shown . Co - localization panels show overlapping mCherry - LC3 and LAMP1 signals . ( f - h ) Quantification of autophagosome - lysosome fusion in HeLa cells . Percentages of autolysosomes ( positive for both mCherry - LC3 and LAMP1 ) to autophagosomes ( positive for mCherry - LC3 and negative for LAMP1 ) were quantified . In ( h ) , we analysed cells treated for 2 h with nocodazole before fixation , which dispersed the perinuclear lysosomal cluster ( see Fig . 2a ) . ( i ) Autophagosomal and autolysosomal numbers in tfLC3 - expressing cells after ARL8B overexpression and knockdown ( Supplementary Fig . S5o shows representative cells . ) . In ( f - i ) we analysed 20 cells per group in three independent experiments . Values are means ± s . e . m of three independent experiments carried out in triplicate . * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - test ; other comparisons not significant ( n . s . ) . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

figf5(b) Serum and amino-acid starvation of HeLa cells for 5 h reduces LC3-II levels (above), but increases autolysosome numbers detected using tfLC3 (below). With tfLC3, GFP- and RFP-positive puncta represent autophagosomes before lysosomal fusion, whereas RFP-positive/GFP-negative puncta represent autolysosomes-GFP is more rapidly quenched by low lysosomal pH (see Methods). Increased autolysosomes indicate enhanced starvation-induced flux of LC3 to lysosomes.(c) ARL8B knockdown increases autophagosomal synthesis. siRNA-transfected HeLa cells were incubated for 48 h, then left untreated or incubated with bafilomycin A1. LC3-II levels versus actin were quantified (bottom graphs). Asterisk: nonspecific band.(d) ARL8B overexpression inhibits autophagosome synthesis and degradation. HeLa cells overexpressing ARL8B or empty vector were analysed as in (c).(e) HeLa cells were co-transfected with either ARL8B overexpression construct or siRNA (non-targeting siRNA and empty peGFP vector were transfection controls) together with mCherry-LC3 for 48 h. After fixation, cells were stained for endogenous LAMP1 and DNA (DAPI). Representative maximum-intensity projections of serial confocal optical sections are shown. Co-localization panels show overlapping mCherry-LC3 and LAMP1 signals. (f-h) Quantification of autophagosome-lysosome fusion in HeLa cells. Percentages of autolysosomes (positive for both mCherry-LC3 and LAMP1) to autophagosomes (positive for mCherry-LC3 and negative for LAMP1) were quantified. In (h), we analysed cells treated for 2 h with nocodazole before fixation, which dispersed the perinuclear lysosomal cluster (see Fig. 2a). (i) Autophagosomal and autolysosomal numbers in tfLC3-expressing cells after ARL8B overexpression and knockdown (Supplementary Fig. S5o shows representative cells.). In (f-i) we analysed 20 cells per group in three independent experiments. Values are means ± s.e.m of three independent experiments carried out in triplicate. *P0.05, **P0.01, ***P0.005 Student's t-test; other comparisons not significant (n.s.). Uncropped images of blots are shown in Supplementary Fig. S7.


Figure 1A Live cell imaging reveals that expression of GFP ‐ KSHV ‐ TK , but not GFP induces contraction of HeLa cells ( see Supplementary Movies S1 and S2 ) . The time from the start of imaging is indicated above each panel , and the scale bars represent 18 μm . Quantification of GFP ‐ KSHV ‐ TK ‐ induced cell contraction . The graphs show cell area ( μm2 ) of cells expressing the indicated protein , and error bars represent SEM from three independent experiments in which n = 224 for GFP and n = 203 for GFP ‐ KSHV ‐ TK . * * * P 0 . 001 . B Phase images taken from Supplementary Movie S3 illustrating GFP ‐ KSHV ‐ TK induced blebbing of contracted cells . The time from the start of imaging is indicated above each panel , and the scale bars represent 18 μm . C Scanning electron microscopy of HeLa cells expressing GFP or GFP ‐ KSHV ‐ TK . D Immunofluorescence microscopy images illustrating the association of GFP ‐ KSHV ‐ TK , but not GFP , with actinstress fibres . Scale bar , 10 μm .

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

Figure 1A Live cell imaging reveals that expression of GFP‐KSHV‐TK, but not GFP induces contraction of HeLa cells (see Supplementary Movies S1 and S2). The time from the start of imaging is indicated above each panel, and the scale bars represent 18 μm. Quantification of GFP‐KSHV‐TK‐induced cell contraction. The graphs show cell area (μm2) of cells expressing the indicated protein, and error bars represent SEM from three independent experiments in which n = 224 for GFP and n = 203 for GFP‐KSHV‐TK. ***P 0.001.B Phase images taken from Supplementary Movie S3 illustrating GFP‐KSHV‐TK induced blebbing of contracted cells. The time from the start of imaging is indicated above each panel, and the scale bars represent 18 μm.C Scanning electron microscopy of HeLa cells expressing GFP or GFP‐KSHV‐TK.D Immunofluorescence microscopy images illustrating the association of GFP‐KSHV‐TK, but not GFP, with actinstress fibres. Scale bar, 10 μm.


Figure 2A Co ‐ expression of GFP ‐ RhoN19 but not GFP inhibits RFP ‐ KSHV ‐ TK ‐ induced cell contraction . Graphs represent cell area ( μm2 ) of KSHV ‐ TK , RhoN19 or KSHV ‐ TK and RhoN19 expressing cells . Error bars represent SEM from 3 independent experiments , n = 204 for KSHV ‐ TK , n = 223 for RhoN19 and n = 189 for KSHV ‐ TK and RhoN19 . B GFP ‐ KSHV ‐ TK ‐ induced cell contraction does not occur in cells treated with the ROCK and myosin II inhibitors Y ‐ 27632 and blebbistatin , respectively . Error bars represent SEM from three independent experiments , n = 213 for DMSO , n = 189 for Y ‐ 27632 and n = 245 . * * * P 0 . 001 . C Immunoblot analysis of rhotekinpull ‐ down ( P . D . ) assays reveals that GFP ‐ KSHV ‐ TK increases the level of GTP ‐ bound RhoA ( GTP ‐ RhoA ) . Actin is included as a loading control , and the input lysates are indicated . D GFP ‐ KSHV ‐ TK but not GFP induces the loss of phosphotyrosine , phospho ‐ FAK Y925 and phospho ‐ paxillin Y31 epitopes from focal adhesions .

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Figure 2A Co‐expression of GFP‐RhoN19 but not GFP inhibits RFP‐KSHV‐TK‐induced cell contraction. Graphs represent cell area (μm2) of KSHV‐TK, RhoN19 or KSHV‐TK and RhoN19 expressing cells. Error bars represent SEM from 3 independent experiments, n = 204 for KSHV‐TK, n = 223 for RhoN19 and n = 189 for KSHV‐TK and RhoN19.B GFP‐KSHV‐TK‐induced cell contraction does not occur in cells treated with the ROCK and myosin II inhibitors Y‐27632 and blebbistatin, respectively. Error bars represent SEM from three independent experiments, n = 213 for DMSO, n = 189 for Y‐27632 and n = 245. ***P 0.001.C Immunoblot analysis of rhotekinpull‐down (P.D.) assays reveals that GFP‐KSHV‐TK increases the level of GTP‐bound RhoA (GTP‐RhoA). Actin is included as a loading control, and the input lysates are indicated.D GFP‐KSHV‐TK but not GFP induces the loss of phosphotyrosine, phospho‐FAK Y925 and phospho‐paxillin Y31 epitopes from focal adhesions.


Figure 3B Immunoblot analysis of BHK cells infected with MuHV ‐ 4 and MuHV ‐ 4 [ g ‐ KSHV ‐ TK ] or uninfected cells using antibodies against phosphotyrosine ( p ‐ Tyr ) , GFP , MuHV ‐ 4 TK , ORF17 ( capsid ) and glycoprotein N . C Immunofluorescence microscopy analysis of MuHV ‐ 4 ‐ and MuHV ‐ 4 [ g ‐ KSHV ‐ TK ] ‐ infected cells with the indicated antibodies reveals GFP ‐ KSHV ‐ TK expressed in the context of lytic infection ( MuHV ‐ 4 [ g ‐ KSHV ‐ TK ] ) induces the loss of phospho ‐ FAK Y925 and paxillin Y31 from focal adhesions . White arrowheads indicate examples of infected cells expressing GFP ‐ KSHV ‐ TK or GFP ( indicating WT MuHV ‐ 4 infection , BAC + virus ) . D Immunofluorescence microscopy analysis reveals that GFP ‐ KSHV ‐ TK expressed in the context of lytic infection ( MuHV ‐ 4 [ g ‐ KSHV ‐ TK ] ) does not alter focal adhesion integrity ( paxillin ) in the presence of the ROCK inhibitor Y ‐ 27632 . E Immunofluorescence microscopy analysis of paxillin in the lytically induced rKSHV . 219 / Vero cell line in the presence or absence of Y ‐ 27632 .

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Figure 3B Immunoblot analysis of BHK cells infected with MuHV‐4 and MuHV‐4 [g‐KSHV‐TK] or uninfected cells using antibodies against phosphotyrosine (p‐Tyr), GFP, MuHV‐4 TK, ORF17 (capsid) and glycoprotein N.C Immunofluorescence microscopy analysis of MuHV‐4‐ and MuHV‐4 [g‐KSHV‐TK]‐infected cells with the indicated antibodies reveals GFP‐KSHV‐TK expressed in the context of lytic infection (MuHV‐4 [g‐KSHV‐TK]) induces the loss of phospho‐FAK Y925 and paxillin Y31 from focal adhesions. White arrowheads indicate examples of infected cells expressing GFP‐KSHV‐TK or GFP (indicating WT MuHV‐4 infection, BAC+ virus).D Immunofluorescence microscopy analysis reveals that GFP‐KSHV‐TK expressed in the context of lytic infection (MuHV‐4 [g‐KSHV‐TK]) does not alter focal adhesion integrity (paxillin) in the presence of the ROCK inhibitor Y‐27632.E Immunofluorescence microscopy analysis of paxillin in the lytically induced rKSHV.219/Vero cell line in the presence or absence of Y‐27632.


Figure 4A Cellular localisation and immunoblot analysis of phosphotyrosine ( upper panel ) and GFP ( lower panel ) in cells expressing wild ‐ type or kinase ‐ dead GFP ‐ KSHV ‐ TK . B Scanning electron microscopy of HeLa cells expressing GFP or GFP ‐ KSHV ‐ TK ‐ DEAD . C Immunofluorescence microscopy analysis of HeLa cells expressing GFP ‐ KSHV ‐ TK ‐ DEAD ( white arrowheads ) with the indicated phospho antibodies . D Immunofluorescence microscopy images of the localisation of FAK and zyxin in HeLa cells expressing GFP ‐ KSHV ‐ TK ‐ DEAD ( white arrowheads ) . E Purified recombinant GST ‐ EBV ‐ TK , GST ‐ MuHV ‐ 4 - TK and GST ‐ KSHV ‐ TKimmunoblotted for phosphotyrosine and GST following a sequential dephosphorylation and kinase reaction . F Purified recombinant GST ‐ KSHV ‐ TK and GST ‐ KSHV ‐ TK ‐ KINASE ‐ DEAD immunoblotted for phosphotyrosine and GST . G Immunoblot analysis demonstrating that the N ‐ terminus of KSHV ‐ TK is only auto ‐ phosphorylated by the kinase domain in cis .

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Figure 4A Cellular localisation and immunoblot analysis of phosphotyrosine (upper panel) and GFP (lower panel) in cells expressing wild‐type or kinase‐dead GFP‐KSHV‐TK.B Scanning electron microscopy of HeLa cells expressing GFP or GFP‐KSHV‐TK‐DEAD.C Immunofluorescence microscopy analysis of HeLa cells expressing GFP‐KSHV‐TK‐DEAD (white arrowheads) with the indicated phospho antibodies.D Immunofluorescence microscopy images of the localisation of FAK and zyxin in HeLa cells expressing GFP‐KSHV‐TK‐DEAD (white arrowheads).E Purified recombinant GST‐EBV‐TK, GST‐MuHV‐4-TK and GST‐KSHV‐TKimmunoblotted for phosphotyrosine and GST following a sequential dephosphorylation and kinase reaction.F Purified recombinant GST‐KSHV‐TK and GST‐KSHV‐TK‐KINASE‐DEAD immunoblotted for phosphotyrosine and GST.G Immunoblot analysis demonstrating that the N‐terminus of KSHV‐TK is only auto‐phosphorylated by the kinase domain in cis.


Figure 5A Immunoblot analysis of N ‐ terminal deletions of KSHV ‐ TK demonstrates that the first 100 amino acids are required for tyrosine phosphorylation . B Immunoblot analysis reveals that mutation of tyrosines 65 , 85 and 120 ablates tyrosine phosphorylation of the KSHV ‐ TK . C - E Immunofluorescence microscopy analysis with the indicated antibodies reveals that mutation of tyrosines 65 , 85 and 120 to phenylalanine abrogates the ability of GFP ‐ KSHV ‐ TK to induce loss of phosphotyrosine epitopes , phospho ‐ paxillin and phospho ‐ FAK from focal adhesions . C - E Immunofluorescence microscopy analysis with the indicated antibodies reveals that mutation of tyrosines 65 , 85 and 120 to phenylalanine abrogates the ability of GFP ‐ KSHV ‐ TK to induce loss of phosphotyrosine epitopes , phospho ‐ paxillin and phospho ‐ FAK from focal adhesions . F Immunoblot analysis of level of GTP ‐ bound and total RhoA in cells expressing the indicated GFP ‐ tagged KSHV ‐ TK proteins . G Immunofluorescence microscopy analysis reveals that localisation of paxillin at focal adhesions is unaffected by KSHV ‐ TK ‐ Y3F expressed in the context of lytic infection .

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Figure 5A Immunoblot analysis of N‐terminal deletions of KSHV‐TK demonstrates that the first 100 amino acids are required for tyrosine phosphorylation.B Immunoblot analysis reveals that mutation of tyrosines 65, 85 and 120 ablates tyrosine phosphorylation of the KSHV‐TK.C-E Immunofluorescence microscopy analysis with the indicated antibodies reveals that mutation of tyrosines 65, 85 and 120 to phenylalanine abrogates the ability of GFP‐KSHV‐TK to induce loss of phosphotyrosine epitopes, phospho‐paxillin and phospho‐FAK from focal adhesions.C-E Immunofluorescence microscopy analysis with the indicated antibodies reveals that mutation of tyrosines 65, 85 and 120 to phenylalanine abrogates the ability of GFP‐KSHV‐TK to induce loss of phosphotyrosine epitopes, phospho‐paxillin and phospho‐FAK from focal adhesions.F Immunoblot analysis of level of GTP‐bound and total RhoA in cells expressing the indicated GFP‐tagged KSHV‐TK proteins.G Immunofluorescence microscopy analysis reveals that localisation of paxillin at focal adhesions is unaffected by KSHV‐TK‐Y3F expressed in the context of lytic infection.


Figure 6A Immunoblot analysis reveals that GFP ‐ KSHV ‐ TK WT , Y2F and Y3F but not GFP ‐ KSHV ‐ TK ‐ DEAD associate with FAK . B GFP ‐ KSHV ‐ TK does not displace paxillin or zyxin from focal adhesions ( white arrowheads ) or induce contraction in the absence of FAK . Scale bar , 20 μm . C GFP ‐ KSHV ‐ TK does not induce loss of zyxin and p ‐ Tyr from focal adhesions or contraction of HeLa cells depleted of paxillin . Error bars represent SEM from three independent experiments , n = 196 for mock siRNA and n = 229 for paxillin siRNA ; * * * P 0 . 001 . Scale bar , 20 μm .

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Figure 6A Immunoblot analysis reveals that GFP‐KSHV‐TK WT, Y2F and Y3F but not GFP‐KSHV‐TK‐DEAD associate with FAK.B GFP‐KSHV‐TK does not displace paxillin or zyxin from focal adhesions (white arrowheads) or induce contraction in the absence of FAK. Scale bar, 20 μm.C GFP‐KSHV‐TK does not induce loss of zyxin and p‐Tyr from focal adhesions or contraction of HeLa cells depleted of paxillin. Error bars represent SEM from three independent experiments, n = 196 for mock siRNA and n = 229 for paxillin siRNA; ***P 0.001. Scale bar, 20 μm.


Figure 7B Immunoblot analysis of GFP antibody immunoprecipitates reveals that GFP ‐ KSHV ‐ TK but not Y2F or GFP associates with CrkI , II and L . C Immunofluorescence microscopy analysis reveals CrkII associates with GFP ‐ KSHV ‐ TK but not the Y2F mutant . Scale bars , 20 μm . D Immunoblot analysis of RFP antibody immunoprecipitates reveals that RFP ‐ KSHV ‐ TK specifically associates with the SH2 domain of CrkII and CrkL . E Immunofluorescence microscopy analysis of phosphotyrosine epitopes in cells expressing GFP ‐ CrkII SH2 , GFP ‐ CrkL SH2 , RFP ‐ KSHV ‐ TK or a combination of CrkII / LSH2 and RFP ‐ KSHV ‐ TK . Arrowheads represent contracted KSHV ‐ TK ‐ expressing cells . The SH2 domains of CrkII or L block KSHV ‐ TK ‐ induced cell rounding . Scale bars , 20 μm .

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Figure 7B Immunoblot analysis of GFP antibody immunoprecipitates reveals that GFP‐KSHV‐TK but not Y2F or GFP associates with CrkI, II and L.C Immunofluorescence microscopy analysis reveals CrkII associates with GFP‐KSHV‐TK but not the Y2F mutant. Scale bars, 20 μm.D Immunoblot analysis of RFP antibody immunoprecipitates reveals that RFP‐KSHV‐TK specifically associates with the SH2 domain of CrkII and CrkL.E Immunofluorescence microscopy analysis of phosphotyrosine epitopes in cells expressing GFP‐CrkII SH2, GFP‐CrkL SH2, RFP‐KSHV‐TK or a combination of CrkII/LSH2 and RFP‐KSHV‐TK. Arrowheads represent contracted KSHV‐TK‐expressing cells. The SH2 domains of CrkII or L block KSHV‐TK‐induced cell rounding. Scale bars, 20 μm.


Figure 8B Immunoblot analysis reveals that GFP ‐ KSHV ‐ TK WT and Y2F but not Y120F associate with p85 of PI3 ‐ Kinase . C Immunoblot analysis of p85PI3 ‐ Kinase antibody immunoprecipitates reveals that GFP ‐ KSHV ‐ TK and its Y2F mutant but not Y120F associate with p85 of PI3 ‐ Kinase . D Immunoblot analysis of CrkII and CrkL antibody immunoprecipitates reveals that GFP ‐ KSHV ‐ TK and the p85PI3 ‐ Kinase associate with CrkII and CrkL . No association is observed with the Y2F , Y3F or kinase ‐ dead TK . E Tyrosine phosphorylation of CrkII and CrkL is only upregulated in cells expressing GFP ‐ KSHV ‐ TK . F Immunoblot analysis reveals that recombinant His ‐ CrkII binds to and is phosphorylated by KSHV ‐ TK but not the Y2F or Y3F mutants .

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Figure 8B Immunoblot analysis reveals that GFP‐KSHV‐TK WT and Y2F but not Y120F associate with p85 of PI3‐Kinase.C Immunoblot analysis of p85PI3‐Kinase antibody immunoprecipitates reveals that GFP‐KSHV‐TK and its Y2F mutant but not Y120F associate with p85 of PI3‐Kinase.D Immunoblot analysis of CrkII and CrkL antibody immunoprecipitates reveals that GFP‐KSHV‐TK and the p85PI3‐Kinase associate with CrkII and CrkL. No association is observed with the Y2F, Y3F or kinase‐dead TK.E Tyrosine phosphorylation of CrkII and CrkL is only upregulated in cells expressing GFP‐KSHV‐TK.F Immunoblot analysis reveals that recombinant His‐CrkII binds to and is phosphorylated by KSHV‐TK but not the Y2F or Y3F mutants.


Figure 1A . Maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA ( red ) combined with immunostaining for L1 ORF1p ( green ) and nuclei stained with DAPI ( blue ) . The grey square marks position of the inset . White arrow heads point to cytoplasmic foci where L1 RNA and ORF1p co - localize . Scatter plot shows the number of co - localized L1 Tf - ORF1 foci in the cytoplasm per cell ( n = 275 WT , 304 Dicer _ KO1 , 311 Dicer _ KO2 cells ) . Red arrow heads point to relatively larger sized L1 RNP foci . Bar graphs are mean values of percentage of cells with large L1 Tf - ORF1 foci co - localizing in the cytoplasm ( n = 3 technical replicates ) . B . Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for L1 ORF1p ( red ) , MOV10 ( green ) and nuclei stained with DAPI ( blue ) . The grey square marks position of inset in the zoomed image . White arrow heads point to cytoplasmic foci where L1 ORF1p and MOV10 co - localize . Scatter plot shows the number of co - localized ORF1 - MOV10 foci in the cytoplasm per cell ( n = 293 WT , 295 Dicer _ KO1 , 295 Dicer _ KO2 cells ) . Red arrow heads point to relatively larger sized L1 ORF1 - MOV10 foci . Bar graphs are mean values of percentage of cells with large ORF1 - MOV10 foci co - localizing in the cytoplasm ( n = 3 technical replicates ) .

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Figure 1A. Maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA (red) combined with immunostaining for L1 ORF1p (green) and nuclei stained with DAPI (blue). The grey square marks position of the inset. White arrow heads point to cytoplasmic foci where L1 RNA and ORF1p co-localize. Scatter plot shows the number of co-localized L1 Tf-ORF1 foci in the cytoplasm per cell (n=275 WT, 304 Dicer_KO1, 311 Dicer_KO2 cells). Red arrow heads point to relatively larger sized L1 RNP foci. Bar graphs are mean values of percentage of cells with large L1 Tf-ORF1 foci co-localizing in the cytoplasm (n=3 technical replicates).B. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for L1 ORF1p (red), MOV10 (green) and nuclei stained with DAPI (blue). The grey square marks position of inset in the zoomed image. White arrow heads point to cytoplasmic foci where L1 ORF1p and MOV10 co-localize. Scatter plot shows the number of co-localized ORF1-MOV10 foci in the cytoplasm per cell (n= 293 WT, 295 Dicer_KO1, 295 Dicer_KO2 cells). Red arrow heads point to relatively larger sized L1 ORF1-MOV10 foci. Bar graphs are mean values of percentage of cells with large ORF1-MOV10 foci co-localizing in the cytoplasm (n=3 technical replicates).


Figure 2A . WT and Dicer _ KO mESCs were treated with 0 . 5mM Sodium Arsenite ( NaAsO2 ) for 20 minutes or left untreated prior to fixation with formaldehyde . Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for G3BP1 ( red ) and MOV10 ( green ) with nuclei stained with DAPI ( blue ) . B . Representative western blots showing low AGO2 protein levels in Dicer _ KO as compared to WT mESCs ( right side ) . LAMINB1 served as loading control . On the left , maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA FISH ( red ) combined with immunostaining for a resident protein of P - bodies , DDX6 ( green ) and nuclei stained with DAPI ( blue ) . The grey square marks position of the inset . Yellow arrow heads point to cytoplasmic foci where L1 RNA and DDX6 protein co - localize .

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Figure 2A. WT and Dicer_KO mESCs were treated with 0.5mM Sodium Arsenite (NaAsO2) for 20 minutes or left untreated prior to fixation with formaldehyde. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for G3BP1 (red) and MOV10 (green) with nuclei stained with DAPI (blue).B. Representative western blots showing low AGO2 protein levels in Dicer_KO as compared to WT mESCs (right side). LAMINB1 served as loading control. On the left, maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA FISH (red) combined with immunostaining for a resident protein of P-bodies, DDX6 (green) and nuclei stained with DAPI (blue). The grey square marks position of the inset. Yellow arrow heads point to cytoplasmic foci where L1 RNA and DDX6 protein co-localize.


Figure 3A . Representative western blots showing L1 ORF1p and MOV10 levels in the indicated cell lines . Immunoblot with antibody recognizing TUBULIN and coomassie stained membranes depict the loading , asterisk marks position of non - specific band in the ORF1 immunoblot ( n = 3 technical replicates ) . B . Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for L1 ORF1p ( green ) combined with RNA FISH for L1 Tf RNA ( red ) and nuclei stained with DAPI ( blue ) ( n = 3 technical replicates ) . C . Representative images of BlastR colonies stained with crystal violet blue of indicated cell lines is shown on the left . Cells were transfected with either mutant reporter plasmid ( L1N21A ) or retrotransposition competent reporter ( L1WT ) as shown in the scheme with timeline for the experiment on the top . Bar graphs on the right depict the average number of BlastR colonies ( n = 3 biological replicates ) .

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Figure 3A. Representative western blots showing L1 ORF1p and MOV10 levels in the indicated cell lines. Immunoblot with antibody recognizing TUBULIN and coomassie stained membranes depict the loading, asterisk marks position of non-specific band in the ORF1 immunoblot (n=3 technical replicates).B. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for L1 ORF1p (green) combined with RNA FISH for L1 Tf RNA (red) and nuclei stained with DAPI (blue) (n=3 technical replicates).C. Representative images of BlastR colonies stained with crystal violet blue of indicated cell lines is shown on the left. Cells were transfected with either mutant reporter plasmid (L1N21A) or retrotransposition competent reporter (L1WT) as shown in the scheme with timeline for the experiment on the top. Bar graphs on the right depict the average number of BlastR colonies (n=3 biological replicates).


Figure 4A . Scheme and quantification of relative luminescence from luciferase assays validating post - transcriptional regulation of MOV10 by miRNAs in HEK293T cells ( n = 4 biological replicates ) . B . Scheme and quantification of relative luminescence from luciferase assays in Hek293T cells to confirm specificity of post - transcriptional regulation of MOV10 by miRNAs with either wild - type ( WT ) or Mutant ( MUT ) reporters where the MicroRNA Response Element ( MRE ) for the depicted miRNAs in the 3 ' UTR of MOV10 were mutated ( n = 3 biological replicates ) . C . Scheme and representative images for western blot analysis and corresponding quantification to assess miRNA mediated regulation on MOV10 expression in mESCs ( n = 3 biological replicates ) . D . Scheme and representative images from colony forming assays for plasmid based retrotransposition analysis where miRNA mediated downregulation of MOV10 in Drosha _ KO mESCs is accompanied by an increase in L1 retrotransposition ( n = 3 biological replicates ) .

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Figure 4A. Scheme and quantification of relative luminescence from luciferase assays validating post-transcriptional regulation of MOV10 by miRNAs in HEK293T cells (n=4 biological replicates).B. Scheme and quantification of relative luminescence from luciferase assays in Hek293T cells to confirm specificity of post-transcriptional regulation of MOV10 by miRNAs with either wild-type (WT) or Mutant (MUT) reporters where the MicroRNA Response Element (MRE) for the depicted miRNAs in the 3'UTR of MOV10 were mutated (n=3 biological replicates).C. Scheme and representative images for western blot analysis and corresponding quantification to assess miRNA mediated regulation on MOV10 expression in mESCs (n=3 biological replicates).D. Scheme and representative images from colony forming assays for plasmid based retrotransposition analysis where miRNA mediated downregulation of MOV10 in Drosha_KO mESCs is accompanied by an increase in L1 retrotransposition (n=3 biological replicates).


Figure 5A . Scheme of experiment and representative images along with quantitation showing induction of L1 RNP aggregate formation upon ectopic MOV10 expression . Images are maximum intensity projections across Z stacks from indicated mESCs stained for L1 ORF1p ( red ) combined with immunostaining for HA ( green ) to detect ectopically expressed MOV10 tagged with HA , and nuclei stained with DAPI ( blue ) . White arrow heads point to cytoplasmic foci where L1 ORF1p and HA - MOV10 co - localize . Data collected from 289 Ctrl , 275 L1UP Cl1 and 296 L1UP Cl2 mESCs ( n = 3 biological replicates ) . B . Scheme and representative images from colony forming assays for plasmid based retrotransposition analysis in Ctrl and L1UP mESCs comparing rate of retrotransposition upon transfection with Empty Vector ( EV ) or ectopic expression of HA - MOV10 ( n = 3 biological replicates ) .

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Figure 5A. Scheme of experiment and representative images along with quantitation showing induction of L1 RNP aggregate formation upon ectopic MOV10 expression. Images are maximum intensity projections across Z stacks from indicated mESCs stained for L1 ORF1p (red) combined with immunostaining for HA (green) to detect ectopically expressed MOV10 tagged with HA, and nuclei stained with DAPI (blue). White arrow heads point to cytoplasmic foci where L1 ORF1p and HA-MOV10 co-localize. Data collected from 289 Ctrl, 275 L1UP Cl1 and 296 L1UP Cl2 mESCs (n=3 biological replicates).B. Scheme and representative images from colony forming assays for plasmid based retrotransposition analysis in Ctrl and L1UP mESCs comparing rate of retrotransposition upon transfection with Empty Vector (EV) or ectopic expression of HA-MOV10 (n=3 biological replicates).


Figure 6A . Scheme of experiment and representative images showing RNP aggregate formation upon ectopic expression of full length L1 and HA - MOV10 in WT mESCs . Images are maximum intensity projections across Z stacks stained for L1 ORF1p ( red ) combined with immunostaining for HA ( green ) to detect ectopically expressed MOV10 , and nuclei stained with DAPI ( blue ) . The bottom panel shows cells where accumulation of ORF1 - MOV10 was observed in relatively large foci compared to cells where co - localization of the two proteins was mostly in relatively small foci ( top panel ) , 68 - 119 transfected cells were analyzed per experiment ( n = 3 biological replicates ) . B . Scheme of experiment and representative images showing RNP aggregate formation upon ectopic expression of HA - ORF1 or HA - ORF1 in conjunction with T7 - MOV10 in WT mESCs . Images are maximum intensity projections across Z stacks stained for HA ( green ) and T7 ( red ) to detect ectopically expressed ORF1p and MOV10 respectively , and nuclei stained with DAPI ( blue ) . 100 - 147 transfected cells were analyzed per experiment ( n = 3 biological replicates ) .

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

Figure 6A. Scheme of experiment and representative images showing RNP aggregate formation upon ectopic expression of full length L1 and HA-MOV10 in WT mESCs. Images are maximum intensity projections across Z stacks stained for L1 ORF1p (red) combined with immunostaining for HA (green) to detect ectopically expressed MOV10, and nuclei stained with DAPI (blue). The bottom panel shows cells where accumulation of ORF1-MOV10 was observed in relatively large foci compared to cells where co-localization of the two proteins was mostly in relatively small foci (top panel), 68-119 transfected cells were analyzed per experiment (n=3 biological replicates).B. Scheme of experiment and representative images showing RNP aggregate formation upon ectopic expression of HA-ORF1 or HA-ORF1 in conjunction with T7-MOV10 in WT mESCs. Images are maximum intensity projections across Z stacks stained for HA (green) and T7 (red) to detect ectopically expressed ORF1p and MOV10 respectively, and nuclei stained with DAPI (blue). 100-147 transfected cells were analyzed per experiment (n=3 biological replicates).


Figure 1D Cruciform unfolding assay with increasing WRN concentrations and cruciform consisting of random inverted repeats ( random - IR ) . Top , quantitation of cruciform unfolding . The amount of linear DNA from the " No protein " lane was subtracted from all other samples . Averages shown ; n = 3 technical replicates ; error bars , SEM . E Cruciform unfolding assays as in ( D ) with WRN , WRN K577M , and WRN E84A ( all used at 10 nM ) , carried out either in the presence or absence of ATP , or with ATP - ƔS . Averages shown ; n ≥ 3 technical replicates ; error bars , SEM . G Representative cruciform unfolding assays with increasing concentrations of WRN , together with human RPA ( hRPA , 30 nM ) , yeast S . cerevisiae RPA ( yRPA , 30 nM ) , or human mitochondrial SSB ( hmtSSB , 50 nM ) . pUC19 with the random - IR cruciform structure was used as a substrate . H Quantitation of assays such as in ( G ) . The amount of linear DNA from the " No protein " lane was subtracted from all other samples . Averages shown ; n ≥ 3 technical replicates ; error bars , SEM .

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

Figure 1D Cruciform unfolding assay with increasing WRN concentrations and cruciform consisting of random inverted repeats (random-IR). Top, quantitation of cruciform unfolding. The amount of linear DNA from the "No protein" lane was subtracted from all other samples. Averages shown; n = 3 technical replicates; error bars, SEM.E Cruciform unfolding assays as in (D) with WRN, WRN K577M, and WRN E84A (all used at 10 nM), carried out either in the presence or absence of ATP, or with ATP-ƔS. Averages shown; n ≥ 3 technical replicates; error bars, SEM.G Representative cruciform unfolding assays with increasing concentrations of WRN, together with human RPA (hRPA, 30 nM), yeast S. cerevisiae RPA (yRPA, 30 nM), or human mitochondrial SSB (hmtSSB, 50 nM). pUC19 with the random-IR cruciform structure was used as a substrate. H Quantitation of assays such as in (G). The amount of linear DNA from the "No protein" lane was subtracted from all other samples. Averages shown; n ≥ 3 technical replicates; error bars, SEM.


Figure 2A Representative assays with cruciform DNA ( consisting of random - IR ) either without or with WRN incubation prior to cruciform detection by SLX1 - SLX4CCD . The ScaI restriction site is 931 bp away from the cruciform site cleaved by SLX1 - SLX4CCD . Combined activity of ScaI and SMX results in 931 and 1795 bp bands indicated by the green arrows . The intensity of these bands decreases upon WRN incubation , resulting predominantly in linear DNA cut by ScaI , suggesting that WRN removes the substrate for SLX1 - SLX4CCD . The catalytic dead version of SLX1 - SLX4CCD ( SLX1CD - SLX4CCD ) , where SLX1 carries an R41A mutations in its nuclease domain , has no activity on the cruciform DNA . B Quantitation of assays such as in ( A ) . Averages shown ; n = 3 technical replicates ; error bars , SEM .

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

Figure 2A Representative assays with cruciform DNA (consisting of random-IR) either without or with WRN incubation prior to cruciform detection by SLX1-SLX4CCD. The ScaI restriction site is 931 bp away from the cruciform site cleaved by SLX1-SLX4CCD. Combined activity of ScaI and SMX results in 931 and 1795 bp bands indicated by the green arrows. The intensity of these bands decreases upon WRN incubation, resulting predominantly in linear DNA cut by ScaI, suggesting that WRN removes the substrate for SLX1-SLX4CCD. The catalytic dead version of SLX1-SLX4CCD (SLX1CD-SLX4CCD), where SLX1 carries an R41A mutations in its nuclease domain, has no activity on the cruciform DNA. B Quantitation of assays such as in (A). Averages shown; n = 3 technical replicates; error bars, SEM.


Figure 3B Mapping of TA repeats cruciform structure . T7 Endonuclease I activity , followed by SspI , cleaves the cruciform within negative supercoiled DNA , producing two bands of the expected size ( 607 bp and 2119 bp , indicated by the green arrows , lane 2 ) . Simultaneous incubation of the substrate with SspI and EcoRI allows EcoRI cutting at the TA repeats sequence ( lane 5 ) , while the extruded cruciform is refractory to EcoRI digestion ( lane 4 , the * indicates scDNA ) . C Representative cruciform detection assays . Plasmid DNA was subjected to Cruciform extrusion procedure , where indicated . Supercoiled DNA ( cruciform , refractory to EcoRI ) and linear DNA ( no cruciform , cleaved by EcoRI ) were resolved on a 1 % agarose gel , stained with GelRed . Top , quantitation of extruded cruciform in % with respect to the amount of total DNA that is present in the lane . Averages shown ; n = 4 technical replicates ; error bars , SEM . D Representative cruciform unfolding assays with increasing WRN concentrations , using pUC19 with either random inverted repeats or TA repeats as a substrate . Top , quantitation of cruciform unfolding . The amount of linear DNA from the " No protein " lane was subtracted from all other samples . Averages shown ; n = 3 technical replicates ; error bars , SEM .

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

Figure 3B Mapping of TA repeats cruciform structure. T7 Endonuclease I activity, followed by SspI, cleaves the cruciform within negative supercoiled DNA, producing two bands of the expected size (607 bp and 2119 bp, indicated by the green arrows, lane 2). Simultaneous incubation of the substrate with SspI and EcoRI allows EcoRI cutting at the TA repeats sequence (lane 5), while the extruded cruciform is refractory to EcoRI digestion (lane 4, the * indicates scDNA).C Representative cruciform detection assays. Plasmid DNA was subjected to Cruciform extrusion procedure, where indicated. Supercoiled DNA (cruciform, refractory to EcoRI) and linear DNA (no cruciform, cleaved by EcoRI) were resolved on a 1% agarose gel, stained with GelRed. Top, quantitation of extruded cruciform in % with respect to the amount of total DNA that is present in the lane. Averages shown; n = 4 technical replicates; error bars, SEM. D Representative cruciform unfolding assays with increasing WRN concentrations, using pUC19 with either random inverted repeats or TA repeats as a substrate. Top, quantitation of cruciform unfolding. The amount of linear DNA from the "No protein" lane was subtracted from all other samples. Averages shown; n = 3 technical replicates; error bars, SEM.


Figure 4A Recombinant BLM and S . cerevisiae Sgs1 used in this study . The gel was stained with Coomassie Brilliant blue . B Representative cruciform unfolding assay with increasing concentrations of WRN , BLM , and Sgs1 . Cruciform DNA consisting of random inverted repeats was used . C Quantitation of assays such as ( B ) . Averages shown ; n ≥ 3 technical replicates ; error bars , SEM . F Representative cruciform unfolding assays with increasing concentrations of WRN and WRN - 3A . Cruciform DNA consisting of random inverted repeats was used . G Quantitation of assays such as in ( F ) . Averages shown ; n = 3 technical replicates ; error bars , SEM .

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Figure 4A Recombinant BLM and S. cerevisiae Sgs1 used in this study. The gel was stained with Coomassie Brilliant blue.B Representative cruciform unfolding assay with increasing concentrations of WRN, BLM, and Sgs1. Cruciform DNA consisting of random inverted repeats was used. C Quantitation of assays such as (B). Averages shown; n ≥ 3 technical replicates; error bars, SEM.F Representative cruciform unfolding assays with increasing concentrations of WRN and WRN-3A. Cruciform DNA consisting of random inverted repeats was used. G Quantitation of assays such as in (F). Averages shown; n = 3 technical replicates; error bars, SEM.


Figure 5B Recombinant full - length and truncation WRN variants used in this study are shown . The gel was stained with Coomassie Brilliant blue . D Cruciform unfolding assays with increasing concentrations of WRN variants . E Quantitation of assays such as shown in ( D ) . Averages shown ; n = 3 technical replicates ; error bars , SEM .

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Figure 5B Recombinant full-length and truncation WRN variants used in this study are shown. The gel was stained with Coomassie Brilliant blue.D Cruciform unfolding assays with increasing concentrations of WRN variants. E Quantitation of assays such as shown in (D). Averages shown; n = 3 technical replicates; error bars, SEM.


Figure 6A MutL homologue heterodimers used in this study : MutLα ( MLH1 - PMS2 ) , MutLβ ( MLH1 - PMS1 ) and MutLγ ( MLH1 - MLH3 ) . B MutS homologue heterodimers used in this study : MutSα ( MSH2 - MSH6 ) , MutSβ ( MSH2 - MSH3 ) and MutSγ ( MSH4 - MSH5 ) . E Representative electrophoretic mobility shift assays with the MutS and MutL homologue heterodimers , using an oligonucleotide - based Holliday junction as a substrate . Red asterisk indicates the position of the radioactive label . F Representative cruciform unfolding assays with MutSβ and MutS ⍺ , carried out without or with ATP , using the random - IR cruciform as a substrate . Top , quantitation of cruciform unfolding . Averages shown ; n = 3 technical replicates ; error bars , SEM . G Representative cruciform unfolding assays with MutSβ and MutS ⍺ , carried out without or with hRPA , using the random - IR cruciform as a substrate . Top , quantitation of cruciform unfolding . Averages shown ; n = 3 technical replicates ; error bars , SEM .

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

Figure 6A MutL homologue heterodimers used in this study: MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1) and MutLγ (MLH1-MLH3). B MutS homologue heterodimers used in this study: MutSα (MSH2-MSH6), MutSβ (MSH2-MSH3) and MutSγ (MSH4-MSH5).E Representative electrophoretic mobility shift assays with the MutS and MutL homologue heterodimers, using an oligonucleotide-based Holliday junction as a substrate. Red asterisk indicates the position of the radioactive label.F Representative cruciform unfolding assays with MutSβ and MutS⍺, carried out without or with ATP, using the random-IR cruciform as a substrate. Top, quantitation of cruciform unfolding. Averages shown; n = 3 technical replicates; error bars, SEM. G Representative cruciform unfolding assays with MutSβ and MutS⍺, carried out without or with hRPA, using the random-IR cruciform as a substrate. Top, quantitation of cruciform unfolding. Averages shown; n = 3 technical replicates; error bars, SEM.


Figure 7A Representative polyacrylamide gel showing recombinant MutSβ , MutSβ - 3K255A ( MSH3 with mutation K255A , deficient in mismatch recognition ) and MutSβ - 3K902A ( MSH3 with mutation K902A , ATPase - deficient ) . The gel was stained with Coomassie Brilliant blue . B Electrophoretic mobility shift assay with MutSβ , MutSβ - 3K255A and MutSβ - 3K902A , using either dsDNA ( 50 bp ) or dsDNA bearing one extrahelical T , as a substrate . 6 % native acrylamide gel was used . G Unfolding of TA cruciform by WRN and MMR proteins . Bottom , representative experiments ; top , quantitation ; averages shown ; n = 4 technical replicates ; error bars , SEM . H Unfolding of TA cruciform by MutLα and wild - type WRN or helicase dead WRN - K577M . Bottom , representative experiments ; top , quantitation , averages shown ; n = 4 technical replicates ; error bars , SEM .

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

Figure 7A Representative polyacrylamide gel showing recombinant MutSβ, MutSβ-3K255A (MSH3 with mutation K255A, deficient in mismatch recognition) and MutSβ-3K902A (MSH3 with mutation K902A, ATPase-deficient). The gel was stained with Coomassie Brilliant blue.B Electrophoretic mobility shift assay with MutSβ, MutSβ-3K255A and MutSβ-3K902A, using either dsDNA (50 bp) or dsDNA bearing one extrahelical T, as a substrate. 6% native acrylamide gel was used.G Unfolding of TA cruciform by WRN and MMR proteins. Bottom, representative experiments; top, quantitation; averages shown; n = 4 technical replicates; error bars, SEM. H Unfolding of TA cruciform by MutLα and wild-type WRN or helicase dead WRN-K577M. Bottom, representative experiments; top, quantitation, averages shown; n = 4 technical replicates; error bars, SEM.


Figure 1 ( 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 . ( 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 . ( 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 . ( 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 . ( 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 .

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

Figure 1(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.(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.(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.(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.(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.


Figure 2 ( 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 . ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.(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.(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.


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

Figure 3(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.(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.(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.(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.


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

Figure 4(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.(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.(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.(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.


Figure 5 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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).(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.