A 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 .

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A 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 .

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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 .

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 .

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D Immunofluorescence microscopy images illustrating the association of GFP‐KSHV‐TK, but not GFP, with actinstress fibres. Scale bar, 10 μm.


A 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 .

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A 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 .

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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 .

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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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D GFP‐KSHV‐TK but not GFP induces the loss of phosphotyrosine, phospho‐FAK Y925 and phospho‐paxillin Y31 epitopes from focal adhesions.


B 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 .

B 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 ) .

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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 .

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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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E Immunofluorescence microscopy analysis of paxillin in the lytically induced rKSHV.219/Vero cell line in the presence or absence of Y‐27632.


A Cellular localisation and immunoblot analysis of phosphotyrosine ( upper panel ) and GFP ( lower panel ) in cells expressing wild ‐ type or kinase ‐ dead GFP ‐ KSHV ‐ TK .

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A 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 .

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 .

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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 ) .

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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 .

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 .

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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 .

G Immunoblot analysis demonstrating that the N‐terminus of KSHV‐TK is only auto‐phosphorylated by the kinase domain in cis.


A Immunoblot analysis of N ‐ terminal deletions of KSHV ‐ TK demonstrates that the first 100 amino acids are required for tyrosine phosphorylation .

A 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 .

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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 .

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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 .

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 .

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.


A Immunoblot analysis reveals that GFP ‐ KSHV ‐ TK WT , Y2F and Y3F but not GFP ‐ KSHV ‐ TK ‐ DEAD associate with FAK .

A 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 .

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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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.


B Immunoblot analysis of GFP antibody immunoprecipitates reveals that GFP ‐ KSHV ‐ TK but not Y2F or GFP associates with CrkI , II and L .

B 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 .

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 .

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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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.


B Immunoblot analysis reveals that GFP ‐ KSHV ‐ TK WT and Y2F but not Y120F associate with p85 of PI3 ‐ Kinase .

B 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 .

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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 .

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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 .

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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F Immunoblot analysis reveals that recombinant His‐CrkII binds to and is phosphorylated by KSHV‐TK but not the Y2F or Y3F mutants.


A . 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 ) .

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A. 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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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).


A . 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 ) .

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A. 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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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.


A . 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 ) .

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A. 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 ) .

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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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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).


A . Scheme and quantification of relative luminescence from luciferase assays validating post - transcriptional regulation of MOV10 by miRNAs in HEK293T cells ( n = 4 biological replicates ) .

A. 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 ) .

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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 ) .

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 ) .

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).


A . 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 ) .

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A. 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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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).


A . 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 ) .

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A. 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 ) .

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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).


D 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 .

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D 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 .

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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 .

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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.


A 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 .

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A 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.


B 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 ) .

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B 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 .

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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.


A Recombinant BLM and S . cerevisiae Sgs1 used in this study . The gel was stained with Coomassie Brilliant blue .

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A 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 .

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 .

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.


B Recombinant full - length and truncation WRN variants used in this study are shown . The gel was stained with Coomassie Brilliant blue .

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B 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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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.


A 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 ) .

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A 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 .

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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 .

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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.


A 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 .

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A 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 .

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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 .

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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.


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

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


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

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


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

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


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

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


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

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


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

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


( G ) HCT116 cells were treated with Leupeptin and Embelin in the indicated combinations . Cell lysates were analysed by western blotting . The data are representative of three biological replicates . The ratio of LCII / LC3I to actin is presented in Supplementary Figure S1D .

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(G) HCT116 cells were treated with Leupeptin and Embelin in the indicated combinations. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1D.


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

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


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

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


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

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


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

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


( B ) HCT116 cells expressing XIAP ‐ specific or control siRNAs , HCT116 XIAP WT and XIAP KO cells were individually transfected with Flag ‐ XIAP or control vector . Cell lysates were analysed by western blotting . The data are representative of three biological replicates . The ratio of LCII / LC3I to actin is presented in Supplementary Figure S2D .

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(B) HCT116 cells expressing XIAP‐specific or control siRNAs, HCT116 XIAP WT and XIAP KO cells were individually transfected with Flag‐XIAP or control vector. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2D.


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

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


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

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(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 .

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


( F ) HCT116 XIAP WT and XIAP KO cells treated with 10 μM Nutlin3 for 6 h . LC3 conversion was evaluated by western blot analysis . The data are representative of three biological replicates . The ratio of LCII / LC3I to actin is presented in Supplementary Figure S2H .

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(F) HCT116 XIAP WT and XIAP KO cells treated with 10 μM Nutlin3 for 6 h. LC3 conversion was evaluated by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2H.


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


( A ) Human osteosarcoma U2OS GFP - LC3 cells were treated with a library of polyphenols and polyamines for 6 h And then GFP - LC3 dots were counted to measure autophagy activity . Top 20 hits were shown in the right frame .

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

(A) Human osteosarcoma U2OS GFP-LC3 cells were treated with a library of polyphenols and polyamines for 6 h And then GFP-LC3 dots were counted to measure autophagy activity. Top 20 hits were shown in the right frame.


Human osteosarcoma U2OS cells ( B or neuroglioma H4 cells ( D stably expressing GFP - LC3 were treated with a library of chalcones ( 30 µM ) as indicated , or were left untreated for 6 h . The cells were then fixed and GFP - LC3 dots were counted as an indicator for autophagy . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) . Representative images are shown in ( B , D ) . Scale bar equals 10 µm . ( C , E ) Acetylation : U2OS and H4 cells were treated as described above , followed by incubation with specific antibodies to block acetylated tubulin ; Thereafter immunofluorescence was conducted with antibodies against acetylated lysine residues and appropriate AlexaFluor conjugated secondary antibodies before the assessment of cytoplasmic fluorescence intensities . Representative images of acetylation are shown in ( C , E ) . Scale bar equals 10 µm .

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

Human osteosarcoma U2OS cells (B or neuroglioma H4 cells (D stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated , or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Representative images are shown in (B,D). Scale bar equals 10 µm. (C,E) Acetylation: U2OS and H4 cells were treated as described above, followed by incubation with specific antibodies to block acetylated tubulin; Thereafter immunofluorescence was conducted with antibodies against acetylated lysine residues and appropriate AlexaFluor conjugated secondary antibodies before the assessment of cytoplasmic fluorescence intensities. Representative images of acetylation are shown in (C,E). Scale bar equals 10 µm.


Human osteosarcoma U2OS cells F ) or neuroglioma H4 cells H ) stably expressing GFP - LC3 were treated with a library of chalcones ( 30 µM ) as indicated or rapamycin ( 10 µM ) , or were left untreated for 6 h . The cells were then fixed and GFP - LC3 dots were counted as an indicator for autophagy . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) . Viability : U2OS and H4 cells were treated with 30 µM of the indicated chalcones for 24 h and then nuclei were stained with Hoechst 33342 and the number of cells harboring normal nuclei ( i . e . non pyknotic , " healthy cells " ) was determined . All chalcones were hierarchically clustered upon z - scoring the following phenotypes : autophagy ( number of GFP - LC3 dots ) , viability ( number of healthy cells ) and acetylation ( cytoplasmic fluorescence intensity of acetylated lysine residues ) in U2OS and H4 cells . Results are reported as a heatmap ( G , I ) .

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

Human osteosarcoma U2OS cells F) or neuroglioma H4 cells H) stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated or rapamycin (10 µM), or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Viability: U2OS and H4 cells were treated with 30 µM of the indicated chalcones for 24 h and then nuclei were stained with Hoechst 33342 and the number of cells harboring normal nuclei (i.e. non pyknotic, "healthy cells") was determined. All chalcones were hierarchically clustered upon z-scoring the following phenotypes: autophagy (number of GFP-LC3 dots), viability (number of healthy cells) and acetylation (cytoplasmic fluorescence intensity of acetylated lysine residues) in U2OS and H4 cells. Results are reported as a heatmap (G, I).


( J , K ) U2OS cells were treated with 3 , 4 - DC in the presence or absence of dichloroacetate ( DCA ) , α - ketoisocaproic acid ( KIC ) , or L - leucine ( Leu ) . GFP - LC3 dots were quantified in ( J ) . Acetylation staining was performed as previously described , and acetylation intensity in the cytoplasm was measured in ( K ) . Data are means ± SD ( * * * = p < 0 . 001 versus DMSO / Ctr ; # # = p < 0 . 01 , # # # = p < 0 . 001 versus 3 , 4 - DC / Ctr ) .

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

(J, K) U2OS cells were treated with 3,4-DC in the presence or absence of dichloroacetate (DCA), α-ketoisocaproic acid (KIC), or L-leucine (Leu). GFP-LC3 dots were quantified in (J). Acetylation staining was performed as previously described, and acetylation intensity in the cytoplasm was measured in (K). Data are means ± SD (*** = p < 0.001 versus DMSO/Ctr; ##=p<0.01, ###=p<0.001 versus 3,4-DC/Ctr).


Hepatoma HepG2 cells were treated with the indicated concentrations of 3 , 4 - DC for 8 h ( A , B ) GAPDH was measured as a loading control . Band intensities of p62 , GAPDH , LC3 - I and LC3 - II were measured and ratios of p62 or LC3 - II versus GAPDH ( LC3 - II / GAPDH , p62 / GAPDH ) and LC3 - II versus LC3 - I ( LC3 - II / LC3 - I ) were calculated in ( B Data are means ± SEM of at least three independent experiments ( LC3 - II / GAPDH : * = p < 0 . 05 , * * = p < 0 . 01 ; p62 / GAPDH : # = p < 0 . 05 , # # # = p < 0 . 001 ; LC3 - II / LC3 - I : $ = p < 0 .

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

Hepatoma HepG2 cells were treated with the indicated concentrations of 3,4-DC for 8 h (A,B) GAPDH was measured as a loading control. Band intensities of p62, GAPDH, LC3-I and LC3-II were measured and ratios of p62 or LC3-II versus GAPDH (LC3-II/GAPDH, p62/GAPDH) and LC3-II versus LC3-I (LC3-II/LC3-I) were calculated in (B Data are means ± SEM of at least three independent experiments (LC3-II/GAPDH: * = p < 0.05, ** = p < 0.01; p62/GAPDH: # = p < 0.05, ### = p < 0.001; LC3-II/LC3-I: $=p<0.05


Hepatoma HepG2 cells were treated with 30 µM 3 , 4 - DC for the indicated time points ( C , D ) . Then , cells were processed to measure LC3 and p62 protein levels by SDS - PAGE and immunoblot GAPDH was measured as a loading control . Band intensities of p62 , GAPDH , LC3 - I and LC3 - II were measured and ratios of p62 or LC3 - II versus GAPDH ( LC3 - II / GAPDH , p62 / GAPDH ) and LC3 - II versus LC3 - I ( LC3 - II / LC3 - I ) were calculated in D ) . Data are means ± SEM of at least three independent experiments ( LC3 - II / GAPDH : * = p < 0 . 05 , * * = p < 0 . 01 ; p62 / GAPDH : # = p < 0 . 05 , # # # = p < 0 . 001 ; LC3 - II / LC3 - I : $ = p < 0 . 05 ) .

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

Hepatoma HepG2 cells were treated with 30 µM 3,4-DC for the indicated time points (C,D). Then, cells were processed to measure LC3 and p62 protein levels by SDS-PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of p62, GAPDH, LC3-I and LC3-II were measured and ratios of p62 or LC3-II versus GAPDH (LC3-II/GAPDH, p62/GAPDH) and LC3-II versus LC3-I (LC3-II/LC3-I) were calculated in D). Data are means ± SEM of at least three independent experiments (LC3-II/GAPDH: * = p < 0.05, ** = p < 0.01; p62/GAPDH: # = p < 0.05, ### = p < 0.001; LC3-II/LC3-I: $=p<0.05).


( E , F ) HepG2 were treated with 30 µM 3 , 4 - DC for 8 h in the presence or absence of chloroquine ( CQ , 50µM ) for 4 h . SDS - PAGE and immunoblot were performed Band intensities of LC3 - II and GAPDH were assessed and their ratio ( LC3 - II / GAPDH ) was calculated in ( F ) . Data are means ± SEM of at least three independent experiments ( * = p < 0 . 05 , * * = p < 0 . 01 versus untreated control ; # # = p < 0 . 01 versus CQ ) .

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

(E,F) HepG2 were treated with 30 µM 3,4-DC for 8 h in the presence or absence of chloroquine (CQ, 50µM) for 4 h. SDS-PAGE and immunoblot were performed Band intensities of LC3-II and GAPDH were assessed and their ratio (LC3-II/GAPDH) was calculated in (F). Data are means ± SEM of at least three independent experiments (* = p < 0.05, ** = p < 0.01 versus untreated control; ##=p<0.01 versus CQ).


( G , H ) U2OS - GFP - LC3 cells were treated with 30 µM 3 , 4 - DC , 10 µM rapamycin , or left untreated for 16 h in the presence or absence of CQ for 4 h . GFP - LC3 dots were measured . Data are means ± SD ( * * * = p < 0 . 001 versus untreated ; # # # = p < 0 . 001 versus CQ ) .

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

(G,H) U2OS-GFP-LC3 cells were treated with 30 µM 3,4-DC, 10 µM rapamycin, or left untreated for 16 h in the presence or absence of CQ for 4 h. GFP-LC3 dots were measured. Data are means ± SD (*** = p < 0.001 versus untreated; ###=p<0.001 versus CQ).


( I , J ) U2OS cells stably expressing LC3 fused with tandem fluorescent GFP - RFP proteins ( GFP - RFP - LC3 ) were treated with the indicated raising doses of 3 , 4 - DC or 50 µM chloroquine ( CQ ) for 16 h . After fixation , GFP - LC3 and RFP - LC3 dots were measured by automated image acquisition and analysis . GFP and RFP double positive LC3 dots indicating autophagosomes ( GFP + ) , and GFP negative but RFP positive LC3 dots indicating autolysosomes ( GFP - ) were counted in ( J ) . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) . Representative images are shown in I . Scale bar equals 10 µm .

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

(I,J) U2OS cells stably expressing LC3 fused with tandem fluorescent GFP-RFP proteins (GFP-RFP-LC3) were treated with the indicated raising doses of 3,4-DC or 50 µM chloroquine (CQ) for 16 h. After fixation, GFP-LC3 and RFP-LC3 dots were measured by automated image acquisition and analysis. GFP and RFP double positive LC3 dots indicating autophagosomes (GFP+), and GFP negative but RFP positive LC3 dots indicating autolysosomes (GFP-) were counted in (J). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001). Representative images are shown in I. Scale bar equals 10 µm.


A , B ) HepG2 cells were treated for the indicated time with 30 µM 3 , 4 - DC ( A ) or indicated dose of 3 , 4 - DC for 8 h ( B ) . Thereafter , cells were collected and SDS - PAGE and immunoblots were performed as described before . Total p70S6K , phosphorylated p70S6K at T389 ( P - p70S6K ) , Atg14 , Lamp1 , LC3 , p62 , and GAPDH protein levels were measured with specific antibodies . Samples for immunoblots in A and B were run in parallel instances , then cut into horizontal stripes and probed separately . Blots probed for P - p70S6K were stripped and reprobed to detect p70 independently from its phosphorylation status . The same applies for LAMP1 and GAPDH .

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

A, B) HepG2 cells were treated for the indicated time with 30 µM 3,4-DC (A) or indicated dose of 3,4-DC for 8 h (B). Thereafter, cells were collected and SDS-PAGE and immunoblots were performed as described before. Total p70S6K, phosphorylated p70S6K at T389 (P-p70S6K), Atg14, Lamp1, LC3, p62, and GAPDH protein levels were measured with specific antibodies. Samples for immunoblots in A and B were run in parallel instances, then cut into horizontal stripes and probed separately. Blots probed for P-p70S6K were stripped and reprobed to detect p70 independently from its phosphorylation status. The same applies for LAMP1 and GAPDH.


( C , D ) H4 - GFP - LC3 cells were treated with 30 µM 3 , 4 - DC in the presence or absence of CHX or AMD or with CQ for 16 h as controls , as indicated . GFP - LC3 dots were quantified in ( D ) . Data are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 versus untreated control ; # = p < 0 . 05 versus untreated with CQ ) .

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

(C, D) H4-GFP-LC3 cells were treated with 30 µM 3,4-DC in the presence or absence of CHX or AMD or with CQ for 16 h as controls, as indicated. GFP-LC3 dots were quantified in (D). Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus untreated control; # = p < 0.05 versus untreated with CQ).


( E ) RNA expression levels 16 h after exposure to 3 , 4 - DC of the indicated genes in HepG2 cells were measured by RT - PCR and normalized to the expression of the housekeeping gene ( GAPDH ) . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) .

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

(E) RNA expression levels 16 h after exposure to 3,4-DC of the indicated genes in HepG2 cells were measured by RT-PCR and normalized to the expression of the housekeeping gene (GAPDH). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001).


, G ) U2OS GFP - LC3 cells were enucleated to obtain cytoplasts , which were further treated with 3 , 4 - DC and torin1 . GFP - LC3 dots in cytoplasts were counted as shown in ( G ) . Data are means ± SD ( * * = p < 0 . 01 ) . Representative images with cytoplast marked by a white square are shown in ( F ) . Scale bar equals 10 µm .

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

,G) U2OS GFP-LC3 cells were enucleated to obtain cytoplasts, which were further treated with 3,4-DC and torin1. GFP-LC3 dots in cytoplasts were counted as shown in (G). Data are means ± SD (** = p < 0.01). Representative images with cytoplast marked by a white square are shown in (F). Scale bar equals 10 µm.


U2OS cells stably expressing GFP - TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h . GFP intensities in nuclei and cytoplasm were measured and the ratio of GFP intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei ( A ) . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) .

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

U2OS cells stably expressing GFP-TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h. GFP intensities in nuclei and cytoplasm were measured and the ratio of GFP intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (A). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001).


U2OS cells stably expressing GFP - TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h . Representative images are shown in ( B ) . Scale bar equals 10 µm .

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

U2OS cells stably expressing GFP-TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h. Representative images are shown in (B). Scale bar equals 10 µm.


( C , D ) U2OS cells were treated with 30 µM 3 , 4 - DC for 6 h , and then endogenous TFEB translocation was assessed by immunostaining ( C ) . TFEB intensities in nuclei and cytoplasm were measured and the ratio of TFEB intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei ( D ) . Data are means ± SD ( * * * = p < 0 . 001 ) . Representative images are shown in ( C ) . Scale bar equals 10 µm .

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

(C, D) U2OS cells were treated with 30 µM 3,4-DC for 6 h, and then endogenous TFEB translocation was assessed by immunostaining (C). TFEB intensities in nuclei and cytoplasm were measured and the ratio of TFEB intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (D). Data are means ± SD (*** = p < 0.001). Representative images are shown in (C). Scale bar equals 10 µm.


( E , F ) U2OS cells were treated with 30 µM 3 , 4 - DC for 6 h or were left untreated . Cytoplasmic and nuclear fractions were assessed for nuclear translocation of the transcription factors TFEB and TFE3 by SDS page .

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

(E, F) U2OS cells were treated with 30 µM 3,4-DC for 6 h or were left untreated. Cytoplasmic and nuclear fractions were assessed for nuclear translocation of the transcription factors TFEB and TFE3 by SDS page.


MEF wild type and TSC2 knockout ( TSC2 KO ) cells were treated with 3 , 4 - DC for 6 h . The cells were fixed and immunofluorescence was performed to detect endogenous TFEB . The ratio of TFEB intensities between nuclei and cytoplasm was calculated in ( H ) . Data are means ± SD ( * * * = p < 0 . 001 versus Ctr ; # # # = p < 0 . 001 versus WT / 3 , 4 - DC ) . Representative images are shown in ( G ) . Scale bar equals 10 µm .

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

MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. The cells were fixed and immunofluorescence was performed to detect endogenous TFEB. The ratio of TFEB intensities between nuclei and cytoplasm was calculated in (H). Data are means ± SD (*** = p < 0.001 versus Ctr; ###=p<0.001 versus WT/3,4-DC). Representative images are shown in (G). Scale bar equals 10 µm.


MEF wild type and TSC2 knockout ( TSC2 KO ) cells were treated with 3 , 4 - DC for 6 h . ( I ) After the treatment , nuclei were isolated and western blot was performed to detect nuclear and cytosol TFEB protein levels .

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

MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. (I) After the treatment, nuclei were isolated and western blot was performed to detect nuclear and cytosol TFEB protein levels.


( J - L ) MEF wild type and TSC2 KO cells were collected and lysed for western blot after the treatment with 3 , 4 - DC . Antibodies against LC3 , p62 , TSC2 , or GAPDH were administrated to detect protein levels , and phosphorylation of mTOR substrate p70 S6K at Threonine 389 ( T389 ) was measured with the phosphorylation specific antibody ( P - p70 ( T389 ) ) and anti - p70 antibody . The bands intensities of LC3 - II , p62 , and GAPDH were measured with Image J , and the ratios of LC3 - II / GAPDH and p62 / GAPDH were calculated in ( K ) and ( L ) . Data are means ± SEM of at least three independent experiments ( * * = p < 0 . 01 , * * * = p < 0 . 001 vs Ctr ; # = p < 0 . 05 , # # = p < 0 . 01 vs WT / 3 , 4 - DC )

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

(J-L) MEF wild type and TSC2 KO cells were collected and lysed for western blot after the treatment with 3,4-DC. Antibodies against LC3, p62, TSC2, or GAPDH were administrated to detect protein levels, and phosphorylation of mTOR substrate p70 S6K at Threonine 389 (T389) was measured with the phosphorylation specific antibody (P-p70(T389)) and anti-p70 antibody. The bands intensities of LC3-II, p62, and GAPDH were measured with Image J, and the ratios of LC3-II/GAPDH and p62/GAPDH were calculated in (K) and (L). Data are means ± SEM of at least three independent experiments (** = p < 0.01, *** = p < 0.001 vs Ctr; #=p<0.05, ##=p<0.01 vs WT/3,4-DC)


( M , N ) U2OS cells treated with 3 , 4 - DC or Torin for 6 h were collected and processed for western blot . TFEB phosphorylation at Serine 211 was checked with the phosphorylation specific antibody ( P - TFEB ( S211 ) ) and total TFEB protein level was also detected with anti - TFEB . The bands intensities of P - TFEB and total TFEB were measured with Image J , and their ratio was calculated in ( N ) . Data are means ± SEM of at least three independent experiments ( * = p < 0 . 05 vs Ctr ) .

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

(M,N) U2OS cells treated with 3,4-DC or Torin for 6 h were collected and processed for western blot. TFEB phosphorylation at Serine 211 was checked with the phosphorylation specific antibody (P-TFEB(S211)) and total TFEB protein level was also detected with anti-TFEB. The bands intensities of P-TFEB and total TFEB were measured with Image J, and their ratio was calculated in (N). Data are means ± SEM of at least three independent experiments (* = p < 0.05 vs Ctr).


U2OS - GFP - LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB ( siTFEB - # 1 , siTFEB - # 2 , siTFEB - # 3 ) or scrambled siRNA ( siCtr ) were treated with 3 , 4 - DC as indicated for 16 h . TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS - PAGE and

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

U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblot


U2OS - GFP - LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB ( siTFEB - # 1 , siTFEB - # 2 , siTFEB - # 3 ) or scrambled siRNA ( siCtr ) were treated with 3 , 4 - DC as indicated for 16 h . GFP - LC3 dots were quantified as indicator of autophagy Data are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 versus siCtr or WT cells treated with 3 , 4 - DC ) . Representative images are shown Scale bar equals 10 µm .

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

U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown Scale bar equals 10 µm.