Figure 4f . Inhibition of CDK12 kinase activity in cycling leads to decreased numbers of actively replicating . Asynchronous AS CDK12 HCT116 were grown for 24 48 h in presence or absence of 3 - MB - PP1 replicating BrdU - stained were quantified by FACS analyses . CTRL = control samples without 3 - MB - PP1 . A representative image of 3 replicates is shown .

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 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 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

Figure 4f. Inhibition of CDK12 kinase activity in cycling cells leads to decreased numbers of actively replicating cells. Asynchronous AS CDK12 HCT116 cells were grown for 24 and 48 h in the presence or absence of 3-MB-PP1 and replicating BrdU-stained cells were quantified by FACS analyses. CTRL=control samples without the 3-MB-PP1. A representative image of 3 replicates is shown.


Figure 4g , h . Prolonged CDK12 inhibition causes chromosomal aberrations in . Specific chromosomal aberrations in treated with 3 - MB - PP1 ( 24 or 48 h ) , 4 mM hydroxyurea ( 5 h ) or control solvent ( CTRL ) were identified by microscopy . A representative image from 3 biological replicates is shown ( g ) . Total numbers of chromosomal aberrations per hundred of representative replicate in g is quantified ( h ) .

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Figure 4g, h. Prolonged CDK12 inhibition causes chromosomal aberrations in cells. Specific chromosomal aberrations in cells treated with 3-MB-PP1 (24 or 48 h), 4 mM hydroxyurea (5 h) or control solvent (CTRL) were identified by microscopy. A representative image from 3 biological replicates is shown (g). Total numbers of chromosomal aberrations per hundred cells of the representative replicate in g is quantified (h).


Figure 5b Genes down - regulated in - seq after CDK12 inhibition have diminished relative occupancy of RNAPII at their 3 ´ ends Metagene analyses of RNAPII ChIP - seq data Each transcript was divided into two parts with fixed ( transcription start site ( TSS ) - 3kb to + 1 . 5kb transcription termination site ( TTS ) - 1 . 5kb to + 3kb ) a central part with variable corresponding to of gene ( shown in % ) . Each part was binned into a fixed number of bins ( 90 / 180 / 90 ) average coverage for each bin was calculated for each transcript in each sample . curve for each transcript was normalized to a sum of one then averaged first across genes second across samples . Dotted lines indicate : TSS , 1500 nucleotides downstream of TSS , 1500 nucleotides upstream of TTS TTS . color track at bottom of each subfigure indicate significance of paired Wilcoxon comparing normalized transcript coverages for each bin between untreated ( CTRL ) treated with 3 - MB - PP1 . P - values are adjusted for multiple testing with Bonferroni method within each subfigure ; color code : red = adjusted p - value ≤ 10 - 15 , orange = adjusted p - value ≤ 10 - 10 , yellow = adjusted p - value ≤ 10 - 3 .

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Figure 5b Genes down-regulated in nuclear RNA-seq after CDK12 inhibition have diminished relative occupancy of RNAPII at their 3´ ends Metagene analyses of RNAPII ChIP-seq data Each transcript was divided into two parts with fixed length (transcription start site (TSS) -3kb to +1.5kb and transcription termination site (TTS) -1.5kb to +3kb) and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/90) and average coverage for each bin was calculated for each transcript in each sample. The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples. Dotted lines indicate: TSS, 1500 nucleotides downstream of TSS, 1500 nucleotides upstream of TTS and TTS. The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated (CTRL) cells and cells treated with 3-MB-PP1. P-values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red=adjusted p-value≤10-15, orange=adjusted p-value≤10-10, yellow=adjusted p-value≤10-3.


Genes down - regulated in - seq after CDK12 inhibition have higher relative occupancy of P - Ser2 in their gene bodies . Metagene analyses of P - Ser2 ChIP - seq data Each transcript was divided into two parts with fixed ( transcription start site ( TSS ) - 3kb to + 1 . 5kb transcription termination site ( TTS ) - 1 . 5kb to + 3kb ) a central part with variable corresponding to of gene ( shown in % ) . Each part was binned into a fixed number of bins ( 90 / 180 / 90 ) average coverage for each bin was calculated for each transcript in each sample . curve for each transcript was normalized to a sum of one then averaged first across genes second across samples . Dotted lines indicate : TSS , 1500 nucleotides downstream of TSS , 1500 nucleotides upstream of TTS TTS . color track at bottom of each subfigure indicate significance of paired Wilcoxon comparing normalized transcript coverages for each bin between untreated ( CTRL ) treated with 3 - MB - PP1 . P - values are adjusted for multiple testing with Bonferroni method within each subfigure ; color code : red = adjusted p - value ≤ 10 - 15 , orange = adjusted p - value ≤ 10 - 10 , yellow = adjusted p - value ≤ 10 - 3 .

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Genes down-regulated in nuclear RNA-seq after CDK12 inhibition have higher relative occupancy of P-Ser2 in their gene bodies. Metagene analyses of P-Ser2 ChIP-seq data Each transcript was divided into two parts with fixed length (transcription start site (TSS) -3kb to +1.5kb and transcription termination site (TTS) -1.5kb to +3kb) and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/90) and average coverage for each bin was calculated for each transcript in each sample. The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples. Dotted lines indicate: TSS, 1500 nucleotides downstream of TSS, 1500 nucleotides upstream of TTS and TTS. The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated (CTRL) cells and cells treated with 3-MB-PP1. P-values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red=adjusted p-value≤10-15, orange=adjusted p-value≤10-10, yellow=adjusted p-value≤10-3.


Figure 5d Examples of genes whose transcription processivity expression is dependent on CDK12 kinase activity . - seq data on respective strand RNAPII , P - Ser2 , P - Ser5 SPT6 ChIP - seq data for MED13 genes from either treated ( red ) or not ( blue , CTRL ) with 3 - MB - PP1 were visualized with Gviz . Read were normalized to total number of mapped reads per sample averaged between replicates . Blue red boxes below - seq data indicate 90 % in control CDK12 inhibited samples , respectively .

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Figure 5d Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity. Nuclear RNA-seq data on the respective strand and RNAPII, P-Ser2, P-Ser5 and SPT6 ChIP-seq data for MED13 genes from cells either treated (red) or not (blue, CTRL) with 3-MB-PP1 were visualized with Gviz. Read counts were normalized to the total number of mapped reads per sample and averaged between replicates. Blue and red boxes below the RNA-seq data indicate the 90% distance in control and CDK12 inhibited samples, respectively.


Figure 5d e . Examples of genes whose transcription processivity expression is dependent on CDK12 kinase activity . - seq data on respective strand RNAPII , P - Ser2 , P - Ser5 SPT6 ChIP - seq data for UBE3C genes from either treated ( red ) or not ( blue , CTRL ) with 3 - MB - PP1 were visualized with Gviz . Read were normalized to total number of mapped reads per sample averaged between replicates . Blue red boxes below - seq data indicate 90 % ( see Fig . 7d 7e corresponding text ) in control CDK12 inhibited samples , respectively .

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Figure 5d e. Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity. Nuclear RNA-seq data on the respective strand and RNAPII, P-Ser2, P-Ser5 and SPT6 ChIP-seq data for UBE3C genes from cells either treated (red) or not (blue, CTRL) with 3-MB-PP1 were visualized with Gviz. Read counts were normalized to the total number of mapped reads per sample and averaged between replicates. Blue and red boxes below the RNA-seq data indicate the 90% distance (see Fig. 7d and 7e and corresponding text) in control and CDK12 inhibited samples, respectively.


Figure 7a . Longer genes tend to have a larger fraction of differentially used exons . Box plot shows fraction of exons significantly differentially used for 9026 expressed genes grouped into deciles on genomic ( including exons introns ) of their longest transcripts . n = 3 replicates .

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Figure 7a. Longer genes tend to have a larger fraction of differentially used exons. Box plot shows the fraction of exons significantly differentially used for 9026 expressed genes grouped into deciles based on the genomic length (including exons and introns) of their longest transcripts. n=3 replicates.


Figure 8b . Transcription elongation rate decreases in bodies of CDK12 - dependent but not CDK12 - independent genes after CDK12 inhibition . Graphs show relative of pre - mRNAs of described genes in AS CDK12 HCT116 either treated with 3 - MB - PP1 or not ( CTRL ) for indicated times after DRB wash off . Pre - mRNA were normalized to samples not treated with DRB ( Unt ) for which value was set as 1 . n = 3 independent experiments , error bars correspond to . Positions of primers ( designed to span exon - intron junctions ) their from transcription start site in kb are indicated in gene structures shown above graphs . Figure 8c . Proposed model . Schema shows groups of genes whose RNAPII processivity is particularly sensitive to CDK12 catalytic activity functions that are especially dependent on optimal expression of these genes . situation in with normal aberrant CDK12 kinase activity is depicted . CDK12 ( green oval ) phosphorylates ( P ) unknown substrate ( s ) ( orange oval ) , possibly including CTD ( blue ) , which results in optimal elongation processivity ( blue arrow ) of RNAPII ( blue oval ) for CDK12 - sensitive genes . Full , functional mRNAs are synthesized ( upper panel ) . Inhibition of CDK12 leads to hyperphosphorylation ( capital P ) of Ser2 ( S2 ) in bodies of CDK12 - sensitive genes which is associated with slower elongation premature termination . Shorter , aberrant mRNAs are made ( lower panel ) . mRNAs are depicted as black lines .

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

Figure 8b. Transcription elongation rate decreases in bodies of CDK12-dependent but not CDK12-independent genes after CDK12 inhibition. Graphs show relative levels of pre-mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3-MB-PP1 or not (CTRL) for indicated times after DRB wash off. Pre-mRNA levels were normalized to the samples not treated with DRB (Unt) for which the value was set as 1. n=3 independent experiments, error bars correspond to SEM. Positions of primers (designed to span exon-intron junctions) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs. Figure 8c. Proposed model. Schema shows groups of genes whose RNAPII processivity is particularly sensitive to CDK12 catalytic activity and cellular functions that are especially dependent on optimal expression of these genes. The situation in cells with normal and aberrant CDK12 kinase activity is depicted. CDK12 (green oval) phosphorylates (P) unknown substrate(s) (orange oval), possibly including the CTD (blue line), which results in optimal elongation and processivity (blue arrow) of RNAPII (blue oval) for CDK12-sensitive genes. Full length, functional mRNAs are synthesized (upper panel). Inhibition of CDK12 leads to hyperphosphorylation (capital P) of Ser2 (S2) in bodies of CDK12-sensitive genes which is associated with slower elongation and premature termination. Shorter, aberrant mRNAs are made (lower panel). mRNAs are depicted as black lines.


Figure 8b . Transcription elongation rate decreases in bodies of CDK12 - dependent but not CDK12 - independent genes after CDK12 inhibition . Graphs show relative of pre - mRNAs of described genes in AS CDK12 HCT116 either treated with 3 - MB - PP1 or not ( CTRL ) for indicated times after DRB wash off . Pre - mRNA were normalized to samples not treated with DRB ( Unt ) for which value was set as 1 . n = 3 independent experiments , error bars correspond to . Positions of primers ( designed to span exon - intron junctions ) their from transcription start site in kb are indicated in gene structures shown above graphs .

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

Figure 8b. Transcription elongation rate decreases in bodies of CDK12-dependent but not CDK12-independent genes after CDK12 inhibition. Graphs show relative levels of pre-mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3-MB-PP1 or not (CTRL) for indicated times after DRB wash off. Pre-mRNA levels were normalized to the samples not treated with DRB (Unt) for which the value was set as 1. n=3 independent experiments, error bars correspond to SEM. Positions of primers (designed to span exon-intron junctions) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs.


Comparison of SARS - CoV - 2 SARS - CoV replication kinetics in Vero E6 . ( a ) Growth curve showing release of infectious viral progeny into medium of infected Vero E6 ( m . o . i . 3 ) , as determined by plaque ( n = 4 ; mean ± sd is presented ) . ( b ) . Comparison of SARS - CoV - 2 Australia / VIC01 / 2020 SARS - CoV Frankfurt - 1 plaque phenotype in Vero E6 .

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Comparison of SARS-CoV-2 and SARS-CoV replication kinetics in Vero E6 cells.(a) Growth curve showing the release of infectious viral progeny into the medium of infected Vero E6 cells (m.o.i. 3), as determined by plaque assay (n = 4; mean ± sd is presented). (b). Comparison of SARS-CoV-2 Australia/VIC01/2020 and SARS-CoV Frankfurt-1 plaque phenotype in Vero E6 cells.


Kinetics of SARS - CoV - 2 SARS - CoV synthesis in infected Vero E6 . ( a ) Hybridization of viral mRNAs isolated from SARS - CoV - 2 - SARS - CoV - infected Vero E6 , separated in an agarose gel probed with a radiolabelled oligonucleotide recognizing genome subgenomic mRNAs of both . Subsequently , gel was re - hybridized to a probe specific for 18S ribosomal , which was used as a loading control . ( b ) of relative abundance of each of SARS - CoV - 2 SARS - CoV transcripts . Phosphorimager was performed for bands of samples isolated at 12 , 14 24 h p . i . , which yielded essentially identical relative abundances . table shows average of these three measurements . SARS - CoV - 2 mRNA sizes were calculated on basis of position of leader transcription - regulatory sequences ( ACGAAC ) in viral genome ( Sawicki Sawicki 1995 , Xu , Hu et al . 2003 ) .

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Kinetics of SARS-CoV-2 and SARS-CoV RNA synthesis in infected Vero E6 cells.(a) Hybridization analysis of viral mRNAs isolated from SARS-CoV-2- and SARS-CoV -infected Vero E6 cells, separated in an agarose gel and probed with a radiolabelled oligonucleotide recognizing the genome and subgenomic mRNAs of both viruses. Subsequently, the gel was re-hybridized to a probe specific for 18S ribosomal RNA, which was used as a loading control. (b) Analysis of the relative abundance of each of the SARS-CoV-2 and SARS-CoV transcripts. Phosphorimager quantification was performed for the bands of the samples isolated at 12, 14 and 24 h p.i., which yielded essentially identical relative abundances. The table shows the average of these three measurements. SARS-CoV-2 mRNA sizes were calculated on the basis of the position of the leader and body transcription-regulatory sequences (ACGAAC) in the viral genome (Sawicki and Sawicki 1995, Xu, Hu et al. 2003).


Cross - reactivity of antisera raised against SARS - CoV structural non - structural . Selected antisera previously raised against SARS - CoV nsps structural cross - react with corresponding SARS - CoV - 2 . SARS - CoV - 2 - infected Vero E6 ( m . o . i . of 0 . 3 ) were fixed at 12 or 24 h p . i . For immunofluorescence microscopy , were ( double ) labelled with ( a ) a rabbit antiserum recognising nsp4 a mouse mAb recognising dsRNA ; ( b ) anti - nsp4 rabbit serum a mouse mAb directed against N ; ( c - e ) rabbit antisera recognising against nsp3 , nsp13 M , respectively . was stained with Hoechst 33258 . Bar , 20 µm .

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Cross-reactivity of antisera raised against SARS-CoV structural and non-structural proteins.Selected antisera previously raised against SARS-CoV nsps and structural proteins cross-react with corresponding SARS-CoV-2 proteins. SARS-CoV-2-infected Vero E6 cells (m.o.i. of 0.3) were fixed at 12 or 24 h p.i. For immunofluorescence microscopy, cells were (double)labelled with (a) a rabbit antiserum recognising nsp4 and a mouse mAb recognising dsRNA; (b) anti-nsp4 rabbit serum and a mouse mAb directed against the N protein; (c-e) rabbit antisera recognising against nsp3, nsp13 and the M protein, respectively. Nuclear DNA was stained with Hoechst 33258. Bar, 20 µm.


to screen for compounds that inhibit SARS - CoV - 2 replication . Inhibition of SARS - CoV - 2 replication ( coloured bars ) was tested in Vero E6 by developing a CPE - reduction evaluating several previously identified inhibitors of SARS - CoV , which was included for comparison ( grey bars ) . For each compound a two - fold serial dilution series in low - micromolar range was tested ; ( a ) Remdesivir , ( b ) chloroquine , ( c ) Alisporivir ( d ) pegylated interferon alpha - 2 . viability was assayed using CellTiter 96 ® Aqueous One Solution proliferation ( MTS ) . Compound toxicity ( solid ) was evaluated in parallel using mock - infected , compound - treated . graphs show results of 3 independent experiments , each performed using quadruplicate samples ( mean ± SD are shown ) .

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Assay to screen for compounds that inhibit SARS-CoV-2 replication.Inhibition of SARS-CoV-2 replication (coloured bars) was tested in Vero E6 cells by developing a CPE-reduction assay and evaluating several previously identified inhibitors of SARS-CoV, which was included for comparison (grey bars). For each compound a two-fold serial dilution series in the low-micromolar range was tested; (a) Remdesivir, (b) chloroquine, (c) Alisporivir and (d) pegylated interferon alpha-2. Cell viability was assayed using the CellTiter 96® Aqueous One Solution cell proliferation assay (MTS assay). Compound toxicity (solid line) was evaluated in parallel using mock-infected, compound-treated cells. The graphs show the results of 3 independent experiments, each performed using quadruplicate samples (mean ± SD are shown).


A . Maximum projections across Z stacks of example from indicated mESCs stained for L1 Tf ( red ) combined with immunostaining for L1 ORF1p ( green ) nuclei stained with DAPI ( blue ) . grey square marks position of inset . arrow heads point to foci where L1 ORF1p co - localize . Scatter plot shows number of co - localized L1 Tf - ORF1 foci in cytoplasm per ( n = 275 WT , 304 Dicer _ KO1 , 311 Dicer _ KO2 ) . Red arrow heads point to relatively larger sized L1 RNP foci . Bar graphs are mean values of of with large L1 Tf - ORF1 foci co - localizing in 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 projections across Z stacks of example from indicated mESCs immunostained for L1 ORF1p ( red ) , MOV10 ( green ) nuclei stained with DAPI ( blue ) . grey square marks position of inset in zoomed image . arrow heads point to foci where L1 ORF1p MOV10 co - localize . Scatter plot shows number of co - localized ORF1 - MOV10 foci in cytoplasm per ( n = 293 WT , 295 Dicer _ KO1 , 295 Dicer _ KO2 ) . Red arrow heads point to relatively larger sized L1 ORF1 - MOV10 foci . Bar graphs are mean values of of with large ORF1 - MOV10 foci co - localizing in 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 Dicer _ KO mESCs were treated with 0 . 5mM Sodium Arsenite ( NaAsO2 ) for 20 minutes or left untreated prior to fixation with formaldehyde . Maximum projections across Z stacks of example from indicated mESCs immunostained for G3BP1 ( red ) 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 in Dicer _ KO as compared to WT mESCs ( right side ) . LAMINB1 served as loading control . On left , maximum projections across Z stacks of example from indicated mESCs stained for L1 Tf FISH ( red ) combined with immunostaining for a resident of P - bodies , DDX6 ( green ) nuclei stained with DAPI ( blue ) . grey square marks position of inset . Yellow arrow heads point to foci where L1 DDX6 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 MOV10 in indicated lines . Immunoblot with antibody recognizing TUBULIN coomassie stained membranes depict loading , asterisk marks position of non - specific band in 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 projections across Z stacks of example from indicated mESCs immunostained for L1 ORF1p ( green ) combined with FISH for L1 Tf ( red ) 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 of BlastR colonies stained with crystal violet blue of indicated lines is shown on left . were transfected with either mutant reporter plasmid ( L1N21A ) or retrotransposition competent reporter ( L1WT ) as shown in scheme with timeline for experiment on top . Bar graphs on right depict 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 of relative luminescence from luciferase assays validating post - transcriptional regulation of MOV10 by miRNAs in HEK293T ( n = 4 biological replicates ) .

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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 of relative luminescence from luciferase assays in Hek293T to confirm specificity of post - transcriptional regulation of MOV10 by miRNAs with either wild - type ( WT ) or Mutant ( MUT ) reporters where MicroRNA Response Element ( MRE ) for depicted miRNAs in 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 representative for western blot corresponding to assess miRNA mediated regulation on MOV10 expression in mESCs ( n = 3 biological replicates ) .

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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 representative from colony forming assays for plasmid retrotransposition where miRNA mediated downregulation of MOV10 in Drosha _ KO mESCs is accompanied by an increase in L1 retrotransposition ( n = 3 biological replicates ) .

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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 representative along with quantitation showing induction of L1 RNP aggregate formation upon ectopic MOV10 expression . are maximum 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 , nuclei stained with DAPI ( blue ) . arrow heads point to foci where L1 ORF1p HA - MOV10 co - localize . Data collected from 289 Ctrl , 275 L1UP Cl1 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 representative from colony forming assays for plasmid retrotransposition in Ctrl 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 representative showing RNP aggregate formation upon ectopic expression of full L1 HA - MOV10 in WT mESCs . are maximum projections across Z stacks stained for L1 ORF1p ( red ) combined with immunostaining for HA ( green ) to detect ectopically expressed MOV10 , nuclei stained with DAPI ( blue ) . bottom panel shows where accumulation of ORF1 - MOV10 was observed in relatively large foci compared to where co - localization of two was mostly in relatively small foci ( top panel ) , 68 - 119 transfected 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 representative showing RNP aggregate formation upon ectopic expression of HA - ORF1 or HA - ORF1 in conjunction with T7 - MOV10 in WT mESCs . are maximum projections across Z stacks stained for HA ( green ) T7 ( red ) to detect ectopically expressed ORF1p MOV10 respectively , nuclei stained with DAPI ( blue ) . 100 - 147 transfected 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).


Heatmaps of AIB1 - YAP co - activated , YAP activated , AIB1 - YAP corepressed , YAP repressed genes shown in panel ( A ) . Red blue indicate high low , respectively . - sequencing was performed in triplicate statistics in Panel ( A - B ) were calculated by EdgeR with Benjamini - Hochberg multiple correction in R .

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Heatmaps of AIB1-YAP co-activated, YAP activated, AIB1-YAP corepressed, and YAP repressed genes shown in panel (A). Red and blue indicate high and low levels, respectively. RNA-sequencing was performed in triplicate and statistics in Panel (A-B) were calculated by EdgeR with Benjamini-Hochberg multiple test correction in R.


Select genes are regulated by YAP AIB1 , others are YAP regulated independently of AIB1 . Endogenous gene expression changes assessed by RT - qPCR .

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Select genes are regulated by YAP and AIB1, others are YAP regulated independently of AIB1. Endogenous gene expression changes assessed by RT-qPCR.


Significant genomic co - occupancy between AIB1 TEAD in normal breast breast cancer . Overlap of AIB1 TEAD4 ChIP - seq peaks in MCF10A MCF7 lines . Select peaks from MCF10A ChIP - seq shown . ChIP - sequencing performed in triplicate .

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Significant genomic co-occupancy between AIB1 and TEAD in normal breast and breast cancer cells. Overlap of AIB1 and TEAD4 ChIP-seq peaks in MCF10A and MCF7 cell lines. Select peaks from MCF10A ChIP-seq shown. ChIP-sequencing performed in triplicate.


TEAD binding motifs are enriched within AIB1 occupied sites across genome . HOMER de novo motif of AIB1 ChIP - seq peaks in MCF10A MCF7 .

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TEAD binding motifs are enriched within AIB1 occupied sites across the genome. HOMER de novo motif analysis of AIB1 ChIP-seq peaks in MCF10A and MCF7 cells.


AIB1 modulates TEAD transcriptional activity in a YAP dependent manner . HEK293T were transfected with CTGF - promoter reporter expression vectors of indicated .

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AIB1 modulates TEAD transcriptional activity in a YAP dependent manner. HEK293T cells were transfected with CTGF-promoter reporter and expression vectors of indicated proteins.


AIB1 is engaged with TEAD on co - activated target promoters in normal . Sequential ChIP - reChIP performed with indicated antibodies in MCF10A . of precipitated assessed by qPCR .

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AIB1 is engaged with TEAD on co-activated target promoters in normal cells. Sequential ChIP-reChIP performed with indicated antibodies in MCF10A cells. Levels of precipitated chromatin assessed by qPCR.


AIB1 is engaged with TEAD on co - activated target promoters in early stage breast cancer . Sequential ChIP - reChIP performed with indicated antibodies in MCFDCIS . of precipitated assessed by qPCR .

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AIB1 is engaged with TEAD on co-activated target promoters in early stage breast cancer cells. Sequential ChIP-reChIP performed with indicated antibodies in MCFDCIS cells. Levels of precipitated chromatin assessed by qPCR.


ANCO1 is recruited to YAP - TEAD by AIB1 . Positive Proximity Ligation ( PLA ) signal ( foci , falsely colored 594nm signal ) shown between ANCO1 - TEAD , ANCO1 - YAP , AIB1 - TEAD , AIB1 - YAP , in control are lost in AIB1 knockdown . Representative of Proximity Ligation ( PLA ) between indicated in MCF10A shGFP ( control ) shAIB1 lines using antibodies ( species ) as indicated ; Scale bars shown indicate 25μm .

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ANCO1 is recruited to YAP-TEAD by AIB1. Positive Proximity Ligation Assay (PLA) signal (white foci, falsely colored 594nm signal) shown between ANCO1-TEAD, ANCO1-YAP, AIB1-TEAD, and AIB1-YAP, in control cells are lost in AIB1 knockdown. Representative images of Proximity Ligation Assay (PLA) between indicated proteins in MCF10A shGFP (control) and shAIB1 cell lines using antibodies (species) as indicated; Scale bars shown indicate 25μm.


YAP overexpression represses , AIB1 knockdown dysregulates , ANCO1 knockdown amplifies genes at 1q21 . 3 locus in MCF10A MCFDCIS . Gene set enrichment of - seq data cDNA array data generated from MCF10A lines as indicated .

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YAP overexpression represses, AIB1 knockdown dysregulates, and ANCO1 knockdown amplifies genes at the 1q21.3 locus in MCF10A and MCFDCIS cells. Gene set enrichment analysis of RNA-seq data and cDNA array data generated from MCF10A cell lines as indicated.


YAP represses 1q21 . 3 localized genes . of endogenous S100 SPPR genes in MCF10A lines following overexpression of YAP / S94A ; were assessed by RT - qPCR .

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YAP represses 1q21.3 localized genes. Levels of endogenous S100 and SPPR genes in MCF10A cell lines following overexpression of YAP/S94A; levels were assessed by RT-qPCR.


ANCO1 knockdown increases expression of S100 SPPR genes in MCF10A MCFDCIS ; were assessed by RT - qPCR .

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ANCO1 knockdown increases expression of S100 and SPPR genes in MCF10A and MCFDCIS cells; levels were assessed by RT-qPCR.


YAP driven suppression of 1q21 genes is reverted upon ANCO1 knockdown . of endogenous 1q21 . 3 genes were assessed in YAP or S94A - expressing , control or shANCO1 MCF10A by RT - qPCR .

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YAP driven suppression of 1q21 genes is reverted upon ANCO1 knockdown. Levels of endogenous 1q21.3 genes were assessed in YAP or S94A-expressing, control or shANCO1 MCF10A cells by RT-qPCR.


TEAD is engaged at annotated enhancer regions within 1q21 . 3 . of TEAD4 ChIP - seq in MDA - MB - 231 ( GSE66081 ) , MCF7 , A549 ( GSE32465 ) at 1q21 . 3 with enhancers scored by GeneHancer . Enhancer regions of interest indicated between dotted lines .

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TEAD is engaged at annotated enhancer regions within 1q21.3. Analysis of TEAD4 ChIP-seq in MDA-MB-231(GSE66081), MCF7, and A549 Cells (GSE32465) at 1q21.3 with enhancers scored by GeneHancer. Enhancer regions of interest indicated between dotted lines.


TEAD , YAP , AIB1 , ANCO1 are engaged at annotated enhancer regions within 1q21 . 3 . Immunoprecipitation ( ChIP ) performed with indicated antibodies in MCF10A .

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TEAD, YAP, AIB1, and ANCO1 are engaged at annotated enhancer regions within 1q21.3. Chromatin Immunoprecipitation (ChIP) performed with indicated antibodies in MCF10A cells.


AIB1 is engaged with TEAD on superenhancer sites at 1q21 . 3 in normal . Sequential ChIP - reChIP performed with indicated antibodies in MCF10A . of precipitated assessed by qPCR .

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AIB1 is engaged with TEAD on superenhancer sites at 1q21.3 in normal cells. Sequential ChIP-reChIP performed with indicated antibodies in MCF10A cells. Levels of precipitated chromatin assessed by qPCR.


ANCO1 is recruited to superenhancers at 1q21 . 3 by AIB1 is significantly reduced at 1q21 . 3 following AIB1 knockdown . ChIP performed with ANCO1 antibodies in control or shAIB1 . of precipitated assessed by qPCR .

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ANCO1 is recruited to superenhancers at 1q21.3 by AIB1 and is significantly reduced at 1q21.3 following AIB1 knockdown. ChIP performed with ANCO1 antibodies in control or shAIB1 cells. Levels of precipitated chromatin assessed by qPCR.


YAP driven aberrant sphere formation of MCF10A lines is ablated by AIB1 knockdown , but further enhanced by ANCO1 knockdown . Indicated lines were plated on Matrigel allowed to form mammospheres for 7 days . 10x 40x magnifications shown , Scale bar = 200μm .

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YAP driven aberrant sphere formation of MCF10A cell lines is ablated by AIB1 knockdown, but further enhanced by ANCO1 knockdown. Indicated cell lines were plated on Matrigel and allowed to form mammospheres for 7 days. 10x and 40x magnifications shown, Scale bar = 200μm.


of aberrant spheres per field displayed in panel ( A ) , n = number of spheres .

Quantification of aberrant spheres per field displayed in panel (A), n= number of spheres.


of ( arbitrary units ) of aberrant spheres per field displayed in panel ( A ) , relative to control YAP S94A shGFP spheres . n = number of spheres .

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Quantification of size (arbitrary units) of aberrant spheres per field displayed in panel (A), relative to control YAP S94A shGFP spheres. n= number of spheres.


MCFDCIS xenograft tumors have increased expression of AIB1 - YAP , EMT , 1q21 . 3 genes during invasive transition . Schematic of experiment shown . Gene set enrichment of - seq data generated from MCFDCIS xenografts at indicated time points .

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MCFDCIS xenograft tumors have increased expression of AIB1-YAP, EMT, and 1q21.3 genes during the invasive transition. Schematic of experiment shown. Gene set enrichment analysis of RNA-seq data generated from MCFDCIS xenografts at indicated time points.


Invasive lesions have significantly less ANCO1 than encapsulated DCIS lesions . Representative of H & E , p63 , ANCO1 stained MCFDCIS xenografts as DCIS invasive lesions . Yellow circles indicate DCIS lesions black arrows indicate invasive lesions . 20x magnification ; Scale bar = 100μm . of ANCO1 positivity in DCIS Invasive lesions shown in panel ( E ) .

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Invasive lesions have significantly less ANCO1 than encapsulated DCIS lesions. Representative images of H&E, p63, and ANCO1 stained MCFDCIS xenografts as DCIS and invasive lesions. Yellow circles indicate DCIS lesions and black arrows indicate invasive lesions. 20x magnification; Scale bar = 100μm. Quantification of ANCO1 positivity in DCIS and Invasive lesions shown in panel (E).


ANCO1 decrease in breast cancer . ANCO1 mRNA expression from normal tissue ( GTEX ) ( TCGA ) for breast samples .

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ANCO1 levels decrease in breast cancer. ANCO1 mRNA expression from normal tissue (GTEX) and Tumor (TCGA) for breast samples.


Representative of ANCO1 in normal human breast DCIS lesions . DCIS lesions can be stratified to either ANCO1 negative ( center panel ) , ANCO1 positive ( right panel ) . Scale bar = 50μm .

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Representative IHC images of ANCO1 staining in normal human breast and DCIS lesions. DCIS lesions can be stratified to either ANCO1 negative (center panel), ANCO1 positive (right panel). Scale bar = 50μm.


High ANCO1 are a good prognostic indicator in Basal Breast Cancer in . KM plot showing progression free survival in with stratified ANCO1 expression . n = 360 .

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High ANCO1 levels are a good prognostic indicator in Basal Breast Cancer in patients. KM plot showing progression free survival in patients with stratified ANCO1 expression. n= 360 patients.


breast samples with reduced ANCO1 have high expression of 1q21 . 3 genes , without altered AIB1 - YAP co - activated gene expression . Dot plots showing TCGA Pan - Cancer data accessed via Xenabroswer . were stratified to high or low expression of ANCO1 by median expression .

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Patient breast tumor samples with reduced ANCO1 levels have high expression of 1q21.3 genes, without altered AIB1-YAP co-activated gene expression. Dot plots showing TCGA Pan-Cancer data accessed via Xenabroswer. Patients were stratified to high or low expression of ANCO1 by median expression.


B : Log2 gene expression fold changes of significantly induced genes ( FDR = 1 % ) across different treatment scenarios . Gene induction of immediate , delayed late responding genes is sustained upon constant activation ( ON scenario ) transient upon two - hour activation ( ON / OFF scenario ) . Genes not significantly induced upon parallel CYHX treatment were considered secondary response genes . Genes were ranked by their model - derived response time .

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B: Log2 gene expression fold changes of significantly induced genes (FDR = 1%) across different treatment scenarios. Gene induction of immediate, delayed and late responding genes is sustained upon constant activation (ON scenario) and transient upon two-hour pulse activation (ON/OFF scenario). Genes not significantly induced upon parallel CYHX treatment were considered secondary response genes. Genes were ranked by their model-derived response time.


B : pERK2 log2 fold change upon sustained activation ( left ) deduced input function ( right ) used for model fitting . Average pERK2 log2 fold change upon 4OHT treatment equals 100 % signalling amplitude .

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B: pERK2 log2 fold change upon sustained activation (left) and deduced input function (right) used for model fitting. Average pERK2 log2 fold change upon 4OHT treatment equals 100% signalling amplitude.


C : Measured gene expression kinetics resulting maximum likelihood fit of gene expression model of significantly induced response genes ( FDR = 1 % ) . Gene expression is shown as of response amplitude . Mean error is calculated as mean of absolute residuals serving as a goodness of fit measure .

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C: Measured gene expression kinetics and the resulting maximum likelihood fit of the gene expression model of all significantly induced primary response genes (FDR = 1%). Gene expression is shown as percentage of response amplitude. Mean error is calculated as the mean of absolute residuals serving as a goodness of fit measure.


B : Signalling input conditions ( left side shows deduced input function , right side shows pERK2 measurements ) : Sustained ERK signalling ( 4OHT ) , two - hour ERK signalling ( 4OHT + U0126 ) , growth factor signalling ( EGF : Epidermal growth factor , FGF : Fibroblast growth factor ) . Deduced input functions : 100 % signalling amplitude corresponds to mean induction in training condition ( 4OHT ) . Growth factor - induced input functions are linear interpolations of pERK2 log2 fold changes relative to mean induction in condition .

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B: Signalling input conditions (left side shows deduced input function, right side shows pERK2 measurements): Sustained ERK signalling (4OHT), two-hour pulse ERK signalling (4OHT+U0126), growth factor signalling (EGF: Epidermal growth factor, FGF: Fibroblast growth factor). Deduced input functions: 100% signalling amplitude corresponds to mean induction in training condition (4OHT). Growth factor-induced input functions are linear interpolations of pERK2 log2 fold changes relative to mean induction in test condition.


C : Predictions are verified with actual gene expression data . Heat maps show log2 fold changes of induced mRNAs . P : Model prediction . D : Gene expression data . E : Mean error = mean of absolute residuals .

C: Predictions are verified with actual gene expression data. Heat maps show log2 fold changes of induced mRNAs. P: Model prediction. D: Gene expression data. E: Mean error = mean of absolute residuals.


Figure 5 - Immediate late genes ( ILGs ) have long mRNA half - lives , are transcribed immediately have GC - rich promoters . A : Boxplot comparison of mRNA half - life estimates on modelling of gene induction ( model - derived ) , transcriptional shutdown ( ActD - derived ) metabolic labelling ( 4SU - derived ) . Estimates from 4OHT - pretreated HEK293 ∆ RAF1 : ER ( ON panel ) are more appropriate to characterise induced genes than estimates from unstimulated ( OFF panel ) . Genes not assigned to any cluster are shown in grey .

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Figure 5 - Immediate late genes (ILGs) have long mRNA half-lives, are transcribed immediately and have GC-rich promoters. A: Boxplot comparison of mRNA half-life estimates based on modelling of gene induction (model-derived), transcriptional shutdown (ActD-derived) and metabolic labelling (4SU-derived). Estimates from 4OHT-pretreated HEK293∆RAF1:ER cells (ON panel) are more appropriate to characterise induced genes than estimates from unstimulated cells (OFF panel). Genes not assigned to any cluster are shown in grey.


C : Log2 fold changes of transcription rate in 4OHT - treated HEK293 ∆ RAF1 : ER derived from metabolic labelling ( 4SU ) - sequencing data document immediate transcription of IEGs ILGs but delayed transcription of DEGs .

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C: Log2 fold changes of transcription rate in 4OHT-treated HEK293∆RAF1:ER cells derived from metabolic labelling (4SU) RNA-sequencing data document immediate transcription of IEGs and ILGs but delayed transcription of DEGs.


Figure 6 - Immediate late genes ( ILGs ) translate signal duration into response amplitude . A : Upper panel : pERK2 log2 fold changes upon different input scenarios ( sustained : 4OHT ; two - hour : 4OHT + U0126 ) . Lower panel : Response amplitude across temporal clusters signal durations . Bold lines show median cluster amplitude at each time point .

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Figure 6 - Immediate late genes (ILGs) translate signal duration into response amplitude. A: Upper panel: pERK2 log2 fold changes upon different input scenarios (sustained: 4OHT; two-hour pulse: 4OHT+U0126). Lower panel: Response amplitude across temporal clusters and signal durations. Bold lines show median cluster amplitude at each time point.


C : qPCR validation to different ERK signal durations . HEK293 ∆ RAF1 : ER were treated with 4OHT U0126 for different periods of time to generate signal duration scenarios of 0 . 5 to 8 hours ( cf . Fig EV1 C ) . mRNAs of IEGs EGR1 FOS relay signal duration to response duration , whereas ILGs CLU FOSL1 decode signal duration to response amplitude ( qPCR data for 17 validated mRNAs is shown in Fig EV6 A ) .

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C: qPCR validation to test different ERK signal durations. HEK293∆RAF1:ER cells were treated with 4OHT and U0126 for different periods of time to generate signal duration scenarios of 0.5 to 8 hours (cf. Fig EV1 C). mRNAs of IEGs EGR1 and FOS relay signal duration to response duration, whereas ILGs CLU and FOSL1 decode signal duration to response amplitude (qPCR data for all 17 validated mRNAs is shown in Fig EV6 A).


E : of western blots to present log2 fold changes of sample genes upon sustained ERK signalling ( 4OHT - induced ) transient ERK signalling ( EGF - induced ) in HEK293 ∆ RAF1 : ER .

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E: Quantification of western blots to present protein log2 fold changes of sample genes upon sustained ERK signalling (4OHT-induced) and transient ERK signalling (EGF-induced) in HEK293∆RAF1:ER cells.


A . Ubiquitin - vinyl sulfone ( UbVS ) assays of RING2 release by addition of pUbl pUb in trans to R0RBR parkin . Assays were performed with incubation of 2 μM wild - type or W403A R0RBR purified in a 1 : 1 with pUb . Crosslinking was initiated by addition of 10 μM UbVS in presence of pUbl or pUb ∆ G76 .

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A. Ubiquitin-vinyl sulfone (UbVS) assays of RING2 release by addition of pUbl and pUb in trans to R0RBR parkin. Assays were performed with incubation of 2 μM wild-type or W403A R0RBR purified in a 1:1 complex with pUb. Crosslinking was initiated by addition of 10 μM UbVS in the presence of pUbl or pUb∆G76.


B . Inhibition of R0RBR activation by K211N mutation .

B. Inhibition of R0RBR activation by the K211N mutation.


C . UbVS assays of RING2 release by various type of pUb .

C. UbVS assays of RING2 release by various type of pUb.


D . Autoubiquitination assays of parkin ligase activity induced by pUbl or pUb ∆ G76 in trans . 2 µM GST - R0RBR in with pUb was incubated with 50 nM E1 , 2 µM UbcH7 , 100 µM ubiquitin 4 mM ATP .

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D. Autoubiquitination assays of parkin ligase activity induced by pUbl or pUb∆G76 in trans. 2 µM GST-R0RBR in complex with pUb was incubated with 50 nM E1, 2 µM UbcH7, 100 µM ubiquitin and 4 mM ATP.


E . UbVS assays of RING2 release with R0RBR purified without pUb bound .

E. UbVS assays of RING2 release with R0RBR purified without pUb bound.


F . Autoubiquitination assays of parkin ligase activity with GST - R0RBR without pUb bound . of unmodified parkin band in each lane is indicated under gel .

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F. Autoubiquitination assays of parkin ligase activity with GST-R0RBR without pUb bound. The percentage of unmodified parkin band in each lane is indicated under the gel.


B . Phosphorylation of wild - type ( WT ) parkin chimeric Ub - R0RBR by PINK1 . Reactions were analyzed on a Phos - tag SDS - PAGE gel to assess level of phosphorylation .

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B. Phosphorylation of wild-type (WT) parkin and chimeric Ub-R0RBR by PINK1. Reactions were analyzed on a Phos-tag SDS-PAGE gel to assess the level of phosphorylation.


C . Release of RING2 by Ub - R0RBR chimera . Ubiquitin - vinyl sulfone assays ( 10 μM UbVS ) were performed with 3 μM phosphorylated non - phosphorylated full - parkin Ub - R0RBR .

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C. Release of RING2 by Ub-R0RBR chimera. Ubiquitin-vinyl sulfone assays (10 μM UbVS) were performed with 3 μM phosphorylated and non-phosphorylated full-length parkin and Ub-R0RBR.


D . Ubiquitination activity of Ub - R0RBR chimera . Autoubiquitination assays of phosphorylated full - parkin Ub - R0RBR ( 3 . 3 µM with 3 µm UbcH7 75 µM S65A ubiquitin ) were performed to assess impact of pUbl substitution by pUb on parkin activity .

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D. Ubiquitination activity of Ub-R0RBR chimera. Autoubiquitination assays of phosphorylated full-length parkin and Ub-R0RBR (3.3 µM with 3 µm UbcH7 and 75 µM S65A ubiquitin) were performed to assess the impact of pUbl substitution by pUb on parkin activity.


B . UbVS assays of release of RING2 by K6 - or K48 - linked ( pUb ) 4 chains . 10 μM UbVS was added to 2 μM R0RBR in presence of increasing amount of K6 ( pUb ) 4 or K48 ( pUb ) 4 chains .

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B. UbVS assays of release of RING2 by K6- or K48-linked (pUb)4 chains. 10 μM UbVS was added to 2 μM R0RBR in the presence of increasing amount of K6(pUb)4 or K48(pUb)4 chains.


C . Autoubiquitination assays of parkin activation by K6 ( pUb ) 4 or K48 ( pUb ) 4 . Assays with 2 µM GST - R0RBR , 50 nM E1 , 2 µM UbcH7 100 µM ubiquitin were performed in presence of increasing amount of K6 ( pUb ) 4 or K48 ( pUb ) 4 chains .

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C. Autoubiquitination assays of parkin activation by K6(pUb)4 or K48(pUb)4. Assays with 2 µM GST-R0RBR, 50 nM E1, 2 µM UbcH7 and 100 µM ubiquitin were performed in the presence of increasing amount of K6(pUb)4 or K48(pUb)4 chains.


D . Competition between K6 ( pUb ) 4 chains monomeric pUb . UbVs ( 10 μM ) was added to 20 nM of WT K211N R0RBR in presence of monomeric pUb / or K6 ( pUb ) 4 chains . Reactions were resolved on SDS - PAGE visualized with anti - parkin antibody by western blotting . of unmodified parkin band in each lane is indicated under gel .

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D. Competition between K6(pUb)4 chains and monomeric pUb. UbVs (10 μM) was added to 20 nM of WT and K211N R0RBR in the presence of monomeric pUb and/or K6(pUb)4 chains. Reactions were resolved on SDS-PAGE and visualized with anti-parkin antibody by western blotting. The percentage of unmodified parkin band in each lane is indicated under the gel.


( a - c ) At 12 - 16 h post - transfection with GFP - LC3 , NIH3T3 ( a , b ) , MEF ( b ) or HCT116 ( c ) containing vector or FLIP gene were treated with Hank ' s solution for 2 - 4 h ( a ) or with 2 μM rapamycin for 1 - 3 h ( b , c ) . Subsequently , GFP - LC3 was detected using an inverted microscope . Scale bar , 10 μm ( a ) . number of GFP - LC3 - positive dots per was counted using a microscope . Data are mean ± s . e . m . ; n = 200 - 300 ; three independent experiments ; * P 0 . 01 ; * * P 0 . 05 ) ( b , c ) . At 60 h post - transfection with control siRNA or cFLIP siRNA , MEF were transfected with GFP - LC3 ( c ) .

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(a-c) At 12-16 h post-transfection with GFP-LC3, NIH3T3 (a, b), MEF (b) or HCT116 (c) cells containing vector or the FLIP gene were treated with Hank's solution for 2-4 h (a) or with 2 μM rapamycin for 1-3 h (b, c). Subsequently, GFP-LC3 was detected using an inverted fluorescence microscope. Scale bar, 10 μm (a). The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P 0.01; **P 0.05) (b, c). At 60 h post-transfection with control siRNA or cFLIP siRNA, MEF cells were transfected with GFP-LC3 (c).


( d ) Autophagosomes were visualized through scanning electron microscopy . Scale bar , 50 nm . arrows indicate autophagosomes . Immunoblots ( IB ) of cFLIP actin expression are shown in Supplementary Information , Fig . S1c .

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(d) Autophagosomes were visualized through scanning electron microscopy. Scale bar, 50 nm. The arrows indicate autophagosomes. Immunoblots (IB) of cFLIP and actin expression are shown in Supplementary Information, Fig. S1c.


( e , f ) NIH3T3 vector NIH3T3 - KSHV - vFLIP ( e ) or TREX - BCBL - Vector TREX - BCBL - vFLIP ( f ) were treated with Hank ' s solution or 2 μM rapamycin for 2 - 4 h , respectively , followed by IB with anti - LC3 anti - actin antibody .

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(e, f) NIH3T3 vector and NIH3T3-KSHV-vFLIP cells (e) or TREX-BCBL-Vector and TREX-BCBL-vFLIP cells (f) were treated with Hank's solution or 2 μM rapamycin for 2-4 h, respectively, followed by IB with anti-LC3 and anti-actin antibody.


( g ) At 12 - 16 h post - transfection with GFP - LC3 , TREX - BCBL - vector TREX - BCBL - vFLIP were treated with doxycycline for 24 h , followed by incubation with 2 μM rapamycin for an additional 12 h . GFP - LC3 was detected using an inverted microscope . Scale bar , 5μm . number of GFP - LC3 - positive dots per was counted using a microscope . Data are mean ± s . e . m . ; n = 200 - 300 ; three independent experiments ; * P 0 . 01 ) .

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(g) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL-vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 2 μM rapamycin for an additional 12 h. GFP-LC3 was detected using an inverted fluorescence microscope. Scale bar, 5μm. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P 0.01).


( h ) Autophagosomes were visualized by scanning electron microscopy . Scale bar , 50 nm .

(h) Autophagosomes were visualized by scanning electron microscopy. Scale bar, 50 nm.


( a ) BCBL were used for immunoprecipitation ( IP ) with mouse IgG or anti - Atg3 , followed by immunoblotting ( IB ) with anti - cFLIP antibody ( left panel ) . NIH3T3 - KSHV - vFLIP were used for IP with mouse IgG or anti - Flag antibody , followed by IB with anti - Atg3 antibody ( right panel ) .

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(a) BCBL cells were used for immunoprecipitation (IP) with mouse IgG or anti-Atg3, followed by immunoblotting (IB) with anti-cFLIP antibody (left panel). NIH3T3-KSHV-vFLIP cells were used for IP with mouse IgG or anti-Flag antibody, followed by IB with anti-Atg3 antibody (right panel).


( b ) FLIP Atg3 interaction after autophagy induction . HCT116 were treated with or without 2 μM rapamycin or Hank ' s solution ( HBSS ) for 4 h before IP with anti - Atg3 antibody , followed by IB with anti - cFLIP antibody .

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(b) FLIP and Atg3 interaction after autophagy induction. HCT116 cells were treated with or without 2 μM rapamycin or Hank's solution (HBSS) for 4 h before IP with anti-Atg3 antibody, followed by IB with anti-cFLIP antibody.


( c ) Schematic diagram of human Atg3 ( top ) . N , terminus ; FR , flexible region ; C , carboxy terminus . At 48 h post - transfection with GST or GST - Atg3 along with Flag - vFLIP ( left ) or GST or GST - Atg3 along with GFP - LC3 ( right ) , HEK293T were used for GST pulldown , followed by IB with indicated antibodies .

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(c) Schematic diagram of human Atg3 (top). N, amino terminus; FR, flexible region; C, carboxy terminus. At 48 h post-transfection with GST or GST-Atg3 along with Flag-vFLIP (left) or GST or GST-Atg3 along with GFP-LC3 (right), HEK293T cells were used for GST pulldown, followed by IB with the indicated antibodies.


( d ) Schematic diagram of KSHV vFLIP ( top ) . black boxes indicate K - α2 K - α4 peptides . At 48 h post - transfection with V5 - Atg3 either GST - vFLIP DED1 mutants ( middle ) or GST - vFLIP DED2 mutants ( bottom ) , HEK293T were used for GST pulldown , followed by IB with anti - V5 antibody .

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(d) Schematic diagram of KSHV vFLIP (top). The black boxes indicate the K-α2 and K-α4 peptides. At 48 h post-transfection with V5-Atg3 and either GST-vFLIP DED1 mutants (middle) or GST-vFLIP DED2 mutants (bottom), HEK293T cells were used for GST pulldown, followed by IB with anti-V5 antibody.


( e ) At 48 h post - transfection with GST , GST - Atg3 , either Flag - vFLIP or Flag - vFLIP mAtg3 , HEK293T were used for GST pulldown , followed by IB with anti - Flag antibody .

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(e) At 48 h post-transfection with GST, GST-Atg3, and either Flag-vFLIP or Flag-vFLIP mAtg3, HEK293T cells were used for GST pulldown, followed by IB with anti-Flag antibody.


( f ) At 48 h post - transfection with GST - Atg3 GFP - LC3 along with increasing amount of Flag - vFLIP , HEK293T were used for GST pulldown , followed by IB with anti - GFP or anti - Flag antibody .

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(f) At 48 h post-transfection with GST-Atg3 and GFP-LC3 along with increasing amount of Flag-vFLIP, HEK293T cells were used for GST pulldown, followed by IB with anti-GFP or anti-Flag antibody.


( g ) NIH3T3 vector NIH3T3 vFLIP were treated with 2 μM rapamycin for 4 h used for IP with anti - Atg3 antibody , followed by IB with anti - LC3 antibody . Finally , whole - lysates ( WCLs ) were used for IB with indicated antibodies to show expression ( a - g ) .

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(g) NIH3T3 vector and NIH3T3 vFLIP cells were treated with 2 μM rapamycin for 4 h and used for IP with anti-Atg3 antibody, followed by IB with anti-LC3 antibody. Finally, whole-cell lysates (WCLs) were used for IB with the indicated antibodies to show expression (a-g).


( a ) TREX - BCBL vector were treated or untreated with 50 nM rapamycin for 6 days in presence of doxycycline subjected to scanning electron microscopy . morphologies of more than 100 dead were examined to quantify of apoptosis , autophagic , apoptosis / autophagic , others forms of .

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(a) TREX-BCBL vector cells were treated or untreated with 50 nM rapamycin for 6 days in the presence of doxycycline and subjected to scanning electron microscopy. The morphologies of more than 100 dead cells were examined to quantify the levels of apoptosis, autophagic death, apoptosis/autophagic death, and others forms of cell death.


( b ) At 12 - 16 h post - transfection with GFP - LC3 , doxycycline - treated TREX - BCBL vector , TREX - BCBL - vFLIP TREX - BCBL - vFLIP mutant were incubated with or without 50 nM rapamycin for 6 days ( top ) or 2 μM rapamycin for 12 h ( bottom ) . of ( trypan blue ) apoptosis level ( annexin V ) of rapamycin - treated was quantified as mean ± s . d . of three independent experiments . number of GFP - LC3 - positive dots per was counted using a microscope . Data are mean ± s . e . m . ; n = 200 - 300 ; three independent experiments ; * P < 0 . 01 .

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(b) At 12-16 h post-transfection with GFP-LC3, doxycycline-treated TREX-BCBL vector, TREX-BCBL-vFLIP and TREX-BCBL-vFLIP mutant cells were incubated with or without 50 nM rapamycin for 6 days (top) or 2 μM rapamycin for 12 h (bottom). The percentage of cell death (trypan blue staining) and apoptosis level (annexin V staining) of rapamycin-treated cells was quantified as mean ± s.d. of three independent experiments. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P<0.01.


( c ) BCBL1 were transfected with control siRNA or Beclin1 siRNA treated with or without rapamycin for 5 days . of was determined by trypan blue . Data are mean ± s . e . m . ; n = 200 - 300 ; three independent experiments ; * P 0 . 05 .

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(c) BCBL1 cells were transfected with control siRNA or Beclin1 siRNA and treated with or without rapamycin for 5 days. The percentage of cell death was determined by trypan blue staining. Data are mean ± s.e.m.; n = 200-300; three independent experiments; *P 0.05.


( d ) TREX - BCBL vector TREX - BCBL - vFLIP were mock - treated or treated with 50 nM rapamycin for indicated periods of time . results were quantified as mean ± s . d . of combined results from three independent experiments ; * P 0 . 001 .

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(d) TREX-BCBL vector and TREX-BCBL-vFLIP cells were mock-treated or treated with 50 nM rapamycin for the indicated periods of time. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.001.


( e ) HEK293 carrying KSHV or KSHVΔvFLIP were treated with 500 nM rapamycin for 6 days . results were quantified as mean ± s . d . of combined results from three independent experiments ; * P 0 . 01 . Trypan blue a Beckman Coulter Z2 Particle analyzer ( BC Z2 CS analyser ) were used to determine growth ( b - e ) .

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(e) HEK293 cells carrying KSHV or KSHVΔvFLIP were treated with 500 nM rapamycin for 6 days. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.01. Trypan blue staining and a Beckman Coulter Z2 Particle Count and size analyzer (BC Z2 CS analyser) were used to determine cell death and cell growth analysis (b-e).


( b ) At 12 - 16 h post - transfection with GFP - LC3 , TREX - BCBL vector TREX - BCBL - vFLIP were treated with doxycycline for 24 h , followed by incubation with 30 μM of TAT , K - α2 or K - α4 peptide ( top left ) or TAT , C - α2 , or C - α4 peptide ( bottom left ) for an additional 12 h . Subsequently , autophagy was quantified as means ± s . d . of combined results from three independent experiments ( 2 μM rapamycin treatment was included as a control ) . TREX - BCBL vector TREX - BCBL - vFLIP were treated with doxycycline for 24 h , followed by incubation with 0 , 30 , or 50 μM of TAT , K - α2 , or K - α4 peptide ( top right ) or TAT , C - α2 , or C - α4 peptide ( bottom right ) for an additional 12 h . Trypan blue was then used to determine ( as a ) . Data are mean ± s . e . m . ; n = 200 - 300 ; three independent experiments ; * P 0 . 01 ; * * P 0 . 05 .

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

(b) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 30 μM of the TAT, K-α2 or K-α4 peptide (top left) or the TAT, C-α2, or C-α4 peptide (bottom left) for an additional 12 h. Subsequently, autophagy was quantified as means ± s.d. of the combined results from three independent experiments (2 μM rapamycin treatment was included as a control). TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 0, 30, or 50 μM of the TAT, K-α2, or K-α4 peptide (top right) or the TAT, C-α2, or C-α4 peptide (bottom right) for an additional 12 h. Trypan blue staining was then used to determine cell death (as a percentage). Data are mean ± s.e.m.; n = 200-300; three independent experiments; *P 0.01; **P 0.05.


( c ) At 12 - 16 h post - transfection with GFP - LC3 , TREX - BCBL vector TREX - BCBL - vFLIP were treated with doxycycline for 24 h , followed by incubation with 30 μM of TAT , K - α2 or K - α4 peptide ( top left ) or TAT , C - α2 , or C - α4 peptide ( bottom left ) for an additional 12 h . Subsequently , autophagy was quantified as means ± s . d . of combined results from three independent experiments ( 2 μM rapamycin treatment was included as a control ) . TREX - BCBL vector TREX - BCBL - vFLIP were treated with doxycycline for 24 h , followed by incubation with 0 , 30 , or 50 μM of TAT , K - α2 , or K - α4 peptide ( top right ) or TAT , C - α2 , or C - α4 peptide ( bottom right ) for an additional 12 h GFP - LC3 were subsequently detected using an inverted microscope . Scale bar , 5 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(c) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 30 μM of the TAT, K-α2 or K-α4 peptide (top left) or the TAT, C-α2, or C-α4 peptide (bottom left) for an additional 12 h. Subsequently, autophagy was quantified as means ± s.d. of the combined results from three independent experiments (2 μM rapamycin treatment was included as a control). TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 0, 30, or 50 μM of the TAT, K-α2, or K-α4 peptide (top right) or the TAT, C-α2, or C-α4 peptide (bottom right) for an additional 12 h and GFP-LC3 puncta were subsequently detected using an inverted fluorescence microscope. Scale bar, 5 μm.


( d ) KSHV - infected BCBL1 were treated with 30 μM of K - α2 or K - α4 peptide for 12 h subjected to scanning electron microscopy . Scale bar , 50 nm .

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

(d) KSHV-infected BCBL1 cells were treated with 30 μM of the K-α2 or the K-α4 peptide for 12 h and subjected to scanning electron microscopy. Scale bar, 50 nm.


( e ) TREX - BCBL - Vector TREX - BCBL - vFLIP were treated with K - α2 K - α4 ( 30 μM each ) peptide for 12 h , followed by immunoblotting ( IB ) with anti - LC3 anti - actin antibody .

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

(e) TREX-BCBL-Vector and TREX-BCBL-vFLIP cells were treated with the K-α2 and the K-α4 (30 μM each) peptide for 12 h, followed by immunoblotting (IB) with anti-LC3 and anti-actin antibody.


( f ) NOD / SCID mice received an injection of 5 × 106 BCBL1 - Luc , followed by intraperitoneal injections with 300 μg TAT , K - α2 or Kα - 4 peptide for three ( top three panels ) . After three , a group of three mice treated with TAT peptide was challenged with 300 μg of K - α2 or Kα - 4 peptide for an additional three ( bottom panel , see Supplementary Information , Fig . S15 ) . Tumours were measured by in vivo bioluminescence imaging .

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

(f) NOD/SCID mice received an injection of 5 × 106 BCBL1-Luc cells, followed by intraperitoneal injections with 300 μg the TAT, K-α2 or Kα-4 peptide for three weeks (top three panels). After three weeks, a group of three mice treated with the TAT peptide was challenged with 300 μg of the K-α2 or Kα-4 peptide for an additional three weeks (bottom panel, see Supplementary Information, Fig. S15). Tumours were measured by in vivo bioluminescence imaging.


( a ) At 16 h post - transfection with GST , GST - K - α2 or GST - K - α4 along with Flag - vFLIP ( top ) or Flag - cFLIPL ( bottom ) , HEK293T were used for GST pulldown , followed by immunoblotting ( IB ) with anti - Flag or anti - FLIP antibody . Whole - lysates ( WCLs ) were used for IB with anti - GST , anti - Flag or anti - FLIP antibody .

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

(a) At 16 h post-transfection with GST, GST-K-α2 or GST-K-α4 along with Flag-vFLIP (top) or Flag-cFLIPL (bottom), HEK293T cells were used for GST pulldown, followed by immunoblotting (IB) with anti-Flag or anti-FLIP antibody. Whole-cell lysates (WCLs) were used for IB with anti-GST, anti-Flag or anti-FLIP antibody.


( b ) At 16 h post - transfection with V5 - Atg3 Flag - vFLIP with increasing amounts of GST - K - α2 or GST - K - α4 , HEK293T were used for GST pulldown , followed by IB with anti - Flag or anti - V5 antibody or for immunoprecipitation ( IP ) with anti - V5 antibody , followed by IB with anti - Flag antibody . WCLs were used for IB with anti - GST , anti - Flag or anti - V5 antibody .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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) At 16 h post-transfection with V5-Atg3 and Flag-vFLIP with increasing amounts of GST-K-α2 or GST-K-α4, HEK293T cells were used for GST pulldown, followed by IB with anti-Flag or anti-V5 antibody or for immunoprecipitation (IP) with anti-V5 antibody, followed by IB with anti-Flag antibody. WCLs were used for IB with anti-GST, anti-Flag or anti-V5 antibody.


( c ) At 12 - 16 h post - transfection with GST - Atg3 along with Flag - vFLIP ( first ) , Flag - cFLIPS ( second ) , or HA - LC3 ( third ) with increasing amounts of K - α2 + K - α4 peptides ( top three panels ) or C - α2 + C - α4 peptides ( bottom panel ) , HEK293T were used for GST pulldown , followed by IB with anti - Flag or anti - HA antibodies . WCLs were used for IB with anti - GST anti - Flag antibody .

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

(c) At 12-16 h post-transfection with GST-Atg3 along with Flag-vFLIP (first), Flag-cFLIPS (second), or HA-LC3 (third) with increasing amounts of the K-α2+K-α4 peptides (top three panels) or C-α2+C-α4 peptides (bottom panel), HEK293T cells were used for GST pulldown, followed by IB with anti-Flag or anti-HA antibodies. WCLs were used for IB with anti-GST and anti-Flag antibody.


( d ) At 12 - 16 h post - transfection with Flag - vFLIP , HA - LC3 V5 - Atg3 , HEK293T were treated with increasing amounts of K - α2 + K - α4 peptides used for IP anti - V5 antibody , followed by IB with anti - Flag or anti - HA antibody . WCLs were used for IB with anti - V5 , anti - HA anti - Flag antibody .

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

(d) At 12-16 h post-transfection with Flag-vFLIP, HA-LC3 and V5-Atg3, HEK293T cells were treated with increasing amounts of the K-α2+K-α4 peptides and used for IP anti-V5 antibody, followed by IB with anti-Flag or anti-HA antibody. WCLs were used for IB with anti-V5, anti-HA and anti-Flag antibody.


( b ) IRGMC mRNA was significantly enriched in miR - 196B complexes . Extracts of expressing FLAG - tagged - AGO1 transfected with biotinylated miR - 196B or control miR - 20 as indicated then with IRGMC or IRGMT plasmids were submitted to tandem affinity purification ( immunoprecipitation with FLAG antibodies followed by affinity purification on streptavidin beads ) . IRGM mRNA variants were quantified using quantitative RT - PCR ; results are presented as between miR - 196B miR - 20 ( non - relevant miRNA ) pull - downs mean of three independent experiments ± standard deviation ( s . d . ) . IP strep , immunoprecipitation straptavidine .

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

(b) IRGMC mRNA was significantly enriched in miR-196B complexes. Extracts of cells expressing FLAG-tagged-AGO1 and transfected with biotinylated miR-196B or control miR-20 as indicated and then with IRGMC or IRGMT plasmids were submitted to tandem affinity purification (immunoprecipitation with FLAG antibodies followed by affinity purification on streptavidin beads). IRGM mRNA variants were quantified using quantitative RT-PCR; results are presented as the ratio between miR-196B and miR-20 (non-relevant miRNA) pull-downs and the mean of three independent experiments ± standard deviation (s.d.). IP strep, immunoprecipitation straptavidine.


( c ) HEK293 ( IRGMC / C ) were transfected with either FLAG - tagged IRGMC or FLAG - tagged IRGMT plasmids co - transfected with miR - 196B . Immunoblotting with an IRGM antibody revealed specificity of downregulation effect mediated by miRNA IRGM mRNA interaction . of immunoblot signals are presented as IRGM expression relative to actin ( mean of at least three independent experiments ± s . d . ) .

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

(c) HEK293 cells (IRGMC/C) were transfected with either FLAG-tagged IRGMC or FLAG-tagged IRGMT plasmids and co-transfected with miR-196B. Immunoblotting with an IRGM antibody revealed the specificity of the downregulation effect mediated by the miRNA IRGM mRNA interaction. Quantification of the immunoblot signals are presented as IRGM expression relative to actin (mean of at least three independent experiments ± s.d.).


( a ) Representative in situ hybridization of frozen sections obtained from colon biopsies of genotyped healthy controls ( n = 40 ) or individuals with Crohn ' s disease ( CD ) ( n = 67 ) without or with active inflammation labeled for miR - 196A . ( L , lumen ; LP , lamina propria ) . Scale bars in upper panel , 25 μm ; scale bars in lower panel , 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

(a) Representative in situ hybridization of frozen sections obtained from colon biopsies of genotyped healthy controls (n = 40) or individuals with Crohn's disease (CD) (n = 67) without or with active inflammation and labeled for miR-196A. (L, lumen; LP, lamina propria). Scale bars in the upper panel, 25 μm; scale bars in the lower panel, 10 μm.


( b ) Epithelial or laminal fractions were captured from sections of biopsies of healthy controls ( n = 8 ) or individuals with Crohn ' s disease with no inflammation ( n = 16 ) , quiescent ( defined as low - grade inflammation ) ( n = 8 ) or acute inflammation ( n = 8 ) using capture microdissection . After extraction , miR - 196A ( black bars ) miR - 196B ( bars ) relative expression was analyzed using RNU19 , 44 U6 . To overcome possible inter - bias , lamina propria fraction value was used for relative . Due to high differences in expression between healthy inflamed tissues , results are presented as a log2 - fold . Error bars indicate s . d . of ΔΔCt values .

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

(b) Epithelial or laminal fractions were captured from sections of biopsies of healthy controls (n = 8) or individuals with Crohn's disease with no inflammation (n = 16), quiescent (defined as low-grade inflammation) (n = 8) or acute inflammation (n = 8) using laser capture microdissection. After RNA extraction, miR-196A (black bars) and miR-196B (white bars) relative expression was analyzed using RNU19, 44 and U6. To overcome possible inter-individual bias, the lamina propria fraction value was used for relative quantification. Due to high differences in expression between healthy and inflamed tissues, the results are presented as a log2-fold ratio. Error bars indicate the s.d. of the ΔΔCt values.


( c ) Representative in situ for IRGM of TMAs from colon biopsies of healthy individuals or individuals with Crohn ' s disease with a defined genotype in a healthy non - inflamed or an acute inflamed phase . Scale bars , 32 μm . ( d ) Mean ( black ) , s . e . m . ( box ) 95 % CI of mean of IRGM expression level for 40 healthy subjects ( 32 individuals with C / C 8 individuals with C / T ) 67 individuals with Crohn ' s disease ( 45 with C / C 22 with C / T ) with quiescent or inflamed colon mucosa . We performed statistical using ANOVA ( P = 0 . 0015 ) an unpaired Student ' s t - ( one tail P value is indicated on figure ) .

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

(c) Representative in situ staining for IRGM of TMAs from colon biopsies of healthy individuals or individuals with Crohn's disease with a defined genotype in a healthy non-inflamed or an acute inflamed phase. Scale bars, 32 μm. (d) Mean (black line), s.e.m. (white box) and 95% CI of the mean of the IRGM expression level for 40 healthy subjects (32 individuals with C/C and 8 individuals with C/T) and 67 individuals with Crohn's disease (45 with C/C and 22 with C/T) with quiescent or inflamed colon mucosa. We performed statistical analysis using ANOVA (P = 0.0015) and an unpaired Student's t-test (the one tail P value is indicated on the figure).


( a ) basal flux of autophagy is affected by IRGM expression level . HEK293 transfected with an IRGM - expressing plasmid , miR - 196 or siIRGM were treated with bafilomycin A1 for 2 h processed for immunoblotting with anti - LC3B . ( b ) of LC3 - II relative to actin ( mean of three independent experiments ± s . d . ) .

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

(a) The basal flux of autophagy is affected by IRGM expression level. HEK293 cells transfected with an IRGM-expressing plasmid, miR-196 or siIRGM were treated with bafilomycin A1 for 2 h and processed for immunoblotting with anti-LC3B. (b) Quantification of LC3-II relative to actin (mean of three independent experiments ± s.d.).


( c ) Downregulation of IRGM expression by miR - 196 abrogates AIEC - mediated autophagy in treated with autophagic inhibitors ( Inh ) or transfected with miR - 196B infected for 4 h with AIEC LF82 ( mean of three independent experiments ± s . d . ) .

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 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

(c) Downregulation of IRGM expression by miR-196 abrogates AIEC-mediated autophagy in cells treated with autophagic inhibitors (Inh) or transfected with miR-196B and infected for 4 h with AIEC LF82 (mean of three independent experiments ± s.d.).


( d ) Confocal microscopic examination of LC3 revealed a significant decrease in of LC3 - associated ( red ) LF82 ( green ) in miR - 196 transfected compared to control ( mean ± s . d . ) .

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

(d) Confocal microscopic examination of LC3 revealed a significant decrease in the percentage of LC3-associated (red) LF82 bacteria (green) in miR-196 transfected cells compared to control cells (mean ± s.d.).