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).


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 .

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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 and 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 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 .

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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 the genome . HOMER de novo motif analysis of AIB1 ChIP - seq peaks in MCF10A and MCF7 cells .

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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 cells were transfected with CTGF - promoter reporter and expression vectors of indicated proteins .

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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 cells . Sequential ChIP - reChIP performed with indicated antibodies in MCF10A cells . Levels of precipitated chromatin 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 cells . Sequential ChIP - reChIP performed with indicated antibodies in MCFDCIS cells . Levels of precipitated chromatin 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 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 .

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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 , 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 .

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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 . Levels of endogenous S100 and SPPR genes in MCF10A cell lines following overexpression of YAP / S94A ; levels 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 and SPPR genes in MCF10A and MCFDCIS cells ; levels 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 . Levels of endogenous 1q21 . 3 genes were assessed in YAP or S94A - expressing , control or shANCO1 MCF10A cells 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 . 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 .

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

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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 cells . Sequential ChIP - reChIP performed with indicated antibodies in MCF10A cells . Levels of precipitated chromatin 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 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 .

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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 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 .

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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.


Quantification 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.


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 .

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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 , 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 .

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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 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 ) .

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

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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 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 .

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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.


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 .

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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 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 .

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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 ) and 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 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 .

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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 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 .

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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 .

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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 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 .

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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 cells derived from metabolic labelling ( 4SU ) RNA - sequencing data document immediate transcription of IEGs and ILGs but delayed transcription of DEGs .

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 pulse : 4OHT + U0126 ) . Lower panel : Response amplitude across temporal clusters and 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 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 ) .

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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 : 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 .

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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 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 .

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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 the 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 complex with pUb was incubated with 50 nM E1 , 2 µM UbcH7 , 100 µM ubiquitin and 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 . The percentage of unmodified parkin band in each lane is indicated under the 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 and chimeric Ub - R0RBR by PINK1 . Reactions were analyzed on a Phos - tag SDS - PAGE gel to assess the 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 and non - phosphorylated full - length parkin and 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 - 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 .

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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 the presence of increasing amount of K6 ( pUb ) 4 or K48 ( pUb ) 4 chains .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 and 100 µM ubiquitin were performed in the 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 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 .

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

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 ) 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 ) .

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

(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 . The arrows indicate autophagosomes . Immunoblots ( IB ) of cFLIP and 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 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 .

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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 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 ) .

O O 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 B-MEASURED_VAR 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

(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 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 ) .

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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 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 .

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

(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 , 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 .

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

(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 ) . 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 .

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

(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 , and either Flag - vFLIP or Flag - vFLIP mAtg3 , HEK293T cells were used for GST pulldown , followed by IB with anti - Flag antibody .

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

(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 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 .

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

(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 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 ) .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

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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 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 .

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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 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 ) .

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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 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 .

O 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 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 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 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 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 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 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 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 .

O 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 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 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 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 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 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 B-MEASURED_VAR O O 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 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 .

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 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 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 .

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(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 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 .

O O O 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 O O O B-CONTROLLED_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

(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 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 .

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 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 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 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 .

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 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 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 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 .

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 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 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 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 .

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

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 O B-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 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 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

(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 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 . ) .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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

(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 ) 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 . ) .

O O O O O O O O O O O B-CONTROLLED_VAR O 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 O O O O O 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 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 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 . ) .

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 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

(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 the percentage of LC3 - associated ( red ) LF82 bacteria ( green ) in miR - 196 transfected cells compared to control cells ( mean ± s . d . ) .

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

(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.).


( e ) miR - 196 transfection leads to increased intracellular LF82 replication . Results are expressed as a fold increase ± s . e . m . of intracellular bacteria .

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

(e) miR-196 transfection leads to increased intracellular LF82 replication. Results are expressed as a fold increase ± s.e.m. of intracellular bacteria.


( f ) IRGM overexpression did not inhibit autophagic flux and it increased LC3 - II accumulation slightly in response to AIEC infection . HEK293 cells were transfected with IRGM - expressing plasmid , treated with autophagic inhibitors and infected with AIEC bacteria for 4 h ( mean of three independent experiments ± s . d . ) .

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

(f) IRGM overexpression did not inhibit autophagic flux and it increased LC3-II accumulation slightly in response to AIEC infection. HEK293 cells were transfected with IRGM-expressing plasmid, treated with autophagic inhibitors and infected with AIEC bacteria for 4 h (mean of three independent experiments ± s.d.).


( g ) Confocal microscopic examination showed an increased percentage of LC3 - associated AIEC bacteria in IRGM cells compared to control cells ( mean ± s . d . ) . ( h ) IRGM overexpression led to a high rate of intracellular replication of LF82 bacteria . ( i ) Most of the bacteria reside in non - acidic vacuoles , as shown with lysotracker at 8 h post infection ( means of three independent experiments ± s . d . ) .

O O 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 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

(g) Confocal microscopic examination showed an increased percentage of LC3-associated AIEC bacteria in IRGM cells compared to control cells (mean ± s.d.). (h) IRGM overexpression led to a high rate of intracellular replication of LF82 bacteria. (i) Most of the bacteria reside in non-acidic vacuoles, as shown with lysotracker at 8 h post infection (means of three independent experiments ± s.d.).


Quantitative expression of Lif mRNA in the colon of DSS - challenged and control mice ( n = 6 per group ) .

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

Quantitative expression of Lif mRNA in the colon of DSS-challenged and control mice (n=6 per group).


LIF measured by ELISA in mouse colon explant supernatants from DSS - challenged and control mice ( n = 4 per group ) .

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

LIF measured by ELISA in mouse colon explant supernatants from DSS-challenged and control mice (n=4 per group).


Quantitative expression of Lif mRNA in LPLs and IECs from DSS - challenged and control mice ( n = 4 per group ) .

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

Quantitative expression of Lif mRNA in LPLs and IECs from DSS-challenged and control mice (n=4 per group).


Quantitative expression of Lif mRNA in the colon of SPF mice treated with or without antibiotics ( Abx ) at the indicated time points during colitis induction ( n = 3 per group ) .

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

Quantitative expression of Lif mRNA in the colon of SPF mice treated with or without antibiotics (Abx) at the indicated time points during colitis induction (n=3 per group).


Quantitative expression of Lif mRNA in LPLs and IECs from mice treated with or without Abx at the indicated time points during colitis induction ( n = 3 per group ) .

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

Quantitative expression of Lif mRNA in LPLs and IECs from mice treated with or without Abx at the indicated time points during colitis induction (n=3 per group).


mRNA expression of the indicated genes in IECs stimulated by LPS ( 2 . 5μg / ml ) , PGN ( 3μg / ml ) or LTA ( 2μg / ml ) for 2 hours .

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

mRNA expression of the indicated genes in IECs stimulated by LPS (2.5μg/ml), PGN (3μg/ml) or LTA (2μg/ml) for 2 hours.


Serum concentrations of LIF in IEC supernatants untreated ( G ) followed by LPS ( 2 . 5μg / ml ) or LTA ( 2μg / ml ) for 12hours , as determined by ELISA .

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

Serum concentrations of LIF in IEC supernatants untreated (G) followed by LPS (2.5μg/ml) or LTA (2μg/ml) for 12hours, as determined by ELISA.


Serum concentrations of LIF in IEC supernatants pretreated with monensin ( H ) followed by LPS ( 2 . 5μg / ml ) or LTA ( 2μg / ml ) for 12hours , as determined by ELISA .

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

Serum concentrations of LIF in IEC supernatants pretreated with monensin (H) followed by LPS (2.5μg/ml) or LTA (2μg/ml) for 12hours, as determined by ELISA.


Immunoblot analysis of the expression and modification of the indicated proteins in the total cell lysate of CD4 + T cells ( I ) and IECs ( J ) treated with LIF ( 20 ng / ml ) at different time points . The lower panel shows the intensity analysis of the bands from three independent experiments .

O O O O O O O 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

Immunoblot analysis of the expression and modification of the indicated proteins in the total cell lysate of CD4+ T cells (I) and IECs (J) treated with LIF (20 ng/ml) at different time points. The lower panel shows the intensity analysis of the bands from three independent experiments.


Stat4 mRNA expression in LPLs and IECs ( n = 6 ) .

Stat4 mRNA expression in LPLs and IECs (n=6).


The proliferation rate of CD4 + T cells ( L ) and DLD - 1 cells ( M ) was determined in the presence or absence of LIF .

O O O 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

The proliferation rate of CD4+ T cells (L) and DLD-1 cells (M) was determined in the presence or absence of LIF.


Weight loss of wild - type ( WT ) ( n = 6 ) or Stat4 knockout ( Stat4 - KO ) ( n = 6 ) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3 % DSS .

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

Weight loss of wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS.


wild - type ( WT ) ( n = 6 ) or Stat4 knockout ( Stat4 - KO ) ( n = 6 ) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3 % DSS . Macroscopic changes in the colon of the mice on day 10 of colitis induction Comparison of the colon length in the mice on day 10 of colitis

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

wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS. Macroscopic changes in the colon of the mice on day 10 of colitis induction Comparison of the colon length in the mice on day 10 of colitis induction


wild - type ( WT ) ( n = 6 ) or Stat4 knockout ( Stat4 - KO ) ( n = 6 ) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3 % DSS . DAI ( stool consistency and bleeding score ) of colitis mice

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

wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS. DAI (stool consistency and bleeding score) of colitis mice treated


wild - type ( WT ) ( n = 6 ) or Stat4 knockout ( Stat4 - KO ) ( n = 6 ) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3 % DSS . H & E histology of representative colons from colitis mice . Scale bar , 20μm .

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

wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS. H&E histology of representative colons from colitis mice. Scale bar, 20μm.


Weight loss of Rag1 - / - ( n = 6 ) mice transferred with CD45RBhi CD4 + T cells and intraperitoneally injected with PBS or LIF for 8 weeks .

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 B-CONTROLLED_VAR O O O O

Weight loss of Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks.


Rag1 - / - ( n = 6 ) mice transferred with CD45RBhi CD4 + T cells and intraperitoneally injected with PBS or LIF for 8 weeks . Macroscopic changes in the colon of Rag1 - / - mice treated Comparison of colon lengths in the Rag1 - / - mice at week 8 ( n = 5 ) .

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 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

Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks. Macroscopic changes in the colon of Rag1-/- mice treated Comparison of colon lengths in the Rag1-/- mice at week 8 (n=5).


Rag1 - / - ( n = 6 ) mice transferred with CD45RBhi CD4 + T cells and intraperitoneally injected with PBS or LIF for 8 weeks . H & E histology of representative mouse colons on day 54 of colitis

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

Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks. H&E histology of representative mouse colons on day 54 of colitis induction


Colons obtained from WT or Stat4 - KO mice on day10 of the colitis model induced as in Figure 2A were immunostained with an antibody against phosphorylated STAT4 . Scale bar , 50μm .

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

Colons obtained from WT or Stat4-KO mice on day10 of the colitis model induced as in Figure 2A were immunostained with an antibody against phosphorylated STAT4. Scale bar, 50μm.


Immunoblot analysis of STAT4 phosphorylation in spleen tissue from colitis mice .

Immunoblot analysis of STAT4 phosphorylation in spleen tissue from colitis mice.


FACS staining of LPLs isolated on day10 from the colon of WT or Stat4 - KO colitis mice receiving PBS or LIF ( n = 4 per group ) . The percentage of IL - 17A + and / or IFNγ + CD4 + T cells in vivo was analyzed .

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FACS staining of LPLs isolated on day10 from the colon of WT or Stat4-KO colitis mice receiving PBS or LIF (n=4 per group). The percentage of IL-17A+ and/or IFNγ+ CD4+ T cells in vivo was analyzed.


Quantitative expression of Il17a mRNA in MLNs and LNs from colitis mice treated


FACS staining of WT and Stat4 - KO naïve CD4 + T cells treated with LIF ( 50 ng / ml ) on day 4 of induction into Th17 cell subsets .

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FACS staining of WT and Stat4-KO naïve CD4+ T cells treated with LIF (50 ng/ml) on day 4 of induction into Th17 cell subsets.


qPCR analysis of Th17 - related genes in WT or Stat4 - KO naïve CD4 + T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF ( n = 3 per group ) .

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qPCR analysis of Th17-related genes in WT or Stat4-KO naïve CD4+ T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF (n=3 per group).


FACS staining of WT or Stat4 - KO naïve CD4 + T cells treated with LIF ( 50 ng / ml ) on day 4 of induction into nonpathogenic or pathogenic Th17 cells .

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FACS staining of WT or Stat4-KO naïve CD4+ T cells treated with LIF (50 ng/ml) on day 4 of induction into nonpathogenic or pathogenic Th17 cells.


Immunoblot analysis of STAT4 phosphorylation in CD4 + T cells pretreated with PBS or LY2784544 ( 1μM ) for 12 hours followed by LIF for the indicated number of minutes .

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Immunoblot analysis of STAT4 phosphorylation in CD4+ T cells pretreated with PBS or LY2784544 (1μM) for 12 hours followed by LIF for the indicated number of minutes.


Immunoblot analysis of CD4 + T cells treated with LIF for the indicated number of minutes followed by immunoprecipitation with an anti - LIFR antibody .

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Immunoblot analysis of CD4+ T cells treated with LIF for the indicated number of minutes followed by immunoprecipitation with an anti-LIFR antibody.


Coimmunoprecipitation and immunoblot analysis of LIF - treated HEK293T cells cotransfected with the indicated constructs . The red asterisks denote phosphorylated STAT bands .

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Coimmunoprecipitation and immunoblot analysis of LIF-treated HEK293T cells cotransfected with the indicated constructs. The red asterisks denote phosphorylated STAT bands.


The mass spectrometry analysis of purified STAT4 protein revealed the phosphorylation of STAT4 at serine residues in the C terminus .

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The mass spectrometry analysis of purified STAT4 protein revealed the phosphorylation of STAT4 at serine residues in the C terminus.


Immunoblot analysis of the indicated protein modifications in the total lysate of CD4 + T cells treated with LIF ( 20 ng / ml ) at different time points .

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Immunoblot analysis of the indicated protein modifications in the total lysate of CD4+ T cells treated with LIF (20 ng/ml) at different time points.


Coimmunoprecipitation and immunoblot analysis of LIF - treated HEK293T cells cotransfected with the indicated constructs .

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Coimmunoprecipitation and immunoblot analysis of LIF-treated HEK293T cells cotransfected with the indicated constructs.


Immunoblot analysis of the distribution of the indicated proteins in the CD4 + T cell fraction .

Immunoblot analysis of the distribution of the indicated proteins in the CD4+ T cell fraction.


Cultured HeLa cells transfected with GFP - tagged STAT constructs were exposed to LIF and imaged 30 minutes later . Scale bar , 50μm .

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Cultured HeLa cells transfected with GFP-tagged STAT constructs were exposed to LIF and imaged 30 minutes later. Scale bar, 50μm.


Heatmap showing Th17 signature genes ( left ) or Th17 regulatory genes ( right ) up - or downregulated in WT or Stat4 - KO LPLs by LIF treatment ( n = 2 ) .

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Heatmap showing Th17 signature genes (left) or Th17 regulatory genes (right) up- or downregulated in WT or Stat4-KO LPLs by LIF treatment (n=2).


Quantitative mRNA expression analysis of the indicated genes in LPLs from colitis mice ( n = 3 per group ) .

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Quantitative mRNA expression analysis of the indicated genes in LPLs from colitis mice (n=3 per group).


Quantitative mRNA expression analysis of the indicated genes in WT or Stat4 - KO naïve CD4 + T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF .

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Quantitative mRNA expression analysis of the indicated genes in WT or Stat4-KO naïve CD4+ T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF.


The GAS - luciferase reporter activity assay was performed in Stat3 - / - MEFs transfected with empty vector ( EV ) or STAT4 constructs and then treated with LIF for 8 hours .

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The GAS-luciferase reporter activity assay was performed in Stat3-/- MEFs transfected with empty vector (EV) or STAT4 constructs and then treated with LIF for 8 hours.


Relative SIE - ( I ) activities in Stat3 - / - MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours .

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Relative SIE-(I) activities in Stat3-/- MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours.


Relative AGG - luciferase reporter activities in Stat3 - / - MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours .

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Relative AGG-luciferase reporter activities in Stat3-/- MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours.


ChIP analysis of naïve CD4 + T cells obtained from IL - 17A - eGFP mice cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days . GFP + cells isolated by flow cytometry were restimulated with IL - 6 in the presence or absence of LIF , crosslinked with formaldehyde and immunoprecipitated with anti - STAT3 ( top ) or anti - STAT4 ( bottom ) antibodies , followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1 - p10 primer pairs . The results are presented relative to the amount of input DNA .

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ChIP analysis of naïve CD4+ T cells obtained from IL-17A-eGFP mice cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. GFP+ cells isolated by flow cytometry were restimulated with IL-6 in the presence or absence of LIF, crosslinked with formaldehyde and immunoprecipitated with anti-STAT3 (top) or anti-STAT4 (bottom) antibodies, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA.


ChIP analysis of naïve WT or Stat4 - KO CD4 + T cells cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days . The cells were restimulated with IL - 6 or LIF for 30 minutes , crosslinked with formaldehyde and immunoprecipitated with the anti - STAT3 antibody , followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1 - p10 primer pairs . The results are presented relative to the amount of input DNA .

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ChIP analysis of naïve WT or Stat4-KO CD4+ T cells cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. The cells were restimulated with IL-6 or LIF for 30 minutes, crosslinked with formaldehyde and immunoprecipitated with the anti-STAT3 antibody, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA.


Relative Il17a - SIE - ( left ) or AGG - luciferase reporter ( right ) activities in Stat3 - / - MEFs transfected with EV , STAT3 or STAT4 or cotransfected with STAT3 and STAT4 and then treated with IL - 6 or LIF for 8 hours .

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Relative Il17a-SIE- (left) or AGG-luciferase reporter (right) activities in Stat3-/- MEFs transfected with EV, STAT3 or STAT4 or cotransfected with STAT3 and STAT4 and then treated with IL-6 or LIF for 8 hours.


Relative Il17a promoter - luciferase reporter activities in Stat3 - / - MEFs transfected with the indicated constructs and then treated with LIF for 8 hours .

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Relative Il17a promoter-luciferase reporter activities in Stat3-/- MEFs transfected with the indicated constructs and then treated with LIF for 8 hours.


Ki67 and cleaved caspase3 staining of representative colons obtained from mice on day10 of colitis induction as described in Figure 2A . The number of proliferating cells per crypt in ( A ) ( upper panel ) or apoptotic cells per field in ( B ) was determined ( n = 5 per group , 5 crypts were counted per mouse ) . Scale bar , 50μm .

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Ki67 and cleaved caspase3 staining of representative colons obtained from mice on day10 of colitis induction as described in Figure 2A. The number of proliferating cells per crypt in (A) (upper panel) or apoptotic cells per field in (B) was determined (n=5 per group, 5 crypts were counted per mouse). Scale bar, 50μm.


Quantitative mRNA expression analysis of the indicated genes in colon tissues obtained from WT or Stat4 - KO colitis mice

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Quantitative mRNA expression analysis of the indicated genes in colon tissues obtained from WT or Stat4-KO colitis mice treated


Immunoblot analysis of the expression and modification of the indicated proteins in DLD - 1 cells treated with LIF ( 20 ng / ml ) for different durations .

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Immunoblot analysis of the expression and modification of the indicated proteins in DLD-1 cells treated with LIF (20 ng/ml) for different durations.


Immunoblot analysis of YAP expression in WT or Stat4 - KO IECs treated with LIF ( 20 ng / ml ) for different durations ; β - actin was used as a loading control ( top ) . The statistical analysis of YAP protein intensity is shown in the bottom panel .

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Immunoblot analysis of YAP expression in WT or Stat4-KO IECs treated with LIF (20 ng/ml) for different durations; β-actin was used as a loading control (top). The statistical analysis of YAP protein intensity is shown in the bottom panel.


Relative YAP promoter - luciferase reporter activities in HEK293T cells transfected with EV , STAT3 or STAT4 and then treated with IL - 6 or LIF for 8 hours .

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Relative YAP promoter-luciferase reporter activities in HEK293T cells transfected with EV, STAT3 or STAT4 and then treated with IL-6 or LIF for 8 hours.


Heatmap showing the expression profile of cell proliferation - related genes in LIF - treated or control IECs ( Ctrl ) ( K ) .

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Heatmap showing the expression profile of cell proliferation-related genes in LIF-treated or control IECs (Ctrl) (K).


Principal coordinate analysis ( PCoA ) of the microbiota composition determined by 16S rRNA gene sequencing of day 0 and day 9 fecal specimens from mice treated Each symbol represents an individual mouse . PC1 and PC2 represent principal components 1 and 2 , respectively .

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Principal coordinate analysis (PCoA) of the microbiota composition determined by 16S rRNA gene sequencing of day 0 and day 9 fecal specimens from mice treated Each symbol represents an individual mouse. PC1 and PC2 represent principal components 1 and 2, respectively.


The bars depict the average relative abundance of the bacterial phyla of the microbiota in fecal specimens , as determined by taxon - based analyses as described in ( A ) ( n = 5 per group ) .

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The bars depict the average relative abundance of the bacterial phyla of the microbiota in fecal specimens, as determined by taxon-based analyses as described in (A) (n=5 per group).


16S rRNA sequencing analyses of the Proteobacteria distribution in feces from WT or Stat4 - KO colitis mice . ( n = 5 per group ) .

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16S rRNA sequencing analyses of the Proteobacteria distribution in feces from WT or Stat4-KO colitis mice. (n=5 per group).


Heatmap depicting the relative abundance of microbes at the family level ( top ) and the genus level ( bottom ) in the fecal specimens described in ( A ) ( n = 3 per group ) .

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Heatmap depicting the relative abundance of microbes at the family level (top) and the genus level (bottom) in the fecal specimens described in (A) (n=3 per group).


qPCR of the 16S rRNA genes of microbes in the phylum Proteobacteria , family Enterobacteriaceae , and genera Escherichia - Shigella in the feces of the indicated mice on day0 and day10 ( n = 6 per group ) of colitis induction .

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qPCR of the 16S rRNA genes of microbes in the phylum Proteobacteria, family Enterobacteriaceae, and genera Escherichia-Shigella in the feces of the indicated mice on day0 and day10 (n=6 per group) of colitis induction.


Quantitative mRNA expression analysis of proinflammatory genes in the colon of mice ( n = 3 per group )

Quantitative mRNA expression analysis of proinflammatory genes in the colon of mice (n=3 per group) treated


E WT and C2βD1212A / D1212A mice were subjected to thromboembolic stroke model . Representatives T2 - weighted MRI of coronal section taken 24 hours after the onset of in situ clot formation by α - thrombin were shown . Graph quantification of infarct volume measurements . Data represent mean ± SEM ( n = 23 - 25 mice per group ; * * p < 0 . 01 , Unpaired t - test ) .

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E WT and C2βD1212A/D1212A mice were subjected to thromboembolic stroke model. Representatives T2-weighted MRI of coronal section taken 24 hours after the onset of in situ clot formation by α-thrombin were shown. Graph quantification of infarct volume measurements. Data represent mean ± SEM (n=23-25 mice per group; **p<0.01, Unpaired t-test).


WT and C2βD1212A / D1212A mice were subjected to tMCAO followed by 24h of reperfusion . ( B ) Protein expression of TNFα , IL - 1β and IL - 6 . Data represent mean ± SEM ( n = 4 mice per group ; * p = 0 . 05 ; Mann - Whitney test ) . Data represent mean ± SEM ( n = 5 - 7 mice per group ; * * p < 0 . 01 ; Mann - Whitney test ) .

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WT and C2βD1212A/D1212A mice were subjected to tMCAO followed by 24h of reperfusion. (B) Protein expression of TNFα, IL-1β and IL-6. Data represent mean ± SEM (n=4 mice per group; *p=0.05; Mann-Whitney test). Data represent mean ± SEM (n=5-7 mice per group; **p<0.01; Mann-Whitney test).


A Gene expression of TNFα ( n = 8 - 10 mice for WT and n = 6 mice for C2βD1212A / D1212A ) , IL - 1β ( n = 8 mice for WT and n = 10 - 12 mice for C2βD1212A / D1212A ) , IL - 6 ( n = 8 mice for WT and n = 10 - 12 mice for C2βD1212A / D1212A ) and VCAM - 1 ( n = 8 - 10 mice for WT and n = 10 mice for C2βD1212A / D1212A ) after 1 hour of ischemia without reperfusion ( right panel ) . Gene expression of IL - 6 ( n = 10 - 12 mice for WT and n = 8 mice for C2βD1212A / D1212A ) and VCAM - 1 ( n = 12 mice for WT and n = 10mice for C2βD1212A / D1212A ) after 1 hour of ischemia followed by 4 hours of reperfusion ( bottom , left panel ) on WT and C2βD1212A / D1212A mice . The mRNA levels are given as the fold increase normalized to PGK1 relative to the healthy contralateral hemisphere of WT mice . Data represent mean ± SEM ( * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , Mann - Whitney test or unpaired t - test , as accordance ) .

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A Gene expression of TNFα (n=8-10 mice for WT and n=6 mice for C2βD1212A/D1212A), IL-1β (n=8 mice for WT and n=10-12 mice for C2βD1212A/D1212A), IL-6 (n=8 mice for WT and n=10-12 mice for C2βD1212A/D1212A) and VCAM-1 (n=8-10 mice for WT and n=10 mice for C2βD1212A/D1212A) after 1 hour of ischemia without reperfusion (right panel). Gene expression of IL-6 (n=10-12 mice for WT and n=8 mice for C2βD1212A/D1212A) and VCAM-1 (n=12 mice for WT and n=10mice for C2βD1212A/D1212A) after 1 hour of ischemia followed by 4 hours of reperfusion (bottom, left panel) on WT and C2βD1212A/D1212A mice. The mRNA levels are given as the fold increase normalized to PGK1 relative to the healthy contralateral hemisphere of WT mice. Data represent mean ± SEM (***p<0.001, **p<0.01, *p<0.05, Mann-Whitney test or unpaired t-test, as accordance).


Representative photographs of coronal brain sections and graph quantification of infarct volume ( dashed white line ) measurements in bone marrow ( BM ) chimeric mice 24 hours after tMCAO . WT > WT : transplantation of WT BM into WT hosts ; C2βD1212A > WT : transplantation of C2βD1212A / D1212A BM into WT hosts ; C2βD1212A > C2βD1212A : transplantation of C2βD1212A / D1212A BM into C2βD1212A / D1212A hosts ; WT > C2βD1212A transplantation of WT BM into kinase - dead hosts . Data represent mean ± SEM ; n = 13 - 16 mice per group ; * p < 0 . 05 * * p < 0 . 01 ; * * * p < 0 . 001 vs WT > WT , Mann - Whitney test or unpaired t - test , as accordance .

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Representative photographs of coronal brain sections and graph quantification of infarct volume (dashed white line) measurements in bone marrow (BM) chimeric mice 24 hours after tMCAO. WT>WT: transplantation of WT BM into WT hosts; C2βD1212A>WT: transplantation of C2βD1212A/D1212A BM into WT hosts; C2βD1212A>C2βD1212A: transplantation of C2βD1212A/D1212A BM into C2βD1212A/D1212A hosts; WT> C2βD1212A transplantation of WT BM into kinase-dead hosts. Data represent mean ± SEM; n=13-16 mice per group; *p<0.05 **p<0.01; ***p<0.001 vs WT>WT, Mann-Whitney test or unpaired t-test, as accordance.


B Analysis of APPL1 , EEA1 , LAMP1 and Rab11a staining in sh - Control and sh - PI3KC2β hCMEC / D3 cells . White dotted lines delineate cells ; Scale bar , 10µm ; nuclei are shown in blue ( DAPI ) .

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B Analysis of APPL1, EEA1, LAMP1 and Rab11a staining in sh-Control and sh-PI3KC2β hCMEC/D3 cells. White dotted lines delineate cells; Scale bar, 10µm; nuclei are shown in blue (DAPI).