Journal: Cell reports

Article Title: Loss of heat shock factor initiates intracellular lipid surveillance by actin destabilization

doi: 10.1016/j.celrep.2022.111493

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The rabbit monoclonal anti-ERM-1 (Developmental Studies Hybridoma Bank ERM1) antibody was used at a 1:1000 dilution.

Techniques: Virus, Recombinant, Electron Microscopy, Protease Inhibitor, Magnetic Beads, Reverse Transcription, SYBR Green Assay, RNA Sequencing, Microarray, ChIP-sequencing, Sequencing, Plasmid Preparation, Control, Software, Microscopy

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) Top panel: cDCs were rested 16 h and stimulated as indicated for 15 min. pSTAT3 was evaluated by flow cytometry. cDCs were gated as CD11c hi cells. Bottom panel: cDCs were rested 1 h, treated with 20 ng ml −1 IL-21, IL-6, IL-10 or Flt3L for 4 h, and intracellular pro-IL-1β analysed by flow cytometry. Shown are data representative of three experiments. ( b ) Summary of three experiments from lower panel of a . ** P values of the untreated sample compared with IL-21, IL-6 and IL-10 treated samples are 0.0002, 0.0017 and 0.0049, respectively; NS, P =0.4; error bars are means±s.e.m. ( c – e ) cDCs were stimulated with 100 ng ml −1 IL-21 or IL-10 for 1 h, then stimulated with 100 ng ml −1 LPS for 4 h, and the expression of Il1b ( c ), Il6 ( d ), and Tnf ( e ) mRNA were determined. Shown are combined results of 3 independent experiments; error bars are means±s.e.m. ( f ) Top panel: BMMs were rested without M-CSF for 16 h, treated with IL-21 or LPS for 4 h, and intracellular pro-IL-1β assessed by flow cytometry. Bottom panel: BMMs (gated as CD11c + F4/80 + cells) were rested and treated with IL-21 or LPS for 15 min. pSTAT3 was evaluated by flow cytometry. Data are representative of two experiments (total of 6 individual samples). ( g ) Top panel: CD4 + T cells were pre-activated with 5 μg ml −1 plate-bound anti-CD3+2 μg ml −1 soluble anti-CD28 for 3 days, washed, rested 16 h, treated with IL-21 for 4 h, and intracellular pro-IL-1β assessed by flow cytometry. Bottom panel: Rested CD4 + T cells were treated with IL-21 for 15 min. pSTAT3 was evaluated by flow cytometry. Data are representative of three experiments. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a – g ), cDCs were rested 1 h, treated with IL-21 for 1 h; pre-activated CD4 + T cells were washed, rested 16 h, treated with IL-21 for 1 h, and ChIP-Seq performed for STAT3, H3K4me1, and H3K27ac. ( a ) Venn diagram showing overlapping and distinctive STAT3 binding sites in cDCs and CD4 + T cells. ( b – g ) For IL-21-induced-STAT3 binding sites that differentially exist in cDCs or CD4 + T cells, there were cell type-specific binding profiles of STAT3 ( b versus c ) and H3K4me1 ( d versus e ) and H3K27ac ( f versus g ) enhancer marks. Shown are normalized read densities near peak summits for cDC- or CD4 + T-cell specific STAT3 binding sites. ‘Dips' at the plot centres ( d – g ) represent open chromatin corresponding to nucleosome depletion. Data are representative of two experiments.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: ChIP-sequencing, Binding Assay

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) Freshly isolated cDCs were rested 1 h, then stimulated with IL-21 for 4 h; CD4 + T cells were pre-activated for 3 days, then washed and rested 16 h, and stimulated with IL-21 for 4 h. Shown are genes differentially regulated by IL-21 in cDCs versus pre-activated CD4 + T cells. For cDCs, gene expression profiling was performed by microarray analysis, where cDCs were pooled from three independent experiments, as described in ref. . For pre-activated CD4 + T cells, gene expression profiling data were generated by RNA-Seq analysis. Shown are data from one of two similar experiments. ( b ) Il1b and Il21 expression in cDCs and CD4 + T cells not treated or stimulated with IL-21 as in a . Data are representative of 3 experiments. Error bars are technical duplicates of the representative experiment. ( c , d ) STAT3 binding, H3K4me1, H3K27ac, H3K4me3, and H3K27me3 marks at the Il1b locus in cDCs ( c ) and CD4 + T cells ( d ). Arrows in c indicate STAT3 binding sites at GAS1, GAS2 and GAS3 regions (GAS1: TTAgggGAA (−155 bp), TACcctGAA (−175 bp), TCCctgGAA (−195 bp); GAS2: TTTgggGAA (−2,452 bp), TTCctcCAA (−2,525 bp), TTCttcAAA (−2,549 bp); GAS3: TTGtgtGAA (−9,761 bp)). Arrows in d indicate the STAT3 binding sites identified in cDCs, but no STAT3 binding was seen at these sites in CD4 + T cells. ( e , f ) STAT3 binding, H3K4me1, H3K27ac, H3K4me3 and H3K27me3 marks at the Il21 gene locus in cDCs ( e ) and CD4 + T cells ( f ). Arrow in f indicates the STAT3 binding site at the GAS motif in the Il21 promoter region. Arrow in e indicates this same site, but no STAT3 binding was seen at this site in cDCs. Data are representative of two experiments. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Isolation, Expressing, Microarray, Generated, RNA Sequencing Assay, Binding Assay

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) cDCs were rested 1 h, treated with 100 ng ml −1 IL-21 or LPS at the indicated time points, and intracellular pro-IL-1β expression was determined. β-actin was used as control. Shown is one of two similar experiments. ( b ) cDCs were treated as in a , with 5 mM ATP added 1 h prior indicated time points in LPS-stimulated samples, and the secretion of IL-1β was determined by enzyme-linked immunosorbent assay (ELISA). Shown are combined results of two independent experiments; error bars are means±s.e.m. ( c ) CD4 + T cells from WT or Il1r −/− mice were cultured in Th17 cell differentiation conditions for 2 days, then supernatant from a 24 h, IL-21-treated cDC culture was added to the Th17 cells and incubated for 2 days, with or without addition of 10 μg ml −1 of anti-IL-1β. Expression of IL-2Rα (MFI) was determined by flow cytometry. The amount of biologically active IL-1β was determined using a standard curve constructed by assaying recombinant IL-1β. Shown are the combined results of two independent experiments with total of six samples. ( d , e ) WT, Casp1 −/− , Nlrp3 −/− and Pycard −/− cDCs were rested 1 h. In d , cDCs were then treated with 100 ng ml −1 LPS for 20–24 h with 5 mM ATP added in the final 1 h, and IL-1β assessed. Data are from two experiments; error bars are means±s.e.m. In e , cDCs were then treated with IL-21 for 20–24 h and IL-1β protein determined. Data are from five experiments. P values of IL-21-treated WT samples as compared with Casp1 −/− , Nlrp3 −/− and Pycard −/− samples are 0.99, 0.22 and 0.96, respectively; error bars are means±s.e.m. ( f ) cDCs from Ripk3 −/− , Ripk3 +/− Casp8 +/− and Ripk3 −/− Casp8 −/− mice were treated as in e , and IL-1β assessed. Data shown are from three experiments. P values of IL-21-treated Ripk3 −/− sample compared with Ripk3 +/− Casp8 +/− and Ripk3 −/− Casp8 −/− samples are 0.57 and 0.93, respectively. In b and d – f , IL-1β production in the culture supernatant was determined by ELISA. Pro-IL-1β induced by IL-21 in the culture supernatant was minimal, based on a pro-IL-1β-specific ELISA. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Cell Differentiation, Incubation, Flow Cytometry, Construct, Recombinant

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) Immunohistochemistry showing reduced methylated H3K79 levels (H3K79me2) that reflect loss of DOT1L activity in damaged areas from osteoarthritic patients (OA) as compared to their corresponding preserved areas and to cartilage from non-OA patients. Images are representative of images from four different patients. Scale bar, 400 μm. ( b ) Heat maps of differential mRNA expression determined by quantitative PCR in chondrocytes treated with DOT1L inhibitor EPZ-5676 (EPZ) or vehicle (V) from passage 0 (P0) until P2, and from preserved versus damaged areas in OA cartilage. The colour code represents the mean expression level of six and four independent patient samples respectively. ( c ) Immunoblot analysis showing decreased methylated H3K79 levels in mouse articular chondrocytes after intra-articular injection of EPZ into C57Bl/6 wild-type mouse knees. The image is representative of one experiment with protein extracts pooled from two or three mice per condition. Unprocessed original scans of blots are shown in . ( d , e ) C57/Bl6 wild-type mouse knees were injected with EPZ (5 mg kg –1 ) or vehicle and killed after 2 or 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( d ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( e ). One experiment was performed with n =10 and 5. Representative images from the 4 week evaluation are shown. * P <0.05 (two-tailed t -test). Error bars indicate mean±s.e.m.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Immunohistochemistry, Methylation, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) KEGG pathway enrichment analysis of microarray data obtained from human articular chondrocytes treated with EPZ-5676 or vehicle. Nominal P values by EASE modified Fisher Exact test using the DAVID analysis tool (see Methods section) are shown. n =5 independent patient-derived cell cultures. ( b ) Co-immunoprecipitation (Co-IP) using an anti-DOT1L antibody showing interaction between DOT1L and β -catenin in human articular chondrocytes, that is increased upon Wnt activation by LiCl and disrupted upon DOT1L inhibition. The image is representative of three experiments. ( c ) TOP/FOP reporter assay in human articular chondrocytes after Wnt stimulation by LiCl and DOT1L inhibition by EPZ. Activity is compared to untreated cells (dotted line). n =3 biologically independent experiments. *** P <0.001 by one-way ANOVA. ( d , e ) LEF1 , TCF1 and c-MYC expression measured by quantitative PCR in chondrocytes treated with EPZ-5676 and LiCl ( d ) or in LiCl-treated chondrocytes transfected with siRNA directed against DOT1L or scrambled siRNA (siDOT1L or siSCR, respectively) ( e ). Data are from one experiment with three technical replicates. ( f ) Immunohistochemistry demonstrating increased TCF1 levels in the articular cartilage of C57/Bl6 wild-type mice after injection of EPZ-5676. The images are representative of three different animals. Scale bar, 200 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Microarray, Modification, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Activation Assay, Inhibition, Reporter Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunohistochemistry, Injection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: All experiments were performed in healthy human articular chondrocytes: treated as indicated with DOT1L inhibitor EPZ-5676, Wnt activator LiCl, SIRT1 antagonist EX527 or SIRT1 agonist SRT1720; or transfected with DOT1L or scrambled siRNA. All data are presented as mean±s.e.m. ( a ) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis of DOT1L and methylated H3K79 and ( b ) acetylated H3K9 (H3K9Ac) and methylated H3K4 (H3K4me3) as markers of active transcription on the transcriptional start site (TSS) of Wnt target genes. Data are from two to five experiments. ( c ) Expression levels of TCF1 Wnt target gene measured by quantitative PCR in chondrocytes transfected with indicated specific or scrambled siRNA (siSCR). Data are from one experiment with technical triplicates. ( d ) TCF1 expression measured by quantitative PCR in the presence of SIRT1 agonist and antagonist. Data from two experiments each with technical triplicates. ( e ) Co-IP analysis using the indicated antibodies demonstrating the interaction of DOT1L and SIRT1. The image is a representative image of three biologically independent experiments. ( f ) SIRT1 activity relative to vehicle-treated cells (dotted line). Data are from three biologically independent experiments. * P <0.05, *** P <0.001 by one-way ANOVA. ( g ) ChIP-qPCR analysis of SIRT1, PPARGC1A, GCN5 and EP300 binding on the TCF1 promoter and ( h ) TCF1 expression after siRNA transfection with indicated specific or scrambled siRNA. Data from two biologically independent experiments.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Methylation, Expressing, Co-Immunoprecipitation Assay, Activity Assay, Binding Assay

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a – c ) Inactivation of SIRT1 protects against DOT1L inhibitor-induced osteoarthritis: ( a ) C57/Bl6 wild-type mouse knees were injected with DOT1L inhibitor EPZ-5676 (5 mg kg −1 ) and SIRT1 inhibitor EX527 (1.25 mg kg –1 ), or vehicle (V) and killed after 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( a ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( b ). One experiment was performed with n =3 (vehicle), 8 (EPZ) and 10 (EPZ+EX527). * P <0.05 by one-way ANOVA. Error bars indicate mean±s.e.m. ( c ) Immunohistochemistry of TCF1 in the indicated groups. TCF1 levels are increased after EPZ treatment and normalized by additional EX527 treatment. The images are representative of three different animals. Scale bar, 200 μm. ( d , e ) Loss of DOT1L function causes severe growth retardation as demonstrated by skeletal staining ( d ) and histology of the growth plate ( e ) of 4-week-old Dot1l fl/fl ;Col2-Cre −/− (Cre-neg) and Dot1l fl/fl ;Col2-Cre +/− (Dot1l Cart-KO ) mice. ( f , g ) Increased TCF1 levels in Dot1l Cart-KO mice as shown by immunohistochemistry in the indicated mice strains in the articular cartilage ( f ) and growth plate ( g ). The images are representative of three different animals. Scale bar, 200 and 100 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Injection, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: Upon Wnt signalling activation, DOT1L-containing complexes bind Wnt target gene chromatin. DOT1L interacts with SIRT1 and inhibits its function, preventing Wnt pathway hyper-activation. When Wnt signalling is activated in the absence of DOT1L function, high SIRT1 activity mediates the recruitment of transcriptional activators to LEF1 and TCF1 genes. High Wnt signalling leads to deleterious downstream effects and loss of cartilage homeostasis.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Activation Assay, Activity Assay

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Workflow showing the selection strategy for KHDRBS1 among DNA-damage response genes transcriptionally activated by Myc and significantly associated to breast cancer prognosis. Venn diagram showing the overlap between Myc-transcriptionally activated genes, DNA-damage response genes and genes associated to breast cancer. Specifically, genes were retrieved from: (i) microarray data of Myc-overexpressing mammospheres (M2) (GSE86407); (ii) published dataset (MD Anderson Human-DNA Repair Genes, https://www.mdanderson.org/documents/Labs/Wood-Laboratory/human-dna-repair-genes.html ), BioRad DNA-damage signaling pathway (SAB Target List H96) and recently published DNA-damage-associated genes (Supplementary Table ); and (iii) breast cancer versus normal breast tissues TCGA BRCA and GTeX gene expression data (Supplementary Table ). Genes were further selected for association to the worse relapse-free survival probability in breast cancer (Supplementary Table ) and novelty in the field, excluding known genes associated with BRCAness . B Box plot representing the distribution of log2 gene expression of KHDRBS1 retrieved from TCGA BRCA ( n = 1212) and GTeX ( n = 179) gene expression data (RNASeq2GeneNorm). p value was calculated with Wilcoxon rank sum test. C Kaplan–Meier plots of relapse-free survival (RFS) probability of BC patients stratified by high or low KHDRBS1 expression levels. D GSEA of DNA-repair gene signatures in IMEC-WT versus M2 ( n =3). E Scheme showing MYC and H3K4me3 PCR amplicons localization (red box) on IMEC-WT and M2 cells and layered H3K27ac signals on KHDRBS1 ( SAM68 ) promoter from ENCODE. Chromatin state was assessed by ChromHMM from ENCODE. MYC-MAX binding on multiple cell lines was assessed by ChIP-seq from ENCODE. F ChIP-qPCR estimating MYC binding at SAM68 promoter in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3). G qRT-PCR analysis of SAM68 gene expression in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3). H ChIP-qPCR of H3K4me3 deposition at KHDRBS1 ( SAM68 ) promoter in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3).

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Selection, Microarray, Gene Expression, Expressing, Binding Assay, ChIP-sequencing, ChIP-qPCR, Quantitative RT-PCR

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Kaplan–Meier plots of distant relapse-free survival (DRFS) of BC patients stratified by high or low Sam68 protein expression levels. Patients were categorized according to all molecular subtypes ( n = 211) and Luminal-A ( n = 91), Luminal-B ( n = 61), HER2 + ( n = 27), TNBC ( n = 32), HER2 + + TNBC ( n = 59) BCs. B Box plot representing the distribution of log2 gene expression of KHDRBS1 retrieved from TCGA BRCA gene expression data (RNASeq2GeneNorm). p value was calculated with Wilcoxon rank sum test. The indicated statistics refer to each molecular subtype versus basal subtypes. * p value ≤ 0.05; ** p value ≤ 0.01; **** p value ≤ 0.0001. C ChIP-qPCR estimating MYC and MAX binding at SAM68 promoter in BCSphCs (#4 and #15). Data are mean ± SEM of two independent experiment for each BCSphCs. D Expression of Myc (green color) and Sam68 (red color) on paraffin-embedded sections on parental BC and corresponding PDX tissue. Nuclei were counterstained with Toto-3 (blue color). Scale bar represents 40 µm. E Relative mRNA expression levels of MYC and KHDRBS1 on BCSphCs (#4, #13, and #21) expressing a MycER fusion protein induced by 50 nM of OHT. Data are represented as fold mRNA level changes of OHT-treated cells over vehicle. Data are represented as mean ± SD of three independent experiments. * p value ≤ 0.05; ** p value ≤ 0.01. F Cell proliferation analysis of ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#1, #4, #13, and #21) transduced with doxycyclin-inducible non-targeting (nt) and short hairpin Sam68 (shSam68). Data are represented as fold variation of shSam68 over scr. ns not significant; ** p value ≤ 0.01. G Size of tumors generated by orthotopic injection of ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#4, #13) in immunocompromised mice (NOD/SCID) at the indicated time points. Data are expressed as mean ± SD ( n = 5 mice per group). ns not significant, *** p value ≤ 0.001.

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Expressing, Gene Expression, ChIP-qPCR, Binding Assay, Transduction, Generated, Injection

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A MYC binding on DNA-damage related genes transcription start sites (TSS) on IMEC-WT and M2 breast cells. B Representative immunofluorescence analysis of Rad51 foci formation in ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC established cell lines and BCSphCs (#4) untreated (UT) and after 6 h of 8 Gy single dose γ-irradiation (IR). Nuclei were counterstained by Toto-3 (blue). Scale bar represents 10 µm. C Waterfall plot analysis of doxorubicin (DOX, 200 nM, left panel ), paclitaxel (PTX, 10 nM, middle panel ) and carboplatin (CARB, 100 µM, left panel ) response at 72 h in ER+ and TNBC BC established cell lines and BCSphCs. D Response rate distribution to chemotherapy for ER+ and TNBC BC established cell lines and BCSphCs treated as in ( C ). Middle line shows the median value of response per group, while single points represent the average value of BC cell response to DOX, PTX and CARB. Data are mean of three independent experiments. Statistical analysis was performed by using Kruskal–Wallis test. Ns not significant, * p value ≤ 0.05; ** p value ≤ 0.01. E Immunoblot analysis of PARP and Sam68 (input) and after immunoprecipitation (IP) with Sam68 antibody in BCSphCs (#15) treated for 4 h with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB). Lamin-B was used as loading control. F Immunoblot analysis of nuclear PAR, PARP, and Sam68 in scramble (scr) and short hairpin Sam68 (shSam68) ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#4) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB) for 4 h. H3 was used as loading control. G Cell proliferation analysis of ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#1, #4, #13, #21) transduced with scramble and short hairpin Sam68 (shSam68) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB) for 72 h. Data are represented as fold variation of shSam68 over scramble. Data are mean ± SD of three independent experiments. ns not significant; * p value ≤ 0.05; ** p value ≤ 0.01. H , I Relative mRNA expression levels of RAD51 (H) and MYC (I) on scramble (scr) and short hairpin Sam68 (shSam68) ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#12 and #13) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX), and carboplatin (CARB) for 24 h. Data are represented as fold mRNA level changes of treated scr and shSam68 cells over vehicle. Data are represented as mean ± SD of three independent experiments. Ns not significant, * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001.

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Binding Assay, Immunofluorescence, Irradiation, Western Blot, Immunoprecipitation, Control, Transduction, Expressing

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Schematic model of DNA-repair signaling pathways mediating the resistance of BC stem-like cells to chemotherapy. B Workflow of purification of sphere cells from serially transplanted BC PDX and their use for in vitro and in vivo drug toxicity testing. C Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs treated with vehicle (veh) and BO2. Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #13, and #21) ± SEM ( n = 5 mice per group). D Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs (#4, #13, #21) treated with vehicle, olaparib, BO2, cisplatin and olaparib plus BO2 and olaparib plus cisplatin and BO2. Arrows indicate the beginning and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #13, and #21) ± SEM ( n = 5 mice per group). **** p value ≤ 0.0001. E Immunoblot analysis of Rad51 in BCSphCs (#15) treated with dinaciclib for 24 h at the indicated concentration. Β-actin was used as loading control. F Cell viability percentage of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs (#4, #13, #15, and #21) treated with vehicle and dinaciclib (10 nM) for 6 days. Data are represented as mean ± SEM ( n = 2). * p value ≤ 0.05; *** p value ≤ 0.001. G Representative images ( left panel ) and quantification of area ( right panel ) of BC sphere cells (#21), transduced with scramble (scr) and short hairpin Sam68 (shSam68) lentiviral vectors, treated with vehicle and dinaciclib for 6 days. Data are represented as mean ± SEM ( n = 3). Ns not significant, ** p value ≤ 0.01; *** p value ≤ 0.001. Scale bar represents 100 µm. H Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs treated with vehicle (veh) and dinaciclib (din). Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #7, #13) ± SEM ( n = 5 mice per group). **** p value ≤ 0.0001. I Cell viability percentage of BCSphCs (#4, #13, #14, #15, #21) treated with vehicle, olaparib and dinaciclib, alone or in combination, at the indicated concentrations for 6 days. Data are represented as mean ± SD ( n = 3). J Synergy plot representing the combination index (CI), computed in CompuSyn by using Chou-Talalay method, for each olaparib and dinaciclib dose pair, calculated from cell viability data of BCSphCs (#13). K Size of tumors generated by orthotopic injection of BCSphCs treated with vehicle, olaparib, dinaciclib and olaparib plus dinaciclib. Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated with BCSphCs (#4, #7, #13) ± SEM ( n = 5 mice per group). *** p value ≤ 0.001.

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Protein-Protein interactions, Purification, In Vitro, In Vivo, Generated, Injection, Western Blot, Concentration Assay, Control, Transduction

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Cell viability percentage of scramble (scr) and short hairpin Sam68 (shSam68) ER+ R (MCF7) BC cell line treated with vehicle and dinaciclib (10 nM) for 6 days. Data are represented as mean ± SEM ( n = 4). * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001. B Relative mRNA expression levels of RAD51 and MYC on scramble (scr) and short hairpin Sam68 (shSam68) ER+ R (MCF7) BC cells treated with vehicle and dinaciclib for 6 days. Data are represented as fold mRNA level changes of treated scr and shSam68 over vehicle ( n = 3). C Cell viability percentage in ER+ R (MCF7) BC cells treated with vehicle, olaparib and dinaciclib, alone or in combination, at the indicated concentrations for 6 days. Data are represented as mean ± SD ( n = 3). D Kaplan–Meier plots of relapse-free survival (RFS) probability of BC patients of all molecular subtypes stratified by high or low MYC , KHDRBS1 , and RAD51 expression levels. E Schematic model showing the persistence of a BC stem-like population, characterized by high expression levels of MYC, SAM68 , and RAD51 , following standard anticancer therapies.

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Expressing

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Representative immunofluorescence images and quantifications showing that 3‐month‐old Hnf1a pKO mice have increased number of KI67 + (red) acinar cell nuclei co‐staining with DAPI (blue) and Amylase (green). Arrows point to KI67 + acinar cells in Hnf1a pKO mouse. Acinar proliferation is represented as the average of the KI67 + /Amylase + cell ratio. Quantifications were performed on 3 random fields from 3 Pdx1 Cre and 3 Hnf1a pKO mice. P ‐values are from two‐tailed Student's t ‐test. Representative H&E stainings of pancreata from Kras G12D and Hnf1a pKO ; Kras G12D mice. B–D Kras G12D and Hnf1a pKO ; Kras G12D mice have normal morphology at 7 days. E–J At 21 days, Hnf1a pKO ; Kras G12D mice show acinar‐to‐ductal metaplasia (dashed encircled areas) and regions with desmoplastic reaction (asterisk), which are not observed in Kras G12D mice (E, H). K–P At 8 weeks, Kras G12D pancreas show occasional abnormal ductal structures (dashed encircled areas in N, which is a magnification of squared dotted box in K) and Hnf1a pKO ; Kras G12D mice (L, M, O, P) present mucinous tubular complexes (black arrows), and more advanced PanINs with luminal budding (open arrows) including foci of spindle cell proliferation (asterisks) and incipient infiltrative growth (black dashed box area in O). Data information: Black dashed boxes in (E, F, K, L and O) indicate magnified areas in (H, G, N, M and P) respectively. Scale bars indicate 200 μm (A), 100 μm (C, E, F, K, L), 50 μm (O), and 20 μm (B, D, G, H–J, M, N, P).

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Breeding strategy to generate Hnf1a aKO and Ptf1a Cre ;Hnf1a +/+ control mice using Ptf1a Cre and Hnf1a LoxP alleles. B Ptf1a Cre deletes HNF1A efficiently in acinar cells but to a lesser extent in islets of Langerhans. HNF1A IHC and hematoxylin staining in pancreas of control and Hnf1a aKO mice. HNF1A is expressed in acinar and islet cells, but not in ductal cells in normal pancreas (left). HNF1A expression is depleted in acinar cells but largely not in islets in Hnf1a aKO pancreas (right). The squared dotted boxes (top) indicate magnified areas (bottom). Arrows point at ducts, arrow head at HNF1A‐positive acinar cell, and open arrow head at HNF1A positive islet cell. The dotted encircled areas indicate islets of Langerhans. Scale bar (top) 300 μm (bottom) 50 μm. C H&E stainings in pancreas of control (left) and Hnf1 aKO mice (right) showing unaltered pancreatic morphology. Scale bar 300 μm. D Expression of acinar differentiation genes in pancreas from Hnf1a aKO and controls, depicted as box plots with median values and IQR of TPM values. Whiskers extend to highest and lowest data points within 1.5× IQR outside box limits. P ‐values were determined by two‐tailed Student's t ‐test and n = 3 replicates per condition. E GSEA showing increased expression of oncogenic pathways in Hnf1a aKO pancreas. F Western blots (top) and quantifications (bottom) showing increased phospho‐p42 levels in Hnf1a aKO pancreas. Quantification of signal intensities of phospho‐p44/p42 normalized to total‐p44/p42 levels. Data are shown as dots with mean and error bars ± SD. P ‐values were determined by two‐tailed Student's t ‐test. G Distribution of pancreatic HNF1A binding sites in annotated genomic regions. H Venn diagrams illustrating that HNF1A‐bound regions are enriched in regions of active promoters and enhancers. P ‐values and odds ratios were calculated by Fisher's exact test. I Enrichment of known HNF1 motifs in the top 500 most significant HNF1A‐bound ChIP‐seq regions and percentage of regions containing each motif. The “union” is the percentage of regions with at least one motif sequence occurrence. Enrichment P ‐values are calculated using the one‐tailed binomial test. J Genome browser track for Fn1 and Timp1 genes showing upregulated expression in Hnf1a aKO and Kdm6a pKO pancreas, and absence of HNF1A or KDM6A binding in adjacent regions. Plots show TPM values normalized to Hprt with mean and error bars ± SD. P ‐values were determined by two‐tailed Student's t ‐test.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Staining, Expressing, Two Tailed Test, Western Blot, Binding Assay, ChIP-sequencing, Sequencing, One-tailed Test

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Fold change (FC) in transcripts in Hnf1a aKO versus control pancreas, plotted against significance (−Log 10 q; genes significant at q < 0.05 are shown as colored dots above the horizontal line). B GSEA showing that genes specific to differentiated acinar cells were downregulated in Hnf1a aKO pancreas, but not genes specific to islets or duct cells. Upregulated genes were enriched in genes specific to mesenchymal cells. Lineage‐enriched genes were obtained from Muraro et al . C Top functional annotations for differentially expressed genes in Hnf1a aKO pancreas. D GSEA revealed that Hnf1a aKO pancreas showed increased transcripts involved in oncogenic pathways such as EMT, MAPK, KRAS, PI3K‐AKT. E HNF1A promotes transcriptional activation of direct target genes. Left: HNF1A‐bound genes were enriched among genes that showed downregulation in Hnf1a mutants, but not among upregulated genes. P ‐values and odds ratios (O.R.) calculated by Fisher's exact test. Right: Venn diagrams showing overlap of HNF1A‐bound genes with genes that were downregulated and upregulated in Hnf1a mutant pancreas.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Functional Assay, Activation Assay, Mutagenesis

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Human orthologs of genes that were up‐ and downregulated in Hnf1a aKO pancreas were also up‐ and downregulated in human pancreas with low versus high HNF1A expression (lowest versus highest expression deciles, respectively). A random list of 717 genes controlled for similar expression levels was used for comparison. Violin plots include median and interquartile ranges. Dots are average values for each gene. Kruskal–Wallis P < 0.0001. B GSEA demonstrates that down‐ or upregulated genes in Hnf1a aKO mice (downward or upward arrows) showed down‐ or upregulation, respectively, in gene lists ranked by differential expression in non‐classical versus classical PDAC molecular subtypes from the TCGA‐PAAD study (Cancer Genome Atlas Research Network, Electronic Address Aadhe, Cancer Genome Atlas Research N, ). All enrichments had GSEA FDR q ‐values < 0.01. C Analysis of HNF1A function in 121 high‐purity cases of the ICGC‐PACA‐AU cohort identified tumors with most pronounced downregulation of direct HNF1A target genes. We performed GSEA with a gene set of 106 human orthologs of HNF1A direct targets showing downregulation in Hnf1a aKO pancreas. For each tumor sample, we performed differential expression against all other samples and used GSEA to ascertain abnormal expression of the mouse HNF1A‐dependent gene set in the tumor. Samples were ranked by the resulting normalized enrichment score (NES) and classified as either HNF1A LoF samples (purple, NES < 0; P < 0.05), or Control 1 (beige, NES < 0; P > 0.05) and Control 2 (gray, NES > 0). HNF1A LoF samples were predominantly non‐classical tumors (Collisson et al , ; Moffitt et al , ; Bailey et al , ). Putative loss‐of‐function KDM6A mutations ( KDM6A LoF mutants) were found in 19% of HNF1A LoF tumors versus 2% of all others (Fisher's P = 0.005). KDM6A mutations were considered functional if classified as “high” functional impact in ICGC (small ≤ 200‐bp deletions/insertions, single base substitutions), or as likely loss‐of‐function structural variants in Bailey et al , all of which were frame‐shift mutations. Other KDM6A mutations were classified as unknown. Heatmaps show Z ‐score‐normalized expression of deregulated genes in Hnf1a aKO pancreas. We confirmed that 85% of 106 downregulated and 60% of genes of 146 upregulated human orthologs showed differential expression across the 3 HNF1A profiles ( q < 0.05, SAM multiclass analysis). D HNF1A mRNA levels differed in HNF1A LoF and control groups (Kruskal–Wallis, P < 0.01), despite considerable variability and overlap between groups. E KDM6A mRNA levels were downregulated in HNF1A LoF tumors (Kruskal–Wallis, P < 0.001). Data information: Box plots in (D and E) show HNF1A and KDM6A expression in HNF1A LoF tumors ( n = 26) and Control 1 ( n = 39) and Control 2 ( n = 57) tumors. The horizontal central line marks the median. Box limits indicate the first and third quartiles, and whiskers extend to highest and lowest data points within 1.5× interquartile range outside box limits.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Expressing, Functional Assay

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Consensus clustered Z‐score‐normalized gene expression heatmaps of high‐purity TCGA‐PAAD and ICGC‐PACA‐AU human PDAC samples. Clustering was performed with non‐negative matrix factorization based on expression of significantly down‐ and upregulated genes in Hnf1a aKO pancreas. This revealed a cluster (HNF1A cluster 3) with concordant up‐ and downregulation of genes in Hnf1a aKO pancreas, which predominantly matched non‐classical PDAC molecular subtypes (quasimesenchymal, basal, squamous‐like, pink in top tracks), as opposed to classical PDAC subtypes (green in top tracks). Multiclass SAM differentially expressed genes ( q < 0.05) between HNF1A clusters are shown. Genes were hierarchically clustered using complete linkage with one minus Pearson correlation metrics. Along the right side of the heatmaps are green and red indicators of down‐ and upregulated genes in Hnf1a aKO pancreas, respectively. B TP63 expression was increased in HNF1A LoF tumors compared to control tumors. RSEM normalized count data are shown as box plots with interquartile range, median, and whiskers. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5× IQR outside box limits. HNF1A LoF ( n = 26), Control 1 ( n = 39), and Control 2 ( n = 57) tumors (P, Kruskal–Wallis). C, D Expression of HNF1A and KDM6A , showing downregulation in non‐classical PDAC subtypes (P, Kruskal–Wallis). Dots are RSEM normalized values presented with mean ± SD. Collisson subtypes: Quasimesenchymal (QM, n = 34) and Classical (CL, n = 54). Moffitt subtypes: Basal (BA, n = 65) and Classical (CL, n = 85). Bailey subtypes: Squamous‐like (SQ‐like, n = 31) and Pancreatic Progenitor (PP, n = 53). E, F HNF1A levels are not lower in high histological grade PDAC (E), while KDM6A levels are (F). To determine whether histological grade of human PDAC was associated with expression levels of HNF1A (E) or KDM6A (F) proteins, we evaluated contingency tables of tumor grades versus staining intensities of each case in tissue microarray (TMA) IHC. Tumor grades were scored as either moderately differentiated (G2) or poorly differentiated/high grade (G3), and staining intensities were expressed as an Immuno Reactivity Score (IRS) reflecting either No, Weak, Moderate, or Strong staining intensities (see material and methods for details). Numbers of cases and percentages (in brackets) out of total cases are indicated for each tumor grade and staining intensity. The Chi‐squared test was used to determine the probability of a significant relationship. Chi‐square and P ‐values are shown. N = 217 patients for HNF1A and N = 208 patients for KDM6A.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Expressing, Staining, Microarray

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Efficient deletion of Kdm6a in the pancreatic epithelium at E15.5. KDM6A (red) is ubiquitously expressed in all pancreatic cells. CDH1 (green) marks epithelial cells. Upon deletion, KDM6A staining is lost specifically in CDH1‐expressing epithelial cells but not in mesenchymal cells or in the stomach epithelium (white arrow heads). Scale bar indicates 100 μm. B Kdm6a mutant mice show normal fasting and fed glycemia. The horizontal stroked line indicates blood glucose levels at 250 mg/dl as a reference. C–H The pancreas of Kdm6a pKO mice were histologically normal until 8 weeks of age. At 8 weeks of age, some signs of acinar cell attrition and fat replacement could be observed, as shown in this representative image. Scale bars: 250 μm (10× magnification), 50 μm (40× magnification). I Representative picture (left) showing increased number of KI67 (red) amylase‐expressing acinar cells (green) in Kdm6a pKO pancreas. Scale bar, 250 μm. Quantifications (right) were performed on three pancreatic sections separated by at least 100 μm from 4 control and 4 Kdm6a pKO mice. Acinar cell proliferation was represented as the average of the KI67 + /Amylase + cell ratio ± SD. P ‐values were determined by two‐tailed Student's t‐ test. J GSEA plots showing enrichment of Oncostatin M and “TNFA signaling via NFKB” gene sets among genes upregulated in Kdm6a pKO pancreas. K Western blots (top) and quantifications (bottom) showing increased phospho‐p44/p42 levels in Kdm6a pKO pancreas. Quantification of signal intensities of phospho‐p44/p42 normalized to total‐p44/p42 levels. Data are shown as dots with mean and error bars ± SD. P ‐values were determined by two‐tailed Student's t ‐test. L Most significantly deranged REACTOME pathways in both KDM6A‐ and HNF1A‐deficient pancreas (see also ). M Kdm6a pKO down‐ and upregulated gene sets showed concordant deregulation in KDM6A LoF mutant tumors versus classical PDAC (based on Bailey et al 's signature) (Bailey et al , ). N Tumors with KDM6A‐deficient phenotypes showed decreased KDM6A mRNA. We created a gene set of human orthologs of Kdm6a pKO downregulated genes, and for each high‐purity tumor sample in the ICGC‐PACA‐AU, we used GSEA to test for enrichment of this gene set in gene lists that were rank‐ordered by differential expression in the individual sample versus all other samples. Samples with NES < 0 and P ‐value < 0.05 were considered as having KDM6A LoF phenotypes and were compared against all other samples. Z ‐score‐normalized count data are shown as box plots with IQR, median, and whiskers. Whiskers extend to highest and lowest data points within 1.5× IQR outside box limits. P ‐values were determined by two‐tailed Student's t ‐test. O Gene sets that showed up‐ or downregulation in non‐classical human PDAC showed concordant enrichment in up‐ or downregulated genes in Kdm6a pKO versus control pancreas. GSEA NES and FDR q‐values are shown. P Genomic distribution of KDM6A binding sites in mouse pancreas. Q, R Top: ChIP‐seq and RNA‐seq tracks in control and Kdm6a pKO pancreas, in two loci harboring downregulated genes ( Kif12 , Gprc5c ) in Kdm6a pKO pancreas. Bottom: ChIP‐qPCR validations for regions highlighted in green (R1, R2, R3), showing that Kdm6a mutants have increased H3K27me3 and decreased H3K27ac in most regions. H3K4me1 was also decreased in mutants in distal sites. Error bars show SD, and P ‐values were determined by two‐tailed Student's t ‐test, n = 3. S KDM6A‐bound regions are enriched in active pancreas promoters and enhancers. P ‐values are calculated by Fisher's exact test. T, U Genome Browser examples (top) of HNF1A and KDM6A binding to genes known as negative regulators of EMT: Gstp1 (T) and Deptor in (U) that are downregulated in Hnf1a aKO and Kdm6a pKO pancreas (bottom). Plots show TPM values normalized to Hprt with mean and error bars ± SD. N = 4 per condition and P ‐values were determined by two‐tailed Student's t ‐test.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Staining, Expressing, Mutagenesis, Two Tailed Test, Western Blot, Binding Assay, ChIP-sequencing, RNA Sequencing Assay

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Motif analysis in functional KDM6A‐bound regions, showing top ten de novo motifs ranked by P ‐value determined by HOMER software. B Co‐binding analysis in functional KDM6A‐bound enhancer and promoter regions revealed that HNF1A was the most enriched co‐bound TF among three other acinar cell TFs. Binding regions of TAL1 in a non‐pancreatic cell type and random binding sites were used as negative controls. P ‐values were determined by Fisher's exact test for peak comparisons using all enhancer and promoter regions as background. C The most downregulated genes in Kdm6a pKO pancreas are shown ranked by q‐value and are almost invariably bound by HNF1A and downregulated in Hnf1a aKO pancreas, or known to be direct HNF1A‐dependent target genes from other studies (red and purple, respectively). D, E GSEA analysis on the Hnf1a aKO and Kdm6a pKO ranked‐ordered gene lists versus their reciprocal up‐ or downregulated gene sets, demonstrated that KDM6A and HNF1A regulate similar genes. F Expression changes in Hnf1a aKO and Kdm6a pKO pancreas, showing that genes bound by KDM6A and downregulated in Kdm6a pKO pancreas (red dots) were generally downregulated in Hnf1a aKO pancreas. G HNF1A and KDM6A co‐occupy the same regions in Pah , which is downregulated in Hnf1a and Kdm6a knock‐out pancreas. H Genes that were co‐bound by KDM6A and HNF1A showed greatest downregulation in Kdm6a pKO pancreas, compared with KDM6A‐bound genes that were not bound by HNF1A. Box plots show median and IQR of Log 2 TPM fold‐changes and whiskers extend to highest and lowest data points within 1.5× IQR outside box limits. P ‐values were determined by two‐tailed Student's t ‐tests and n = 4 replicates per condition.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Functional Assay, Software, Binding Assay, Expressing, Knock-Out, Two Tailed Test

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A–D Left: Genome Browser examples of loci co‐bound by HNF1A and KDM6A, showing loss of KDM6A binding in HNF1A‐deficient pancreas (region highlighted in green) and decreased RNA levels in HNF1A‐deficient pancreas. Right: ChIP‐qPCRs showing loss of KDM6A and HNF1A binding in highlighted regions in left and qPCRs show downregulation of target genes in Hnf1a ‐KO pancreas. Error bars show SD, and P ‐values were determined by two‐tailed Student's t ‐test, n = 4 for ChIP‐qPCRs and n = 3 for qPCRs.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Binding Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A HNF1A binding to chromatin is unaffected in Kdm6a pKO pancreas. Scatterplot showing unchanged HNF1A binding in Kdm6a pKO versus control pancreas (e.g., see also Appendix Fig S6A–D ). B Western blot showing KDM6A depletion in two clones from Kdm6a ‐KO acinar cell lines. C–F qPCR in Kdm6a ‐KO acinar cell lines (KO1 and KO2) shows reduced expression of HNF1A bound genes, while ChIP‐qPCRs for HNF1A show that its binding to those genes is unchanged when KDM6A is depleted. Selected genes and HNF1A binding regions were from Appendix Fig S6A–D . qPCR data are relative mRNA expression of indicated genes versus Hprt . ChIP‐qPCR values indicate fold enrichment relative to control region. Error bars show ± SD, and P ‐values were determined by two‐tailed Student's t ‐test. G GSEA analysis on ranked‐ordered gene list from Kras G12D ;Kdm6a pKO data from Andricovich et al versus gene sets from up‐ or downregulated genes in Hnf1a aKO and Kdm6a pKO pancreas demonstrates that KDM6A and HNF1A regulate similar genes in normal and Kras G12D ‐transformed pancreas. H GSEA, comparing rank‐ordered expression data from Kras G12D ;Kdm6a pKO , Hnf1a aKO , and Kdm6a pKO mice with gene sets from Andricovich et al , shows that most of the pathways that are enriched in Kdm6a ‐deficient pancreatic cancer are dependent on HNF1A and KDM6A function in the non‐tumoral pancreas.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Binding Assay, Western Blot, Clone Assay, Expressing, Two Tailed Test, Transformation Assay

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Co‐immunoprecipitation of endogenous HNF1A and KDM6A followed by Western blot demonstrated that HNF1A is in the same complex as KDM6A. B Western blot showing loss of HNF1A and unchanged KDM6A in Hnf1a −/− pancreas. C Differential binding analysis of KDM6A in Hnf1a −/− versus wild‐type pancreas. Pink dots below zero (1,873 sites) show regions with reduced KDM6A binding, and pink dots above zero (118 sites) are regions with increased binding at FDR < 0.05. D, E Regions that show reduced KDM6A binding in Hnf1a −/− chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs. P ‐values in (D) were calculated with two‐tailed Mann–Whitney U‐test and in (E) with Fisher's exact test. F KDM6A binding is markedly reduced in HNF1A‐ and KDM6A‐co‐bound regions in Hnf1a −/− pancreas, but not in other KDM6A‐bound regions. G Genes that loose KDM6A binding in Hnf1a ‐mutant pancreas are predominantly downregulated in Hnf1a aKO pancreas and are direct HNF1A target genes (red dots). H Summary model depicting that HNF1A recruits KDM6A to genomic binding sites, activating an acinar differentiation program that indirectly suppresses core oncogenic pathways. Defective HNF1A or KDM6A function results in failure of this shared program, with increased activity of pathways that, in the presence of KRAS mutations, promote high‐grade non‐classical PDAC with sarcomatoid features.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Two Tailed Test, MANN-WHITNEY, Mutagenesis, Activity Assay

Journal: Cell Death & Disease

Article Title: The oncogene Etv5 promotes MET in somatic reprogramming and orchestrates epiblast/primitive endoderm specification during mESCs differentiation

doi: 10.1038/s41419-018-0335-1

Figure Lengend Snippet: a Comparison of Etv5 expression levels between mESC lines and somatic cell lines. The relative expression was based on the microarray data from BioGPS database. b The interactions between pluripotency relevant regulators and Etv5 . ChIP-seq and ChIP-chip data with Etv5 as target were extracted from ESCAPE database and used for drawing these interactions. c Growth curve of J1 mESCs stably infected with shCtrl and Etv5 shRNA (shEtv5-7). d RT-qPCR analysis of Etv5 and Tet2 in mESCs stably infected with shCtrl, Etv5 shRNA (shEtv5-7), and shEtv5-7 plus lentiviral Etv5 . Data are shown as mean ± SD ( n = 3). * P < 0.05, *** P < 0.001. Two-way ANOVA with Sidak’s multiple comparisons test was used for c . One-way ANOVA with Dunnett’s multiple comparisons test for d . e Western blotting of TET2 in mESCs stably infected with shCtrl, shEtv5-7, and shEtv5-7 + Etv5 . GAPDH was used as internal control. The relative quantification is also shown. f Dot blot of global 5hmC in mESCs stably infected with shCtrl, shEtv5-7, and shEtv5-7 plus lentiviral Etv5 . The blotting result of serially diluted genomic DNA (100-3.125 ng) was shown (left panel). The same membrane stained with methylene blue as DNA loading control was also presented (right panel)

Article Snippet: For transgenes integration detection, genomic DNA of mouse iPSCs was extracted using TIANamp Genomic DNA Kit (TIANGEN).

Techniques: Comparison, Expressing, Microarray, ChIP-sequencing, ChIP-chip, Stable Transfection, Infection, shRNA, Quantitative RT-PCR, Western Blot, Control, Quantitative Proteomics, Dot Blot, Membrane, Staining