Journal: Oncogene

Article Title: Epigenomic mapping identifies an enhancer repertoire that regulates cell identity in bladder cancer through distinct transcription factor networks

doi: 10.1038/s41388-023-02662-1

Figure Lengend Snippet: A Cell viability in RT112 and SCaBER under siRNA treatment against FOXA1. B Venn diagram comparing differentially expressed genes in RT112 and SCaBER FOXA1 KD. C GSEA plot of Msig Hallmark GSEA Analysis of genes differentially regulated in RT112 and SCaBER cell lines upon FOXA1 siRNA (2 independent siRNA, 2 replicates). D Heatmap of genes in Hallmark interferon gamma response genes that are differentially regulated in FOXA1 KD vs Ct (min Fold Change = 1,5). E Heatmap of Top Luminal TFs expression in RT112 and SCaBER cell lines upon FOXA1 KD. F PCA projection of TCGA Tumours and CRispR mutant clones on the Basal/Luminal signatures. G GSVA analysis of FOXA1 CRispR mutant clones on Urothelial differentiation signature from Eriksson et al. H GSVA analysis of FOXA1 CRispR mutant clones on Basal TFs identified in Fig. I Overrepresentation analysis of DEG in FOXA1 mutant vs Controls. J Volcano plot of Deseq2 RNA-seq analysis comparing pooled CRispR mutant FOXA1 clones in SD48 and RT112 versus controls. K Transient overexpression of HA-FOXA1 in mutant FOXA1 CRispR clones, wildtype RT112 and SCaBER. qPCR expression of ZBED2 after transfection of HA-FOXA1 relative to control plasmid, 4 days post transfection including 24 h of Puromycin selection ( n = 3 for CrispR clones, n = 2 for WT RT112 and SCaBER). Significance was calculated using 2way ANOVA test ( p -value < 0.05 = *).

Article Snippet: The human bladder cancer-derived cell lines RT112, 5637, KK47, and SCaBER were obtained from DSMZ (Heidelberg, Germany).

Techniques: Expressing, CRISPR, Mutagenesis, Clone Assay, RNA Sequencing Assay, Over Expression, Transfection, Plasmid Preparation, Selection

Journal: Oncogene

Article Title: Epigenomic mapping identifies an enhancer repertoire that regulates cell identity in bladder cancer through distinct transcription factor networks

doi: 10.1038/s41388-023-02662-1

Figure Lengend Snippet: A TCGA expression of ZBED2 by Subtypes. B TCGA expression Heatmap of ZBED2 and FOXA1 and TCGA correlation between ZBED2 and FOXA1. C Expression of FOXA1 and ZBED2 in single-cell transcriptomics from bladder cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE), highlighting the nearly mutually exclusive expression of these genes. D Genome browser view of ZBED2 and FOXA1 loci in SD48 and 5637 cell lines. E GSEA analysis (Hallmark) of ZBED2 correlated genes in basal cells population of GSM4307111 scRNA-seq Tumour. F GSEA analysis (Hallmark) of gene expression upon siZBED2 KD in RT112 (siZBED2-1 and siZBED2-2). G 3’seq STAT2 and CD274 (PD-L1) expression in RT112 and SCaBER after siZBED2 and siFOXA1.

Article Snippet: The human bladder cancer-derived cell lines RT112, 5637, KK47, and SCaBER were obtained from DSMZ (Heidelberg, Germany).

Techniques: Expressing, Single-cell Transcriptomics

Journal: Clinical Epigenetics

Article Title: Promoter methylation of DNA damage repair (DDR) genes in human tumor entities: RBBP8 / CtIP is almost exclusively methylated in bladder cancer

doi: 10.1186/s13148-018-0447-6

Figure Lengend Snippet: RBBP8 promoter methylation in human cancer cell lines. a Schematic map of the human RBBP8 gene including the relative positions and median β values of 17 CpG sites based on 450K methylation array profiling in bladder cancer (TCGA dataset) within a predicted CpG island (between base − 353 and + 648). Colored boxes present methylation level (mean ß -values for each CpG site) of the TCGA data set. Red, high methylation; blue, low methylation. + 1, RBBP8 transcription start site (TSS) of variant #1. The dots indicating the methylation sites closer to where they are depicted. CpG sites analyzed by MSP (black arrows) were indicated within the upstream promoter region close to the TSS. The relative position of the promoter area analyzed by bisulfite-pyrosequencing that comprises eight single CpG sites (gray dots) is shown as a black line. Orange boxes illustrate gene transcription-relevant regulatory core and ubiquitous elements statistically identified by using Genomatix software ( http://www.genomatix.de/ ). A — core promoter motif ten elements (− 200 to − 179); B — activator protein 2 (− 109 to − 94); C — activator-, mediator-, and TBP-dependent core promoter element for RNA polymerase II transcription from TATA-less promoters (+ 28 to + 39). b Representative MSP results of the RBBP8 promoter methylation status in cell lines of bladder, kidney, colon, lung, prostate, and breast cancer. Bands labeled with U and M reflect unmethylated and methylated DNA, respectively. Bisulfite-converted unmethylated, genomic (U-co), and polymethylated, genomic (M-co) DNA were used as positive controls. NTC, non-template control. c RBBP8 mean methylation values of analyzed CpG sites (1 to 8) using bisulfite-pyrosequencing of bladder cancer cell lines (RT4, RT112, and J82). d RBBP8 mRNA expression in normal urothelial cells (UROtsa) and bladder cancer cell lines (RT4, RT112, J82, and EJ28) arranged in relation to their RBBP8 promoter methylation status. U, unmethylated; M, methylated. Error bars: + s.e.m. e qPCR analysis for RBBP8 mRNA expression after in vitro demethylation analysis demonstrating a clear RBBP8 re-expression after treatment with both DAC (+) and TSA (+) only in RT4 with methylated RBBP8 promoter status (M) whereas in J82 cells without any RBBP8 methylation (U) RBBP8 expression was not further inducible. Non-treated cells served as controls and were set to 1. Error bars: + s.e.m.

Article Snippet: Strong RBBP8 promoter methylation in RT4 (median methylation level 90.5%) and RT112 bladder cancer cells (median methylation level: 28.5%) was confirmed by using bisulfite-pyrosequencing covering the MSP product sequence (Fig. ).

Techniques: Methylation, Variant Assay, Software, Labeling, Control, Expressing, In Vitro