Journal: Nucleic acids research

Article Title: Human HELQ regulates DNA end resection at DNA double-strand breaks and stalled replication forks.

doi: 10.1093/nar/gkad940

Figure Lengend Snippet: Figure 7. HELQ functions in parallel to CtIP to preserve fork integrity. ( A ) Fork degradation assays were performed in HELQ KO #1 cells expressing the indicated shRNAs. ( B ) HU (2 mM, 4 h) induced RPA2 phosphorylation assa y s w ere perf ormed in wild type (WT) and HELQ KO #1 cells expressing shCtIP or vector (Ctrl). ( C ) HCT116 cells (WT) and HCT116 CtIP KO cells (CtIP KO) expressing shHELQ or vector (Ctrl) were treated with 2 mM HU for 4 h and subjected to metaphase spread assay. The chromosomal aberrations per chromosome spread are plotted. ( D ) The HU (2 mM, 4 h) induced RAD51-nascent DNA (EdU) interaction w as analyz ed b y PLA in U2OS cells (WT) and HELQ KO #1 cells expressing shCtIP or v ector (Ctrl). R epresentativ e images and quantification of the a v erage number of PLA foci per nucleus are shown. Scale bar = 5 μm. ( E ) WT and HELQ KO #1 cells expressing shCtIP or vector (Ctrl) were treated with the indicated concentration of HU for 48 h, then a cell viability assay was performed. ( F ) The viability of WT and HELQ KO #1 cells expressing shCtIP or vector (Ctrl) was analyzed by colony formation assay. Representative images (left) and quantifications of the relative survival (right) relative to vector (Ctrl) infected wild type U2OS cells (WT), which was arbitrarily set to 100%, are shown. In panels A, C, D, E and F, the data represent the means ± SD from at least three independent experiments. * P < 0.05, *** P < 0.001, **** P < 0.0001, n.s.: not significant. Mann–Whitney test was used in A, C and D; Student’s t -test was used in E and F.

Article Snippet: Antibodies used in this study included the following: FLAG (F1804, Sigma-Aldrich), HELQ (PA5-65181, Sigma-Aldrich and 19436s, Cell Signaling Technology), Biotin (200-002-211, Jackson Immunoresearch and A150-109A, Bethyl), γH2AX (05–636, Millipore), CtIP (A300-488A, Bethyl), RPA2 S4 / 8p (A300-245A, Bethyl), RPA2 (A300-244A, Bethyl), BRCA1 (9010s, Cell Signaling Technology), BRCA2 (10741S, Cell Signaling Technology), MRE11 (ab208020, Abcam), RAD51 (ab133534, Abcam), BrdU (ab6326, Abcam and 347580, BD), MUS81(ab247136, Abcam), BLM (ab2179, Abcam), DNA2 (18727-1-AP , Proteintech), EXO1 (16253-1-AP , Proteintech), HLTF (Ab17984, Abcam), SMARCAL1 (sc-376377, Santa Cruze), ZRANB3 (A303-033A, Bethyl).

Techniques: Expressing, Phospho-proteomics, Plasmid Preparation, Concentration Assay, Viability Assay, Colony Assay, Infection, MANN-WHITNEY

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 bladder cancer of the TCGA data set. a Visualization of the promoter methylation of the RBBP8 gene as a scatterplot. The β values for each sample were jittered around the probe location and plotted as points. The per sample type 90% quantiles of methylation are shown as smoothed lines. The colors represent different sample groups (BLCA, bladder cancer; HNSC, head-neck squamous cell carcinoma). b The Pearson correlation coefficients ( ρ ) for probe pairs in the core region of the promoter are shown as heatmap demonstrating a high degree of correlation between probes ( ρ > 0.7). c The beta values of all probes of the promoter region were summarized by their median value, stratified by the sample as well as tissue type, and visualized as a box plot. For RBBP8 gene loci, a frequent hypermethylation ( β > 0.25 in > 5% of cases) was only observable in 37, 10, and 7% of bladder urothelial carcinoma (BLCA), head-neck squamous cell carcinoma (HNSC), and lung squamous cell carcinoma (LUSC), respectively. d Inverse correlation between RBBP8 methylation (defined CpG gene set) and RBBP8 mRNA expression in primary bladder cancer (BLCA), neck squamous cell carcinoma (HNSC), and lung squamous carcinoma (LUSC) samples of the TCGA data portal. Spearman correlation BLCA: − 0.32, p < 0.001; Spearman correlation HNSC: − 0.04, p = ns; Spearman correlation LUSC: 0.08, p = ns. e Box plot illustrates significant downregulation of RBBP8 methylation in primary tumors featuring increased RBBP8 promoter methylation ( β value > 0.4). Horizontal lines — grouped medians. Boxes — 25 to 75% quartiles. Vertical lines — range, peak, and minimum. * p < 0.05, ** p < 0.01, ns: not significant. f Box plot shows RBBP8 methylation in primary tumors classified by intrinsic subtypes. g Kaplan-Meier survival curves display overall survival (OS) of patients with high RBBP8 methylation ( β value > 0.4, dark gray curve) compared to low RBBP8 methylation ( β value ≤ 0.4, gray curve) based on TCGA datasets

Article Snippet: Primary anti-RBBP8 antibody (dilution 1:200) (Atlas Antibodies, HPA039890, Sigma-Aldrich, Germany) was linked with DAKO EnVisionTMFLEX system and visualized with DAKO Liquid DAB Substrate Chromogen System in a DAKO Autostainer plus (K8024, K3468, DAKO).

Techniques: Methylation, Expressing

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: Clinicopathological parameters in relation to RBBP8 promoter methylation of the BLCA TCGA dataset

Article Snippet: Primary anti-RBBP8 antibody (dilution 1:200) (Atlas Antibodies, HPA039890, Sigma-Aldrich, Germany) was linked with DAKO EnVisionTMFLEX system and visualized with DAKO Liquid DAB Substrate Chromogen System in a DAKO Autostainer plus (K8024, K3468, DAKO).

Techniques: Methylation

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: Univariate analysis of clinicopathological parameters influencing OS

Article Snippet: Primary anti-RBBP8 antibody (dilution 1:200) (Atlas Antibodies, HPA039890, Sigma-Aldrich, Germany) was linked with DAKO EnVisionTMFLEX system and visualized with DAKO Liquid DAB Substrate Chromogen System in a DAKO Autostainer plus (K8024, K3468, DAKO).

Techniques: Methylation

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: Primary anti-RBBP8 antibody (dilution 1:200) (Atlas Antibodies, HPA039890, Sigma-Aldrich, Germany) was linked with DAKO EnVisionTMFLEX system and visualized with DAKO Liquid DAB Substrate Chromogen System in a DAKO Autostainer plus (K8024, K3468, DAKO).

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

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: Validation of RBBP8 promoter methylation in primary bladder tumors. a Representative MSP analysis shows the RBBP8 promoter methylation status of normal urothelium (NU) and both papillary (Pap) and invasive (Inv) primary bladder cancer tissues. Band labels with U and M represent an unmethylated and methylated DNA locus, respectively bisulfite-converted unmethylated, genomic (U-co) and polymethylated, genomic (M-co) DNA were used as positive controls. NTC: non-template control. b RBBP8 mean methylation values of analyzed CpG sites (1 to 8) of healthy controls and bladder tumors demonstrating tumors-specific hypermethylation. c to d Box plot analysis of RBBP8 methylation in primary bladder tumors is based on mean values of pyrosequenced CpG sites 1–8. c RBBP8 methylation shows no significant differences between the two bladder cancer pathways (papillary and invasive tumors). d Significant enrichment of RBBP8 methylation is demonstrated in high-grade bladder tumors. Horizontal lines—grouped medians. Boxes—25 to 75% quartiles. Vertical lines—range, peak, and minimum; * p < 0.05. Horizontal lines—grouped medians. Boxes—25 to 75% quartiles. Vertical lines—range, peak, and minimum; ns, not significant, * p < 0.05

Article Snippet: Primary anti-RBBP8 antibody (dilution 1:200) (Atlas Antibodies, HPA039890, Sigma-Aldrich, Germany) was linked with DAKO EnVisionTMFLEX system and visualized with DAKO Liquid DAB Substrate Chromogen System in a DAKO Autostainer plus (K8024, K3468, DAKO).

Techniques: Biomarker Discovery, Methylation, Control

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 protein loss in nuclei of bladder tumors. Immunohistochemical RBBP8 protein staining of representative tissues are shown. a Strong RBBP8 immunoreactivity was detected in the cytoplasm and in the nuclei of a healthy urothelium, Scale bar: 100 μm. b Negative control of urothelial cell layers. The application of primary antibody was omitted. c Strong RBBP8 immunoreactivity in the cytoplasm of high grade, invasive tumor cells which completely lack nuclear staining. d Moderate cytoplasmatic and heterogeneously nuclear RBBP8 protein staining in invasive tumor cells. e Low RBBP8 protein expression in the cytoplasm of invasive bladder cancer showing strong RBBP8 staining in the nucleus. f Strong nuclear and cytoplasmic RBBP8 staining in non-invasive, papillary tumor cells. g Box plot demonstrating overall significant loss of RBBP8 protein only in the nucleus of bladder tumors. h Box plot graph illustrates the loss of RBBP8 protein within the nuclei of high-grade invasive bladder tumors. i Box plot shows a significant RBBP8 protein loss in tumors harboring RBBP8 promoter methylation. U, unmethylated; M, methylated. Horizontal lines — grouped medians. Boxes — 25 to 75% quartiles. Vertical lines — range, peak, and minimum; ns, not significant, * p < 0.05, *** p < 0.001

Article Snippet: Primary anti-RBBP8 antibody (dilution 1:200) (Atlas Antibodies, HPA039890, Sigma-Aldrich, Germany) was linked with DAKO EnVisionTMFLEX system and visualized with DAKO Liquid DAB Substrate Chromogen System in a DAKO Autostainer plus (K8024, K3468, DAKO).

Techniques: Immunohistochemical staining, Staining, Negative Control, Expressing, Methylation

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: Clinicopathological parameters in relation to nuclear RBBP8 protein expression

Article Snippet: Primary anti-RBBP8 antibody (dilution 1:200) (Atlas Antibodies, HPA039890, Sigma-Aldrich, Germany) was linked with DAKO EnVisionTMFLEX system and visualized with DAKO Liquid DAB Substrate Chromogen System in a DAKO Autostainer plus (K8024, K3468, DAKO).

Techniques: Biomarker Discovery, Methylation

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 is detectable in urines from bladder cancer patients. a Representative MSP analysis shows the RBBP8 promoter methylation status of human urine samples derived from healthy controls (Co) and bladder tumors (Ur-T). Band labels with U and M represent an unmethylated and methylated DNA locus. Bisulfite-converted unmethylated, genomic (U-co), and polymethylated, genomic (M-co) DNA were used as positive controls. NTC, non-template control. b Upper graph: RBBP8 mean methylation values of analyzed CpG sites (1 to 8) in cancerous bladder diseases (BLCA-derived) and two control cohorts (benign: control urines #1 and malignant: control urines #2) using pyrosequencing . Lower heatmap: Differences of RBBP8 methylation between BLCA-derived urines and both control conditions (benign and malignant) highlighting GpG sites (#7 and #8) with the strongest impact for discrimination (see green arrows). c Box plot demonstrating a significant increase of RBBP8 methylation in high-grade bladder tumors. Horizontal lines — grouped medians. Boxes — 25 to 75% quartiles. Vertical lines — range, peak, and minimum; * p < 0.05. d RBBP8 mean methylation values of analyzed CpG sites (1 to 8) of controls classified by diseases . BPH, begin prostate hyperplasia; TGCT, testicular germ cell tumors; PRAD, prostate adenocarcinoma

Article Snippet: Primary anti-RBBP8 antibody (dilution 1:200) (Atlas Antibodies, HPA039890, Sigma-Aldrich, Germany) was linked with DAKO EnVisionTMFLEX system and visualized with DAKO Liquid DAB Substrate Chromogen System in a DAKO Autostainer plus (K8024, K3468, DAKO).

Techniques: Methylation, Derivative Assay, Control

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: Clinicopathological parameters in relation to RBBP8 promoter methylation in BLCA associated urine samples

Article Snippet: Primary anti-RBBP8 antibody (dilution 1:200) (Atlas Antibodies, HPA039890, Sigma-Aldrich, Germany) was linked with DAKO EnVisionTMFLEX system and visualized with DAKO Liquid DAB Substrate Chromogen System in a DAKO Autostainer plus (K8024, K3468, DAKO).

Techniques: Methylation

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: Biomarker performance of RBBP8 methylation based on a non-invasive approach. a RBBP8 methylation enables significant discrimination of cancerous bladder diseases from two control conditions (benign and malignant) using urine samples. The scatterplot shows the mean methylation values of the CpG sites #7 and #8; ns, not significant; *** p < 0.0001. b to c ROC curve analysis illustrating RBBP8 single-biomarker performance based on all analyzed CpG sites (green curve) and CpG site #7 and #8 (red curve). b ROC curve in benign disease controls. Red curve (CpG #7 and #8): the cutoff value of 1.25% methylation was defined for positive detection of disease; in that case, RBBP8 methylation achieved a specificity of 90.9% and a sensitivity of 51.9%. Area under the curve (AUC) 0.730 (95% CI, 0.616 to 0.844), p = 0.002. c ROC curve in malignant (prostate and testicular cancer) disease controls. Red curve (CpG #7 and #8): the cutoff value of 4.00% methylation was defined for positive detection of disease; in that case, RBBP8 methylation achieved a specificity of 90.2% and a sensitivity of 40.4%. Area under the curve (AUC) 0.686 (95% CI, 0.583 to 0.789), p = 0.001

Article Snippet: Primary anti-RBBP8 antibody (dilution 1:200) (Atlas Antibodies, HPA039890, Sigma-Aldrich, Germany) was linked with DAKO EnVisionTMFLEX system and visualized with DAKO Liquid DAB Substrate Chromogen System in a DAKO Autostainer plus (K8024, K3468, DAKO).

Techniques: Biomarker Discovery, Methylation, Control

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 biomarker performance

Article Snippet: Primary anti-RBBP8 antibody (dilution 1:200) (Atlas Antibodies, HPA039890, Sigma-Aldrich, Germany) was linked with DAKO EnVisionTMFLEX system and visualized with DAKO Liquid DAB Substrate Chromogen System in a DAKO Autostainer plus (K8024, K3468, DAKO).

Techniques: Biomarker Discovery, Control