Journal: bioRxiv

Article Title: BAP1 loss impairs Non-Homologous End Joining DNA repair promoting genomic instability

doi: 10.64898/2026.02.17.706330

Figure Lengend Snippet: (A) Frequency of BAP1 genetic alterations in tumors from the BAP1 cancer predisposition syndrome and in pan-cancer cohorts of metastatic tumors. Patient cohorts were retrieved from QIMR Oncotarget 2016 and TCGA Firehose Legacy (UM), TCGA PanCancer Atlas (mesothelioma and kidney renal clear cell carcinoma), MSK Hepatology 2021 (intrahepatic cholangiocarcinoma), and MSK Cell 2021 (pan-cancer) datasets via the cBioPortal platform. Data are shown as bar plots. (B, C) Kaplan–Meier survival curves for overall survival (B) and disease-free survival (C) in UM patients with BAP1 wild-type (green) or BAP1-mutated (red) tumors. P-values are indicated. Data were retrieved from QIMR Oncotarget 2016 and TCGA Firehose Legacy. (D, E) Violin plots showing tumor basal diameter, tumor thickness (D), aneuploidy score, fraction of genome altered and microsatellite instability (MSI) score (E) in BAP1 wild-type versus BAP1-mutated UM patients. Orange line: median. **p < 0 . 01, *p < 0 . 05, ns: not significant . P-values were calculated using an unpaired t-test. (F) Western blot analysis of BAP1 silenced MP41 cells. (G) Western blot analysis of γH2AX in MP41 cells following BAP1 silencing. MP41 cells were treated with bleomycin (50 µg/ml, 24 h) and collected at the indicated recovery time points. (H, I) Representative immunofluorescence images of pan-nuclear γH2AX+ MP41 cells (red) at the indicated recovery time points after bleomycin treatment upon BAP1 silencing. DAPI stains DNA (blue) (H). Violin plot quantifying the percentage of pan-nuclear γH2AX+ cells per field across 3 independent experiments (I). Each dot represents a field. Red line: median. Scale bar: 10 µm. ***p < 0 . 001, **p < 0 . 01, *p < 0 . 05, ns: not significant . P-values were determined by one-way ANOVA with Tukey’s correction.

Article Snippet: The following primary antibodies were incubated overnight at 4°C: rabbit BAP1 (D7W70) mAB (Cell Signaling Technology, 13271S), mouse Anti-β-Actin (Sigma Aldrich, A1978-200UL), mouse BRCA1 (D-9) monoclonal IgG (Santa Cruz Biotechnology, sc-6954), mouse monoclonal anti-GAPDH, clone GAPDH-71.1 (Sigma Aldrich, G8795-200UL), mouse Anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Merck/Sigma, 05-636-I), rabbit H3 (D1H2) mAb (Cell Signaling, # 4499), Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Cell Signaling, #8240) and anti-Histone H2A (Abcam, ab18255).

Techniques: Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: BAP1 loss impairs Non-Homologous End Joining DNA repair promoting genomic instability

doi: 10.64898/2026.02.17.706330

Figure Lengend Snippet: (A) Western blot analysis of H2AK119ub levels upon BAP1 depletion in G1 cells. MP41 were irradiated (5 Gy) at 6 hours from Nocodazole release and collected 3 hours post IR (B, C) Scatter dot plots quantifying RPA foci (green in C) during recovery from bleomycin treatment in MP41 cells depleted of BAP1. Representative immunofluorescence images are shown in B. DAPI stains DNA. Scale bar: 10 µm. Red line: mean. ****p < 0.0001, **p < 0.01, ns: not significant. P-values by one-way ANOVA with Tukey’s correction (D–F) Scatter dot plots quantifying RPA foci in Cyclin A2+ cells (panel D), Cyclin A2– cells (G1 phase, panel E) in MP41 cells depleted of BAP1. Representative immunofluorescence images are shown in F. Red line: mean. *p < 0.05, **p < 0.01, ns: not significant. P-values by one-way ANOVA with Tukey’s correction.

Article Snippet: The following primary antibodies were incubated overnight at 4°C: rabbit BAP1 (D7W70) mAB (Cell Signaling Technology, 13271S), mouse Anti-β-Actin (Sigma Aldrich, A1978-200UL), mouse BRCA1 (D-9) monoclonal IgG (Santa Cruz Biotechnology, sc-6954), mouse monoclonal anti-GAPDH, clone GAPDH-71.1 (Sigma Aldrich, G8795-200UL), mouse Anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Merck/Sigma, 05-636-I), rabbit H3 (D1H2) mAb (Cell Signaling, # 4499), Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Cell Signaling, #8240) and anti-Histone H2A (Abcam, ab18255).

Techniques: Western Blot, Irradiation, Immunofluorescence

Journal: bioRxiv

Article Title: BAP1 loss impairs Non-Homologous End Joining DNA repair promoting genomic instability

doi: 10.64898/2026.02.17.706330

Figure Lengend Snippet: (A) Representative immunofluorescence images of RPA (green) and Cyclin A2 (red) in BAP1-depleted MP41 cells ± PTC-209 treatment. DAPI stains DNA. Cells were treated with PTC-209 (0.1 µM, 48 h) prior to irradiation and collected at 3 h post-IR. Arrows mark Cyclin A2– (G1 phase) cells with RPA foci. Scale bar: 10 µm. (B, C) Scatter dot plots showing (B) the percentage of RPA+ Cyclin A2– cells and (C) the number of RPA foci per Cyclin A2– cell at 3 h post-IR. Red line: mean. ****p < 0.0001, ***p < 0.001, **p < 0.01, ns: not significant. P-values by one-way ANOVA with Tukey’s correction. (D) Representative immunofluorescence images of 53BP1 (green) in BAP1-depleted MP41 cells ± PTC-209 treatment at 1 and 3 h post-IR. DAPI stains DNA. Scale bar: 10 µm. (E) Scatter dot plot quantifying 53BP1 foci per cell. Red line: median. ****p < 0.0001, **p < 0.01, ns: not significant. P-values by one-way ANOVA with Tukey’s correction. (F) Schematic model. In BAP1-proficient cells, BAP1 deubiquitylates H2AK119ub deposited by PRC1, permitting 53BP1 recruitment and NHEJ execution in G1. In BAP1-depleted cells, H2AK119ub accumulates, blocking 53BP1 loading, and driving aberrant DNA end resection with RPA recruitment in G1, where HR is not available.

Article Snippet: The following primary antibodies were incubated overnight at 4°C: rabbit BAP1 (D7W70) mAB (Cell Signaling Technology, 13271S), mouse Anti-β-Actin (Sigma Aldrich, A1978-200UL), mouse BRCA1 (D-9) monoclonal IgG (Santa Cruz Biotechnology, sc-6954), mouse monoclonal anti-GAPDH, clone GAPDH-71.1 (Sigma Aldrich, G8795-200UL), mouse Anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Merck/Sigma, 05-636-I), rabbit H3 (D1H2) mAb (Cell Signaling, # 4499), Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Cell Signaling, #8240) and anti-Histone H2A (Abcam, ab18255).

Techniques: Immunofluorescence, Irradiation, Blocking Assay