Journal: bioRxiv

Article Title: Genome-wide CRISPR screens identify DNA repair and R-loop suppression as regulators of the cellular sensitivity to environmentally relevant Bisphenol A exposure

doi: 10.64898/2026.04.13.718249

Figure Lengend Snippet: ( A, B ) DDX21 immunofluorescence showing that treatment with 0.5 μM BPA for 72 hours increases DDX21 foci formation in in RPE1 ( A ) and HeLa ( B ) cells. At least 105 cells were quantified for each condition. The mean value is represented on the graphs, and asterisks indicate statistical significance (t-test two-tailed, unpaired). Representative micrographs, with scale bars representing 10 and 20 µm are shown. ( C ) Western blot showing that HeLa and RPE1 cells do not express the estrogen receptor (ER). ER-expressing MCF7 cells used as a positive control. ( D ) Proximity ligation assay showing that treatment with 0.5 μM BPA for 72 hours or 0.5 μM Cisplatin for 24 hours increases the S9.6-dsDNA co-localization in HeLa cells. At least 101 cells were quantified for each condition. The mean value is represented on the graphs, and asterisks indicate statistical significance (t-test two-tailed, unpaired). ( E ) Representative PLA experiment showing an increase in the number of S9.6-dsDNA foci in sgDDX21 cells, as compared to control HeLa cells, after 0.5 μM BPA for 72 hours. The mean values are represented on the graphs, and asterisks indicate statistical significance (t-test two-tailed, unpaired). Representative micrographs, with scale bars representing 10 µm are shown. ( F ) The means of 3 independent S9.6-dsDNA PLA experiments showing an increase in the mean number of foci in sgDDX21 cells, normalized to control HeLa cells, after 0.5 μM BPA for 72 hours across three independent replicates. The mean values are represented on the graphs, and asterisks indicate statistical significance (t-test two-tailed, unpaired).

Article Snippet: Antibodies used were: S9.6 (Kerafast ENH001), dsDNA (Novus Biologicals NBP3-07302).

Techniques: Immunofluorescence, Two Tailed Test, Western Blot, Expressing, Positive Control, Proximity Ligation Assay, Control

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

doi: 10.1093/nar/gkag226

Figure Lengend Snippet: EXO1 and CHAF1A are recruited to R-loops and synergistically suppress R-loop accumulation. ( A ) BrdU alkaline comet assay showing that CHAF1A depletion causes increased accumulation of replication-associated single stranded DNA lesions in both EXO1-knockout lines compared to WT HeLa cells. At least 100 nuclei were quantified for each condition. The median values are marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are shown at the top. ( B, C ) S9.6 PLA experiments showing increased EXO1 ( B ) and CHAF1A ( C ) recruitment to R-loops in HeLa cells. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). ( D, E ) S9.6-dsDNA PLA experiments showing increased R-loops in HeLa cells with concomitant inactivation of EXO1 and CHAF1A. RNAseH1 overexpression suppresses the PLA signal, indicating that it derives from R-loops. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired).

Article Snippet: Antibodies used were: S9.6 (Kerafast ENH001), double-stranded DNA (dsDNA; Novus NBP3-07302), γH2AX (Abcam ab2893), EXO1 (Novus NBP2-16391), and CHAF1A (Cell Signaling Technology 5480s).

Techniques: Alkaline Single Cell Gel Electrophoresis, Knock-Out, MANN-WHITNEY, Two Tailed Test, Over Expression

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

doi: 10.1093/nar/gkag226

Figure Lengend Snippet: Concomitant depletion of EXO1 and CHAF1A causes the accumulation of R-loop-associated DNA damage. ( A ) S9.6-γH2AX PLA experiments showing increased R-loop-associated DNA damage in HeLa cells with concomitant inactivation of EXO1 and CHAF1A. RNAseH1 overexpression suppresses the PLA signal, indicating that it derives from R-loops. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). The siCHAF1A/siControl mean ratios are also presented. ( B–D ) γH2AX immunofluorescence showing that CHAF1A depletion causes increased DNA damage in both EXO1-knockout lines compared to WT HeLa cells, which is suppressed by RNaseH1 overexpression. Quantifications ( B, D ) and representative micrographs with scale bars representing 10 µm ( C ) are shown. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). ( E–G ) Neutral comet assays showing that CHAF1A depletion causes increased DSB formation in both EXO1-knockout lines compared to WT HeLa cells, which is suppressed by RNaseH1 overexpression. Quantifications ( E, G ) and representative micrographs with scale bars representing 10 µm ( F ) are shown. At least 100 comets were quantified for each sample. The median values are marked on the graph, and asterisks indicate statistical significance (Mann–Whitney, two-tailed).

Article Snippet: Antibodies used were: S9.6 (Kerafast ENH001), double-stranded DNA (dsDNA; Novus NBP3-07302), γH2AX (Abcam ab2893), EXO1 (Novus NBP2-16391), and CHAF1A (Cell Signaling Technology 5480s).

Techniques: Over Expression, Two Tailed Test, Immunofluorescence, Knock-Out, MANN-WHITNEY