Fig 1: BRD4770-induced activation of the FA pathway does not occur via the direct induction of DNA damage or altered cell cycle progression(A) BJ-TERT cells were incubated with (+) and without (-) 200 nM mitomycin C (MMC) in the absence (NT) or presence of 10 μM BRD4770 for 24 h. Cells were fixed and stained with mouse monoclonal anti-γH2AX antibody and counterstained with DAPI, and the number of nuclei with >5 γH2AX foci were scored. At least 300 nuclei were scored for each treatment and this experiment was performed three times with similar results. Error bars represent the standard errors of the means from three independent experiments. (B) BJ-TERT cells were incubated with (+) and without (-) 0.4 μM etoposide (VP-16), in the absence or presence of 10 μM BRD4770, 5 μM GSK-J1 and 2.5 μM BIX01294, for 24 h. Whole-cell lysates were immunoblotted with anti-γH2AX and anti-PCNA (loading control) antibodies. (C) U2OS cells were incubated in the absence (NT) or presence of 200 nM MMC or 5 and 10 μM BRD4770 for 24 h. Whole-cell lysates were prepared and immunoblotted with anti-FANCD2, anti-FANCA, anti-UBE2T, anti-USP1, anti-CHK1 pS345, anti-RPA, anti-RPA pS4/8, and anti-α-Tubulin antibodies. L:S Ratio, ratio of monoubiquitinated to nonubiquitinated FANCD2; RBI, relative band intensity. (D) U2OS cells were incubated in the absence or presence of 2, 5, and 10 μM BRD4770 for 24, 48, or 72 h. Cells were fixed in ice-cold ethanol, stained with propidium iodide, and analyzed by flow cytometry. Cell cycle stage distributions were determined using FlowJo v10.2.