Extended Data Fig. 2: Time course of MEF growth inhibition upon treatment with WM-8014, and requirement for INK4A/ARF and p53 for WM-8014-induced cell cycle arrest.
From: Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth
a, MEF proliferation after treatment with three high concentrations of WM-8014. MEFs were treated either continuously for 15 days, or treatment was discontinued after 1, 2, 4 or 8 days to determine whether cells could re-enter the cell cycle. b, Phase-contrast images of MEFs after 15 days of treatment with 10 µM WM-8014 or 10 µM WM-2474. Note cells with senescence morphology; that is, large nuclei indicating endoreplication without cell division and extensive cytoplasm (WM-8014 panel). c, Flow-cytometry gating strategy for the cell cycle analysis using incorporation of the nucleotide analogue BrdU to mark cells in S phase and 7-aminoactinomycin D (7-AAD) to determine 2N (G0/G1) and 4N (G2/M) DNA content. d, Flow-cytometry gating strategy for the cell cycle analysis of transgenic Fucci cells that express Azami Green in mid-S phase, G2 and M, Kusabira Orange in mid–late G1, are double-positive yellow in early S phase and double-negative in early G1. e, Cell cycle analysis of Cdkn2a null (Ink4a−/−Arf−/−) and littermate control cells after treatment for 8 days with WM-8014, vehicle and the inactive compound WM-2474. MEFs were exposed to BrdU for 1 h before flow-cytometry analysis of BrdU incorporation during DNA synthesis (S phase) and DNA content of 2N (G0/G1) compared with 4N (G2/M) using 7-AAD. f, Senescence-associated β-galactosidase activity in Cdkn2a−/− and control MEFs after treatment for 15 days with 10 μM WM-8014, 10 µM WM-2474 or DMSO vehicle control. g, Cell cycle analysis of Trp53 null MEFs (Trp53−/−) and littermate control cells after treatment with WM-8014, vehicle and inactive compound WM-2474, as in c. n = 3 MEF isolates per genotype (a–e). Data are mean ± s.e.m., and were analysed by two-way ANOVA within duration of treatment with concentration and days of culture as the independent factors (a), or by one-way ANOVA followed by Bonferroni post hoc test (e–g).
