Fig. 3: PKMYT1 inhibition causes unscheduled CDK1 activation and mitotic entry in CCNE1-high cells.
From: CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition
a, QIBC analysis of γH2AX nuclear intensity, EdU incorporation and DNA content (measured with DAPI) in FT282-hTERT TP53R175H cell lines. Representative QIBC (right) and quantification (left) of cells with pan-γH2AX. b, Representative micrograph showing EdU staining, γH2AX localization and nuclear morphology of FT282 CCNE1-high cells. Representative of n = 3 experiments. c, QIBC quantification of HCC1569 cells with pan-γH2AX as a function of RP-6306 dose. d, Quantification of FT282-hTERT TP53R175H CCNE1-high cells transfected with siRNAs targeting cyclin B1 (siCCNB1) or non-targeting siRNA (siCTRL) with pan-γH2AX as a function of RP-6306 dose. e, QIBC quantification of FT282-hTERT TP53R175H cells of the indicated genotype positive for EdU and cyclin B (CCNB1) pS126 as a function of RP-6306 dose. f, g, RP-6306 induces chromosome pulverization. Representative micrographs of metaphase spreads of FT282 parental (WT) and CCNE1-high cells left untreated or following treatment with either RP-6306 (500 nM) for 24 h (f, left) and quantification of FT282 cells (f, right) and HCC1569 cells (g) with pulverized chromosomes with at least 40 metaphases counted per replicate. h, i, Quantification of the first observed G2 phase (h) and the number of nuclear envelope breakdowns (NEBDs) before the first observed cell division (i) using time-lapse imaging of FT282-hTERT TP53R175H PCNA–chromobody–TagRFP (WT) and CCNE1-high (CCNE1) cells treated with DMSO or RP-6306 (500 nM) for 23 h. QIBC validation is shown in Extended Data Figs. 3a, 4a, e, f, h, i. Data in a, c–i are mean ± s.d. (n = 3).
