Fig. 7: Escapees are prone to DNA damage and outgrow non-escapees over extended drug treatment. | Nature Communications

Fig. 7: Escapees are prone to DNA damage and outgrow non-escapees over extended drug treatment.

From: Melanoma subpopulations that rapidly escape MAPK pathway inhibition incur DNA damage and rely on stress signalling

Fig. 7: Escapees are prone to DNA damage and outgrow non-escapees over extended drug treatment.The alternative text for this image may have been generated using AI.

a Images of A375 cells treated with 1-µM dabrafenib for 72 h stained for EdU and γ-H2AX. Quantification of γ-H2AX puncta for escapees (E) and non-escapees (NE) is plotted as split violins. n = 2 biologically independent experiments. b Neutral comet assay in dabrafenib-treated A375 cells. Gels were co-stained for EdU incorporation. Plots indicate percent tail intensity over each entire comet, with mean values displayed as a horizontal line. Left panel: 0, 3 and 7 days, n = 708, 375 and 394 cells. Right panel: 0, 3 and 7 days, n = 40, 7 and 13 cells. c Images of A375 cells treated with 1-µM dabrafenib for 72 h stained for EdU and FANCD2. Quantification of FANCD2 intensity for escapees and non-escapees is plotted as split violins. n = 2 biologically independent experiments. d Flow cytometric analysis of DNA replication licensing determined by chromatin-bound MCM2 in A375 cells treated with BRAF and/or MEK inhibitors. Cells appearing under the dashed line show reduced MCM2 loading at the start of S phase. Right-most plot shows percent under-licensed cells relative to all early S phase cells; mean ± std of three replicate samples. e CDK2 activity heatmap for 482 dabrafenib-treated single cells tracked over 12 days. Cell behaviour was classified based on the first 96 h, and subsequent mitosis events are plotted as black dots. Bar plot quantifies the average number of mitoses per cell in the final 7 or 8 days of filming. Error bars: mean ± SEM. n (E; NE) = 248; 234 cells. f Escapees (Gem+) and non-escapees (Gem−) cells were separated by FACS after a 72 h of 1-µM dabrafenib treatment and were then continuously cultured in 1-µM dabrafenib for 4 weeks. Representative wells for each condition at different time points are shown. Normalized cell count for each well was quantified and plotted for each condition; mean ± SEM of seven replicate wells. The p value summary represents the comparison between E and NE replicates by unpaired t-test. Source data are provided as a Source Data file.

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