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. 2019 Nov 1;4(21):e121951. doi: 10.1172/jci.insight.121951

Figure 3. mTOR inhibition and consequent 4E-BP1 activation impedes DNA replication and DNA repair.

Figure 3

(A) Asynchronous shScramble or sh4E-BP1 MiaPaca-2 cells were incubated with PP242 (5 μM) for 3 hours and labeled with EdU. Nuclear DNA was counterstained by DAPI. (B) The proportions of replicating cells are shown as means and SDs and were generated from at least 3 independent experiments. P values were calculated using 2-way ANOVA (***P < 0.001). (C) Quantitative image-based cytometry (QIBC) single-cell analysis of EdU-labeled cells. Total EdU and DAPI values from A are plotted in a scatter diagram. Percentages indicate the proportion of replicating cells. Quantification of (D) γH2AX-positive and (E) p-53BP1–positive cells in S-phase. Asynchronous cells were pulsed 30 minutes with EdU (10 μM) prior to 2 hours mitomycin C treatment (MMC, 10 μg/ml) and released for 4 hours. Mean γH2AX and p-53BP1 intensity was plotted for every EdU-positive cells in a scatter diagram. P values were calculated using Student’s t test (***P < 0.001). Representative of 3 independent experiments.