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. Author manuscript; available in PMC: 2019 Apr 9.
Published in final edited form as: Cancer Discov. 2017 Sep 18;8(1):74–93. doi: 10.1158/2159-8290.CD-17-0682

Figure 3.

Figure 3

Impairing DNA damage repair augments MAPKi-addiction. (A) Western blot (WB) analysis of p-H2AX levels in slow-cycling predominant R-lines on or off MAPKi(s), with or without ATMi and/or PARPi treatment for 3 d. (B–C) Clonogenic growth (B) or percentages of annexin V/PI-positive dead cells (C) in slow-cycling predominant R-lines on or off MAPKi(s) for 5 d, with or without ATMi and/or PARPi treatment. (D) WB analysis of BRCA1 and p-H2AX levels in in slow-cycling predominant R-lines, transduced with control (V) or BRCA1-specific shRNA lentiviruses, on or off MAPKi(s), with or without PARPi treatment for 3 d. (E–F) R-line cells from D were subjected to the same assays as in B and C, respectively. For WBs, TUBULIN, loading control. (G) Levels of indicated proteins by WBs in mitochondrial (MITO), nuclear (NUCL) subcellular fractions or whole-cell lysates (WCL) of slow-cycling versus cell-death predominant R-lines, on and off MAPKi(s), with or without ATMi and PARPi treatment for 3 d. HSP60, mitochondrial fraction control; Histone H3, nuclear fraction control. (H–I) Clonogenic growth (H) or percentages of annexin V/PI-positive dead cells (I) in cell-death predominant R-lines on or off MAPKi(s) for 6 d and 16 (only in H), with or without ATMi and/or PARPi treatment. (J–L) Levels of caspase-3 activity (J, K) or clonogenic growth (L) with indicated MAPKi(s) treatment, with or without ATMi and PARPi, with or without caspase-3 inhibitor, Z-DEVD-FMK (20 µM), in slow-cycling predominant R-lines (J) for 3, 6 d or cell-death predominant R-lines (K) for 3 d. For J, K, n=5; mean ± SDs; ***p <0.001 based on ANOVA. Staurosporine (1 µM) used to induce caspase-3 activity and death.