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. 2018 Mar 30;20(5):478–488. doi: 10.1016/j.neo.2018.03.003

Figure 2.

Figure 2

AZD6738 targets DDR activated by AZD1775 and enhances AZD1775-induced replication stress in TNBC cancer cells.A, MDA-231 or Hs578t cells were treated with 50 nM or 100 nM of AZD1775 with or without 500 nM of AZD6738 for 24 h. After treatment, western blot was performed using anti-γH2AX, anti-pCHK1 (S345), anti-CHK1, anti-pCDC25c (S216), anti-CDC25c, anti-pHH3, anti-RAD51 anti-pRPA32 (S4/S8), anti-cleaved-caspase 3, and anti-GAPDH antibodies. B, MDA-231 and Hs578t cells were treated for 24 hours with 100 nM of AZD1775, 500 nM of AZD6738 or both. Flow cytometry was used to identify the population of cells positive for γH2AX after treatment. Quantitative data of γH2AX-positive cell population are shown (n = 3, mean ± SD) (*P < .05, **P < .01, and ***P < .001). C, MDA-231 cells were treated with 50 nM or 100 nM of AZD1775 with or without 500 nM of AZD6738 for 24 h. Cells were probed with anti-γH2AX and anti-RAD51 antibodies. Scale bar: 5 μm. D, Quantitative data of the γH2AX-positive (five or more foci per cell) cells and pan-nuclear γH2AX signal after indicated treatments in the MDA-231 and Hs578t cells are shown (n = 3, mean ± SD). E, MDA-231 cells were treated with AZD1775 or AZD6738 alone or in combination for 24 h. Nuclear expression of pRPA32 (S4/S8) was determined by immunofluorescent imaging. Scale bar: 10 μm. F, Quantitative data of pRPA32 (S4/S8) positive cells after the indicated treatments are shown in MDA-231 and Hs578t cells (n = 3, mean ± SD) (**P < .01 and ***P < .001).