Fig. 7: TTK activate the ATR signaling pathway through RPA2.

A OVCAR8 and OV90 cells were transfected with PLKO.NC or RPA2 shRNA (shRPA2 1# and shRPA2 2#). Western blot assays were conducted to detect the levels of RPA2, pATR, ATR, and β-actin. B OVCAR8 and OV90 cells were transfected with pcDNA3.1, pcDNA3.1-RPA2, and pcDNA3.1-RPA2 S33A plasmids for 48 h. Western blot analysis was used to measure the protein levels of RPA2, pATR, ATR, and β-actin. C OVCAR8 and OV90 cells were transfected with PLKO.NC or TTK shRNA (shTTK1 and shTTK2). Western blot assays were performed to assess the levels of TTK, pATR, ATR, and β-actin. D OVCAR8 and OV90 cells were treated with CFI for 48 h, then the levels of TTK, pATR, ATR, and β-actin were evaluated. E OVCAR8 and OV90 cells transfected with PCMV-TTK were treated with or without olaparib. Western blot assays were used to detect the protein levels of TTK, pRPA2 S33, RPA2, pATR, ATR, and β-actin. F OVCAR8 and OV90 cells were treated with CFI for 24 h, followed by transfection with pcDNA3.1 or pcDNA3.1-RPA2. The protein levels of TTK, pRPA2 S33, RPA2, pATR, ATR, and β-actin were assessed. G, H OVCAR8, and OV90 cells were transfected with shTTK2 and RPA2 for 48 h, followed by treatment with or without VE-821 (an ATR inhibitor) for 24 h. Afterward, olaparib was added for 72 h. MTT and colony survival assays were performed to demonstrate that TTK inhibition overcame olaparib resistance in HR-proficient OC cells, partly through the RPA2/ATR axis. Data are mean ± SD, n = 3, one-way ANOVA. (Data are mean ± SD, *P < 0.05, **P < 0.01, n = 3).