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. 2019 Oct 7;20(19):4943. doi: 10.3390/ijms20194943

Figure 3.

Figure 3

RB1 downregulation via siRNA or CDK4/6 inhibitor does not increase proton RBE in MDA-MB-231 cells. (A) Western blot analysis confirms siRNA-mediated RB1 knockdown in MDA-MB-231 cells. β-Actin was used as a loading control. Neither radiation treatment nor RB1 knockdown affected the expression of cyclin D1 (upstream effector) or E2F1 (downstream of RB1). (B) Effect of RB1 depletion on the clonogenic survival of MDA-MB-231 cells after X-ray or proton irradiation. Data are presented as the mean ± SEM of two independent experiments and the differences are evaluated by a two-way ANOVA; *** p < 0.001. (C) The RB1 depletion augmented radiation-induced apoptosis. Apoptotic cell death was assessed by flow cytometry as described in the materials and methods. Data are presented as the mean ± SEM of two independent experiments. The differences were evaluated by a two-way ANOVA; ** p < 0.01; *** p < 0.001. (D) PD-0332991, a selective CDK4/6 inhibitor, decreased total RB and its phosphorylated form in a dose-dependent manner. β-Actin was used as a loading control. (E) The effect of PD-0332991 on clonogenic survival. MDA-MB-231 cells were pre-treated with 100 nM PD-0332991 for 3 h, followed by irradiation with the indicated doses of X-rays or protons. Data are presented as the mean ± SEM of two independent experiments and the differences are evaluated by a two-way ANOVA; * p < 0.05; *** p < 0.001. (F) PD-0332991 enhanced proton radiation-induced apoptosis. MDA-MB-231 cells were pre-treated with 500 nM PD-0332991, followed by exposure to 4 Gy of X-rays or protons. Data are presented as the mean ± SEM of three independent experiments and the differences are evaluated by a two-way ANOVA; * p < 0.05; ** p < 0.01; *** p < 0.001.