Figure 6.

Brusatol selectively suppressed SDHB^KD cells. (A) Dose-response curve showed that SDHB^KD MPC cells were more vulnerable to Brusatol treatment. (B) BrdU (red) incorporation assay shows that Brusatol selectively inhibited the proliferation of SDHB^KD MPC cells. Cell nuclei were labeled with Hoechst 33342 (blue). Bar = 20 μm. (C) Quantification of BrdU-positive cells in Figure 6B. ** p < 0.01; *** p < 0.001. (D) Long-term colony formation assay and quantification showed that Brusatol inhibited cluster formation of SDHB^KD MPC cells. *** p < 0.001. (E) Caspase 3/7-Glo assay showed significantly increased Caspase 3/7 activity in SDHB^KD MPC cells with Brusatol. Luminescence was measured and normalized to protein quantification. *** p < 0.001. (F) Immunoblotting showed increased cleaved-poly (ADP-ribose) polymerase (PARP) and γH2A.X after Brusatol treatment. β-actin was used as internal control. (G) Annexin V/PI apoptosis analysis showed that SDHB^KD MPC cells exhibited more apoptotic cells under Brusatol treatment compared with SDHB^WT cells. ROS scavengers reduced the number of apoptotic cells. (H) Quantification of apoptotic cells from Figure 6G. ** p < 0.01.