Fig. 4.

ROS generated by CYPs is indispensable for IR-induced cytotoxicity in cells with NUPR1 inactivation. a, b Representative images of colony formation were displayed in NUPR1-overexpressing MHCC-97H/MHCC-97L (3000 cells) and NUPR1-knockdown QGY-7701/Hep3B (500 cells and 3000 cells, respectively) pretreated with 5 mM NAC followed by exposure to 6 Gy of IR (left). The survival data were normalized to those of unirradiated control cells (right). c Quantification of cell death was employed in LV-NC/LV-NUPR1 or sh-NC/sh-NUPR1 cell lines pretreated with or without 5 mM NAC followed by exposure to 8 Gy of IR. d Western blot analysis was utilized to determine the expression levels of total/cleaved caspase-3 and total/cleaved PARP in the indicated cells pretreated with or without NAC upon IR (8 Gy). e Relative ROS levels were measured in NUPR1 overexpressing or knockdown cell lines pretreated with or without 20 μM alizarin followed by 8 Gy of IR. f Colony formation assays were applied in stably transfected NUPR1 overexpression or knockdown cells after IR (6 Gy) or combination with 20 μM alizarin treatment. Data are the mean of biological triplicates and are shown as the mean ± SD. P values: *P < 0.05; **P < 0.01; ***P < 0.001 and ns, not significant by Student’s t-test