Figure 6. Induction of reactive oxygen species (ROS) and necroptosis are necessary for chelerythrine chloride induced death in Tsc2-null cells.
A) ROS levels in Tsc2−/− and Tsc2+/+ MEFs treated with 3 uM chelerythrine chloride and/or 2 mM n-acetylcysteine (NAC) for 4 h. ROS levels were normalized to control (vehicle) treated cells. B) Phase contrast analysis of Tsc2-null MEFs treated with chelerythrine chloride (2 uM) and/or NAC (2 mM) or Necrostatin-1 (Necro, 100 uM) for 20 hours. C) Crystal violet analysis of 621-101 cells treated with chelerythrine chloride (5 uM) and/or NAC (2 mM) or Necrostatin-1 (100 uM) for 20 hours (n=4, *p<0.005). D) Crystal violet analysis of Tsc2-null MEFs treated with chelerythrine chloride (2 uM) and/or mitotempo (Mito, 60 uM) for 20 hours (n=4, *p<0.05, **p<0.001). E) Immunoblot analysis of cleaved PARP levels in Tsc2+/+ and Tsc2−/− MEFs treated with chelerythine (2 uM) and/or NAC (2 mM) or Necrostatin-1 (100 uM) for 3 hours.