Fig. 3.

Ectopic E1A expression in PC3 cancer cells synergizes with TSC2 knockdown to produce increased ROS and cell death in stressed conditions. (A) PC3 cells were treated with E1A lentivirus or control and puromycin-selected, and transfected with an E2F-Luciferase reporter and assayed for luciferase activity. (B) PC3 cells were selected for ectopic E1A expression and treated with shTSC2 and control viruses, seeded into soft agar, and colonies counted. (C) PC3 cells with ectopic E1A and lacking TSC2 were incubated in low serum media and assayed for cell death using trypan blue staining. (D) PC3 with ectopic E1A were incubated in 1 mM H₂O₂ for 1 day and cell death was assayed by trypan blue staining. (E–H) PC3 cells with ectopic E1A expression were treated with control or shTSC2 virus, and incubated in low serum media. Cells were then trypsinized and stained with DHE before imaging. (I) Images from DHE staining were quantified for average luminescence per cell. (J) PC3 cells selected for ectopic E1A expression and treated with shTSC2 or control were seeded into soft agar with or without 12 mM NAC and colonies were counted. (K) E1A-selected PC3 cells were treated with control or Rb lentivirus, and then control or shTSC2, and incubated in 1mM H₂O₂ for 1 day and cell death was assayed by trypan blue staining.