Fig. 1.

Cyclin D overexpression in PC3 cancer cells synergizes with TSC2 knockdown to produce increased ROS and cell death in stressed conditions. (A) PC3 cells were infected with control or Cyclin D1 lentivirus, selected with puromycin, and western blotting was performed on cell lysates to assess Cyclin D1 and Actin protein levels. (B) PC3 cells were treated with Cyclin D1 lentivirus or control and puromycin-selected, and transfected with an E2F-Luciferase reporter and assayed for luciferase activity. (C) PC3 cells were treated with Cyclin D1 lentivirus and puromycin-selected, and treated with shTSC2 and control viruses, seeded into soft agar, and colonies counted. (D) PC3 cells overexpressing Cyclin D1 and lacking TSC2 were incubated in low serum media and assayed for cell death using trypan blue staining. (E–H) PC3 cells overexpressing Cyclin D1 were treated with control or shTSC2 virus, incubated in low serum media, trypsinized, stained with DHE and imaged. (I) Images from DHE staining were quantified for average luminescence per cell. (J) Cyclin D1 overexpressing PC3 cells were incubated in 1 mM H₂O₂ for 2 days and cell death was assayed by trypan blue staining. (K) PC3 cells overexpressing cyclin D1 were treated with shTSC2 or control were seeded into soft agar with or without 12 mM NAC and colonies were counted.