Fig 5.

Senescence induction in p53⁻ cells requires p21 induction via a pathway involving reactive oxygen species (ROS) production. (A) The percentages of SA-β-gal⁺ HCT116 p21⁻ cells expressing shControl, shMcl-1, alone, or treated with roscovitine after 6 days of culture in media with or without doxorubicin (Dox). (Inset) Western blot verifying knockdown of Mcl-1 in HCT116 p21⁻ cells. (B) Western blot analysis of the indicated proteins in HCT116 p21⁻ shControl or shMcl-1 cells at the indicated days after treatment with doxorubicin. (C) HCT116 p53⁻ shControl or shMcl-1 cells alone or also expressing exogenous Mcl-1ΔC, Mcl-1 BH3 mutant, or Mcl-1 were pretreated with 5 μM NAC for 24 h followed by doxorubicin treatment for 6 days (magnification, ×20). Cells were then stained with RedoxSensor Red to visualize ROS production. (D) Cells were pretreated with NAC or left untreated for 24 h, followed by treatment with doxorubicin. After 6 days of treatment, lysates were prepared and assayed for p21 expression by Western blotting. (E) The percentages of SA-β-gal⁺ HCT116 p53⁻ cells after 6 days of culture with or without doxorubicin in NAC pretreated or untreated cells.