Fig. 3. Senescence reversal correlates inversely with p16 expression. (A) p16 expression. p16 and actin (control) protein levels were assessed in pre-senescent (P) and senescent (S) BJ and WI-38 (WI) cells by western blotting (WB). The labeling index of the cultures is shown below the blot (% LN). (B) p16 immunostaining. Pre-senescent (P) and senescent (S) BJ and WI-38 cells were immunostained for p16, and nuclei stained with DAPI, as described in Materials and methods. HeLa cells served as a positive control. (C) p16 and p21 levels after rescue from senescence by GSE-22. S-BJ cells were infected with control (GFP) or GSE-22-expressing (rescued GSE) lentivirus. Rescued cells were harvested while proliferating (16 PDs) or after proliferation ceased (23 PDs), and analyzed for p21, p16 and actin (control) by western blotting. (D) Senescence reversal in HMECs. Post-selection HMECs were infected with lentiviruses expressing the indicated proteins, and monitored for growth, as described in Materials and methods. Shown are cells 72 h following infection.