Fig. 6. The silencing of p16 in 2BS cells prevented the appearance of senescence-associated features and the growth-inhibition effects of PPAR agonists. 2BS cells transfected with the expression plasmids pSilencer 2.1-U6 neo (RNAi vector) or pSilencer-p16 (sip16) were analyzed for the relative senescence markers (all at PD51). Cells were treated with 20 µM troglitazone or DMSO (vehicle) as indicated. (A) Western blot analysis of p16 silencing in sip16-transfected cells compared with RNAi-vector-transfected cells. Western blotting was performed using specific antibodies against p16^INK4 as indicated. The β-actin lane serves as a loading control. (B) RNAi-vector-transfected and sip16-transfected cells were stained for SA-β-gal activity (blue), a classical marker of senescence. (C) Growth curves of RNAi-vector-transfected and sip16-transfected 2BS cells were determined by the MTT assay. Values are the mean ± s.d. of triplicate points from a representative experiment (n=3), which was repeated three times with similar results. Values accompanied by different symbols are statistically significantly different from each other. (D) Flow-cytometry analysis of RNAi-vector-transfected and sip16-transfected 2BS cells. Each experiment was performed at least three times. The table shows the representative data. The graph depicts data from three independent experiments (means ± s.d.). ^*P0.05 vs vehicle-treated RNAi-vector cells.