Research Paper Volume 1, Issue 6 pp 542—556

Figure 4. Impact of p16 ^INK4a or p21^Waf1 expression on the cellular senescence program. WMM1175 melanoma cells were cotransfected with plasmids encoding p16^INK4a, p21^Waf1 or the melanoma-associated p16^INK4a_R24P along with pCMV-EGFPN1, which was used as a marker of transfection. Five days post transfection cells were fixed, permeabilized and analyzed. (A) Cell proliferation was monitored by Ki67 immunostaining and the percentage of transfected WMM1175 cells with positive Ki67 staining is indicated and was determined from at least two separate transfection experiments and from a total of at least 300 cells. All standard deviations were less than ±5% (bar=100μm). (B) Transfected WMM1175 cells were analyzed for SA-β-Gal activity, and the percentage of positive SA-β-Gal transfected cells is indicated, and was determined as detailed above (bar=100μm). (C) The appearance of SAHF was analyzed by immunostaining with antibodies to H3K9Me and co-staining DNA with DAPI. The percentage of transfected cells with detectable foci is indicated, and was determined as detailed above (bar=100μm).