Figure 6

The ∆PS mutant of BMI1 promotes proliferation and bypasses senescence more efficiently than the wild type BMI1 in HDFs. (A). WI-38 cells expressing wild type BMI1 or ∆PS mutant of BMI1, and control WI-38-B0 cells were plated at 1 × 10⁴ cells/well in multi well plates, and harvested and counted after indicated number of days. Proliferation curves were generated by plotting the number of cells against the number of days. (B). Mid passage control B0, and wild type BMI1- and ΔPS mutant-expressing cells were generated and serially passaged in culture to determine the effect of the mutant BMI1 on the long term replicative life span of WI-38 cells. After selection, at each passage 10⁵ cells were passaged in a T25 and cultures were grown until 70-80% confluence. At each passage, cell numbers were counted and the proliferation curve was generated by plotting cumulative cell number vs days. (C). WI-38-derived cells were analyzed for the expression of p16INK4a, BMI1 (wild type and mutant), and α-tubulin (loading control) by western blot analysis as described in the Materials and Methods section. (D). The number of senescent cells in WI-38-B0, WI-38-BMI1 WT and WI-38-BMI1 ∆PS culture at passage 3 (after selection) was determined using SA-β-gal staining as described in the Materials and Methods section. After staining, the cells were photographed (10X) under phase contrast using a light microscope. The number of senescent cells and total number of cells in each culture were counted in multiple fields to determine the percentage of senescent cells.