Figure 3

Deletion of the PS domain of BMI1 augments its pro-proliferative activity and promotes EMT in MCF10A cells. (A). Wild type BMI1 and ΔPS mutant of BMI1 upregulates H2A K119Ub activity of PRC1. The relative expression of H2A K119Ub in MCF10A control, MCF10A-BMI1WT and MCF10A-BMI1ΔPS cells was determined by western blot analysis and densitometry as described in the Materials and Methods section and Fig. 1B. (B). MCF10A-derived cells expressing wild type and mutant BMI1 were plated in P100 plastic dishes (5 × 10⁵/plate). Cells were harvested at the indicated day (days 1-5) and counted using a hemocytometer. Proliferation curves were generated by plotting the number of cells against number of days. (C). Increase in AKT activity (phospho-AKT and phospho-GSK3β), and the expression of AKT/GSK3β targets cyclin D1 and CDK4 was determined by western blot analysis using antibody specific to each protein as described in the Materials and Methods section. β-actin was used as a loading control. The relative expression of phospho-AKT (normalized to total AKT), Phospho-GSK3β (normalized to total GSK3β), CDK4 (normalized to β-actin) and cyclin D1 (normalized to β-actin) was quantified by densitometry as described in Fig. 1B. (D). MCF10A-derived cells expressing wild type BMI1 or the ∆PS mutant were analyzed for the expression of E-cadherin, fibronectin and vimentin using western blot analysis. α-tubulin was used as a loading control. (E). The expression of E-cadherin, vimentin and fibronectin in control, and wild type BMI1- and ∆PS mutant-overexpressing cells was determined by immunostaining as described in the Materials and Methods section. After immunostaining, cells were photographed (10X) using a Nikon Eclipse 80i confocal microscope.