Fig. 5

Physiological and clinical relevance of CK2α mediated BMI1 phosphophorylation: a Re-expression of exogenous BMI1 in a BMI1 silenced cells. CP20 and OV90 cells are co transfected with FLAG tagged plasmids for WT (wild-type) -BMI1or Mut (S110A) -BMI1 or EV (empty vector) along with siBMI1 (siRNA against the 3’UTR of the BMI1) or siC (scrambled siRNA). Expression of the exogenous and the endogenous BMI1 were probed by immunoblotting with the FLAG and BMI 1 antibodies respectively. HSP60 is used to indicate equal protein loading. b, c Clonal growth. CP20 and OV90 cells were co-transfected with plasmid and siRNA as described in (A) and 48 h post transfection, cells were recounted and plated as single cells in 35 mm dishes. After 10 (CP20) or 14 (OV90) days, colonies were stained with crystal violet, imaged and 9 images from 3 independent experiments were quantified using ImageJ in a blinded fashion. Top panel depicts a graphical representation of data presented as Relative clonal growth against control (siC + EV) which is considered as 1. Lower panel shows representative images of the colonies. P < 0.05 is considered statistically significant and represented with * (SiC + EV vs EV + siBMI1); # (EV + siBMI1 vs WT-BMI1 + siBMI1); @ (WT-BMI1 + siBMI1 vs Mut-BMI1 + siBMI1). d Coexpression of CK2α and BMI1 in primary tissues. Protein was extracted from the 20 primary tumors (T1 to T20) and 2 normal fallopian tube epithelial (FTE 1, 2) samples as described in the material and methods section and were quantified by densitometry analysis using Image J. Expression of CK2α and BMI1 were individually normalized to their respective α-tubulin levels. Left Panel represents immunograph for BMI1 and CK2α expression in primary tissues. Right panel represents scatterplot of CK2α vs. BMI1 with overlaid linear regression line among tumor tissue samples (N = 19). Spearman correlation coefficient is 0.62 (p = 0.0021)