Figure 5

Bmi1 positively regulates Nanog expression in mammary epithelial cells. A. Graph showing normalized mRNA levels (in arbitrary units) of Bmi1 and Nanog in primary breast tissues. X-axis shows tumors categorized into Bmi1-high, moderate, and low groups. B. The images show immunohistochemical staining for Nanog and CD44 on tumor tissues that showed high, medium and low Bmi1 levels (also refer to Figure 1C). C. The pictographs show Nanog levels assessed by RT-PCR in indicated cell types (HMEC-primary normal human mammary epithelial cells). D. Graph represents luciferase assay in HEK293T cells to assess the Nanog promoter activity (pGL3-Nanog-Luc) on co-transfection with pRL-TK (Renilla luciferase), pLKO1, pBABEpuro-Bmi1 and pLKO1-shBmi1 vectors. After 48 hrs of culturing Firefly luciferase activity was measured and normalized to Renilla luciferase activity and represented as relative luciferase units (RLU), n =3; error bars indicate s.d, (statistical significance was calculated using unpaired t-test between relative luciferase activity between vector control and Bmi1 overexpression; and between vector control and Bmi1 knockdown, * = p <0.05). E. Graph represents luciferase assay to assess the NFκB promoter activity in HEK293T cells upon Bmi1 overexpression and Bmi1 knockdown compared to the vector (pLKO1) alone cells. After 48 hrs of culturing Firefly luciferase activity was measured and normalized to Renilla luciferase activity and represented as relative luciferase units (RLU), n =3; error bars indicate s.d, (statistical significance was calculated using unpaired t-test between relative luciferase activity between vector control and Bmi1 overexpression; and between vector control and Bmi1 knockdown, * = p <0.05, ** = p <0.01). F. Graphs show fold change of NFκB pathway genes (normalized to β2M mRNA levels) with control vector or Bmi1 overexpression or down-modulation in HMLE, NBLE, and MDAMB231 cells, n =3; error bars indicate s.d).