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. Author manuscript; available in PMC: 2015 Feb 9.
Published in final edited form as: Stem Cells. 2014 Jun;32(6):1437–1450. doi: 10.1002/stem.1707

Figure 4. YB-1 transcriptionally regulated BMI1 to enhance self-renewal capacity.

Figure 4

(a) ChIP analysis of HTRY cells pre-treated with DMSO or anacardic acid (AA) for 4 hours prior to induction. DNA templates were pulled down with YB-1 or nonimmune IgG antibody and different promoter regions (ChIP a and ChIP b) were amplified using primers flanking YB-1 binding sites in the BMI1, CD44, and CD49f promoters. (b) Immunoblot analysis of HTRY cells treated with RSK1/2 siRNA for 96 hours or BI-D1870 for 24 hours. (c) The percentage of cells in each phase of the cell cycle at the indicated time points after plating was quantified by DNA content based on Hoechst 33342 intensity using an ArrayScan VTI. (d) Cell cycle profile of HTRY cells treated with scrambled control (scr) or BMI1 siRNA for 96 hours was measured using an ArrayScan VTI. Uninduced (UN) cells served as a control. Immunoblotting confirmed BMI1 knockdown. (e) Uninduced (UN) HTRY cells transfected with empty vector (EV) or BMI expression plasmid and induced HTRY cells treated with scrambled (scr) or BMI1 siRNA for 96 hours were grown in mammosphere cultures. BMI1 over-expression and knockdown was confirmed by immunoblotting. Data represented as mean ± SEM. P values were determined using t test. **, P < 0.01.