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. 2006 Feb 28;103(10):3645–3650. doi: 10.1073/pnas.0600069103

Fig. 2.

Fig. 2.

bmi-1 is a direct transcriptional target of c-Myc. (A) Bmi-1 mRNA expression in mid- and late-passage c-myc+/+ cells, and in mid-passage and hTERT-expressing late-passage c-myc+/− cells analyzed by qPCR (Left). Bmi-1 mRNA expression after ectopic overexpression of c-Myc in c-myc+/+ cells (Right). c-Myc cDNA was introduced with pBabe-puro retrovirus vector, and cells were selected with puromycin and harvested 6 days after infection. EV, empty vector. (B) Knockdown of c-Myc in normal LF1 HDF leads to down-regulation of Bmi-1 mRNA. Cells were transfected with c-Myc or nonspecific (Contr) siRNAs, RNA was extracted 48 h after transfection, and c-Myc and Bmi-1 mRNA levels were quantified by qPCR. (C) Ectopic expression of Bmi-1 prevents the up-regulation of p16 elicited by c-Myc knockdown. Mid-passage c-myc+/+ (LF1) cells were infected with pBabe-puro expressing Bmi-1 (BP-Bmi) or empty vector (BP). Drug-resistant pools of cells were subsequently infected with c-Myc shRNA-expressing lentivirus (shMyc) or empty vector control (EV). RNA was extracted 3 days after infection, and p16 mRNA levels were quantified by qPCR. The c-Myc shRNA used resulted in ≈70% knockdown of c-Myc mRNA. (D) c-Myc binds directly to the bmi-1 promoter. ChIP was performed by using LF1 HDF cells, either serum-deprived to turn off c-Myc expression (0 h), or serum-stimulated to induce c-Myc expression (4 h). Immunoprecipitating antibodies (IP-Ab) were against c-Myc, and GST as a negative control. In addition to primers (Prim) to the bmi-1 promoter (Bmi-1), primers to a known c-Myc target (nucleolin) and to a promoter without E-boxes (No E-box) were used as positive and negative controls, respectively (29). Pull downs were quantified by qPCR.