Figure 3.

Simultaneous knockdown of HIF-1α and SP1 abrogates p21 induction and partially rescues G₁ accumulation. (A) U2OS cells were transfected using nontargeted, HIF-1α, SP1 or both SP1 and HIF-1α siRNA prior to extraction of mRNA. Subsequently, p21, HIF-1α and SP1 mRNA were analyzed using quantitative PCR. Graph depicts mRNA levels normalized to actin. Student t-tests (two tailed) were performed and p-values calculated. *p ≤ 0.05 and **p ≤ 0.01. (B) U2OS cells were transfected as in A prior to lysis. WCL were analyzed by western blot using the indicated antibodies. (C) U2OS were transfected with control and HIF-1α siRNA oligonucleotides prior to fixation and lysis. ChIP were performed using SP1 and control IgG antibodies. p21 promoter regions were amplified using specific primers and levels of SP1 recruitment were analyzed by qPCR. (D) U2OS cells were treated and processed as in (C), but levels of RNA Polymerase II present at the p21 promoter were analyzed. (E) U2OS cells were transfected as in (A) prior to harvesting for cell cycle analysis using the propidium iodide staining protocol. Student's t-tests (two tailed) were performed and p-values calculated. *p ≤ 0.05 and **p ≤ 0.01. Data refers to a minimum of three independent experiments.