Figure 4.

p21 induction and G₁ accumulation also occurs in HIF-1α stable knockdowns and non-transformed cells. (A) Two sequences (1 and 2) of shRNA were used to generate U2OS-HIF-1α stable knockdown cells as reported previously in reference ⁶¹. Confirmation of HIF-1α knockdown in HIF-1α stable cells was performed using western blot analysis. WCL were harvested following 2 h of DFX treatment from non-targeted and HIF-1α stable cell lines. (B) WCLs from U2OS-HIF-1α stable knockdown cell lines were analyzed by western blot using the specific antibodies indicated. (C) U2OS-HIF-1α stable knockdown were harvested for cell cycle analysis using propidium iodide staining protocol. Student's t-tests (two tailed) were performed and p-values calculated. *p ≤ 0.05 and **p ≤ 0.01. (D) MCF10A cells and (E) HFF cells were transfected with non-targeted or HIF-1α siRNA oligonucleotides prior to mRNA extraction. HIF-1α, p21, p27, SP1 and GLUT3 mRNA were analyzed using quantitative PCR. Graphs depict mRNA levels normalized to actin. 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.