FIGURE 1:

ISWI depletion results in an increase in HIF luciferase reporter activity. (A–D) Stable cell lines were generated in U2OS (A, C) and HeLa (B, D), using an HRE-luciferase reporter plasmid. In each respective cell line, nontargeting (NT), HIF-1α, or ISWI siRNA oligonucleotides were used with or without the hypoxia-mimetic DFX (200 μM, 24 h) (A, B) or with or without hypoxia (1% O₂ for 24 h; HPX) (C, D). Data were normalized to DFX- or HPX-treated NT samples. (E) A stable cell line expressing the PHD2 promoter fused to luciferase was exposed to DFX along with NT, HIF-1α, or ISWI siRNA oligonucleotides. Data were normalized to DFX-treated NT samples. (F) A stable cell line expressing NF-κB response elements fused to luciferase, treated with TNF-α, and depleted of either RelA or ISWI using siRNA oligonucleotides. For all experiments bars represent the average of at least three independent biological replicates, and error bars represent the SE. The p values were calculated using Student's t test (*p < 0.05; **p < 0.01; ***p < 0.001). (G) Western blot of U2OS cells depleted of HIF-1α and ISWI and treated with or without DFX showing HIF-1α, hSNF2h, and actin as a loading control.