NFκB activation is essential for synergistic
activation in OVSAYO cells. a) Western blot analysis of phospho-RelA (Ser536) or total RelA expression following ICAM1 induction in cells cultured under the indicated conditions for 16 h. N and H indicate normoxia (20% O2) and hypoxia (1% O2), respectively. b) Diagram of the ICAM1 promoter region and the positions of the Sp1, NFκB, and AP1 binding sites. A black bar indicates the PCR amplicon for the ChIP assay. c) Quantitative ChIP analysis of RelA binding to the ICAM1 promoter region by real-time PCR, using cells cultured for 16 h under the indicated conditions. RelA binding levels are represented as fold-increases relative to negative control experiments using IgG. Data shown are the mean (n = 3) ± SD. *P = 0.02. d) Effect of RelA on synergistic ICAM1 activation in cells cultured for 16 h under the indicated conditions. Data are the mean (n = 3) ± SD. Inset: western blot analysis of ICAM1 expression in cells transfected with indicated siRNAs and cultured under SSH for 24 h. e) Western blotting against RelA and HIFs in lysates from cells transfected with the indicated siRNAs. HIF levels shown are based on band intensities quantified using ImageJ software.