Figure 3.

Synergistic activation of the ICAM1 gene is independent of ARNT. a) ChIP analysis of ARNT binding to gene regulatory regions of the indicated genes. Cells were cultured for 4 h under stimulation with CoCl₂ (500 μM) or treatment with a vehicle control. I: Input control lysate without immunoprecipitation. IgG: negative control using IgG. b) Results in a) were quantitatively estimated by real-time PCR analysis. c) ChIP analysis of HIF2 binding to the ICAM1 promoter region. Cells were treated as in a). d) The ICAM1 promoter construct (Figure 2e) was co-transfected with the indicated expression vectors. After 24 h, expression of indicated proteins was confirmed by western blotting. e) Luciferase activities were measured at 24 h post-transfection. Luciferase activities measured in cells transfected with the empty vector were defined as 100%. Data shown are the mean (n = 3) ± SD. f) Western blot analysis of lysates from OVSAYO cells transfected with non-specific (NS) or ARNT siRNA and then cultured under SSH for an additional 16 h. g) Real-time RT-PCR analysis of ICAM1 and VEGF gene expressions in OVSAYO cells transfected with NS or ARNT siRNA and then cultured under SSH conditions for an additional 16 h. Data shown are the mean (n = 3) ± SD.