Fig. 3

The HIF-1α/GPER signaling is involved in the expression of IL-1β induced by hypoxia. mRNA (a) and protein (b) expression of HIF-1α and IL-1β evaluated respectively by real-time PCR and immunoblotting in MDA-MB-231 breast cancer cells cultured upon normoxia or hypoxia (2% O2). In RNA experiments, values are normalized to the actin beta (ACTB) expression and shown as fold changes of mRNA expression induced by hypoxia compared to normoxic cells. c IL-1β levels evaluated by ELISA in the supernatants collected from MDA-MB-231 cells cultured upon normoxia or hypoxia. d The up-regulation of IL-1β observed in MDA-MB-231 cells cultured upon hypoxia is no longer evident silencing HIF-1α. mRNA (e) and protein (f) expression of GPER evaluated in MDA-MB-231 cells cultured upon normoxia or hypoxia, as evaluated by real-time PCR and immunoblotting, respectively. g Recruitment of HIF-1α to the HRE site located within the GPER promoter sequence in MDA-MB-231 cells cultured upon hypoxia. In control samples nonspecific IgGs were used instead of the primary antibody. The amplified sequences were evaluated by real-time PCR. h The up-regulation of GPER observed in MDA-MB-231 cells exposed to hypoxia was abrogated silencing HIF-1α. i Immunoblots of HIF-1α and IL-1β from GPER-silenced MDA-MB-231 cells exposed to hypoxia. Side panels show densitometric analysis of the blots normalized to β-actin. Values represent the mean ± SD of three independent experiments performed in triplicate. (*) and (○) indicate p < 0.05 for cells cultured under normoxia versus cells cultured under hypoxia