Fig. 4

The ERK1/2 and AKT transduction pathways along with c-fos, HIF-1α and GPER are involved in the up-regulation of IL-1β induced by hypoxia. a ROS generation in MDA-MB-231 cells cultured upon normoxia or 15 min hypoxia (2% O2) in the presence or absence of the free radical scavenger NAC, as evaluated using the fluorescent probe DCF-DA. Scale bar 200 μM. Side panel shows the quantitative measurement of intracellular ROS (DCF fluorescence obtained in normoxic cells was set as one-fold induction upon which ROS levels induced by hypoxia was calculated). Values represent the mean ± SD of three independent experiments performed in triplicate. b The ERK1/2 and AKT activation induced by 15 min hypoxia in MDA-MB-231 cells is no longer evident in the presence of NAC. Side panels show densitometric analysis of the blots normalized to ERK2 and AKT that served as loading control, as indicated. c Immunoblot of c-fos from MDA-MB-231 cells cultured upon normoxia or hypoxia and in the presence of MEK inhibitor PD98059 (PD) or PI3K inhibitor LY294,002 (LY). Immunoblots of c-fos (d), HIF-1α, GPER and IL-1β (e) in MDA-MB-231 cells upon normoxia or hypoxia in the presence or absence of NAC. f Immunoblots of HIF-1α, GPER and IL-1β in MDA-MB-231 cells upon normoxia or hypoxia and in the presence of PD or LY. g Immunoblots of HIF-1α, GPER and IL-1β in MDA-MB-231 cells transfected with a scramble or a dominant-negative c-fos construct (DN/c-fos) and thereafter exposed to normoxia or hypoxia. h The 26S proteasome inhibitor MG132 rescued the HIF-1α repression observed in MDA-MB-231 cells transfected for 24 h with the DN/c-fos construct and exposed to low oxygen tension (2%). Side panels show densitometric analysis of the blots normalized to β-actin. Values represent the mean ± SD of three independent experiments performed in triplicate. i-j Co-immunoprecipitation assays performed in MDA-MB-231 cells cultured in normoxia or hypoxia, as indicated. Cell lysates were immunoprecipitated with anti-c-fos (i) or anti-GPER (j) antibodies. Immunocomplexes were analyzed by immunoblot with antibodies against the indicated proteins. In control samples, nonspecific IgG was used instead of the primary antibody. An equal amount of the total lysates (input) was blotted for β-actin as loading control. k GPER expression evaluated by immunofluorescence assays in MDA-MB-231 cells cultured upon normoxia or hypoxia. Nuclei were stained by DAPI (blue signal). Fluorescence intensities were quantified in 20 random fields for each condition and results are expressed as fold change of relative fluorescence units (RFU) over cells cultured upon normoxia (set as one-fold induction). Enlarged details are shown in the separate box. l Immunoblots of nuclear fraction lysates derived from MDA-MB-231 cells cultured upon normoxia or hypoxia. Side panel shows densitometric analysis of the blots normalized to laminin, which served as a nuclear marker. β-actin served as a cytoplasmic marker. m Recruitment of HIF-1α and GPER to the HRE site located within the IL-1β promoter sequence in MDA-MB-231 cells exposed to hypoxia. In control samples nonspecific IgG was used instead of the primary antibody. The amplified sequences were evaluated by real-time PCR. Values represent the mean ± SD of three independent experiments performed in triplicate. (*), (○) p < 0.05 for cells cultured upon normoxia versus cells cultured upon hypoxia