Fig. 7

HIF-1α negatively regulates PCK2 expression. a HIF-1α and HIF-2α expression in normoxic or hypoxic MCF-7 TRCs were analyzed by western blotting. b MCF-7 cells transfected with HIF-1α siRNA or HIF-1α-overexpressing plasmid were cultured in 90 Pa soft 3D fibrin gels in hypoxia or normoxia for 5 days, HIF-1α and PCK2 expression was analyzed by western blotting. c–e MCF-7 cells transfected with HIF-1α siRNA were seeded in 90 Pa soft 3D fibrin gels and cultured for 4 days in hypoxic condition, and then incubated with ¹³C-glucose for another 24 h, LC-MS was performed for m + 2 or m + 4 labeled citrate, fumarate and malate detection. f, g Schematic representation of the promoter region which located at -489 to -482 bp on the upstream of the transcription start site of Pck2. MCF-7 TRCs were cultured in 21% (N) or 1% (H) O₂ condition and then HIF-1α enrichment around the promoter of Pck2 were analyzed by CHIP-PCR as described in methods and IgG was used as a negative control (f). CHIP-qPCR were used to Pck2 quantitative detection and total genomic DNA was used as input (g). h MCF-7 TRCs were transfected with HIF-1α siRNA or overexpress HIF-1α plasmid, and then the luciferase activity was detected as described in methods. i MCF-7 TRCs were treated with different concentrations of DMF (0, 10 μM, 20 μM) in normoxia, and then HIF-1α and PCK2 expression were determined by western blotting. j MCF-7 cells were cultured in 90 Pa soft 3D fibrin gels without or with 20 μΜ DMF in normoxia for five days and the colony size was analyzed. Data shown are representative of three independent experiments and error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001