Fig. 4

HIF-1α induces SSP gene expression in response to serine/glycine deprivation. A Indicated cells were cultured with or without S/G(-) media for the indicated periods of time. Whole cell lysates (WCL) and nuclear fractions were prepared, and immunoblotting analyses were then performed with the indicated antibodies. B HRE luciferase activities were measured in the indicated cells culturing with or without S/G(-) media for the indicated periods of time. C Indicated cells were cultured with or without S/G(-) media for the indicated periods of time. Quantitative real-time PCR analyses were performed with the indicated primers. D Indicated cells were cultured with or without S/G(-) media for the indicated periods of time. Immunoblotting analyses were performed with the indicated antibodies. E Indicated cells were cultured with or without S/G(-) media for 24 h, and then treated with CHX (100 μg⋅mL⁻¹) for the indicated periods of time. Immunoblotting analyses were performed with the indicated antibodies. Quantification of HIF-1α levels relative to tubulin is shown. Data represent the mean ± s.d. of three independent experiments. F Indicated cells were cultured with S/G(-) media in the presence or absence of PX-478 (10 μM) for 24 h. Quantitative real-time PCR analyses were performed with the indicated primers. G Schematics of the putative HIF-1α binding site on the PHGDH, PSAT1, and PSPH promoter regions, respectively. H Indicated cells were cultured with or without S/G(-) media for the indicated periods of time. ChIP assays were performed with anti-HIF-1α antibody, and quantitative real-time PCR analyses were performed with primers against the PHGDH, PSAT1, and PSPH promoters. I and J Indicated cells with or without the depletion of HIF-1α were cultured with or without S/G(-) media for the indicated periods of time. Quantitative real-time PCR (I) and immunoblotting (J) analyses were performed with the indicated primers and antibodies, respectively. K HRE luciferase activities were measured in the indicated cells cultured in complete media with or without CoCl₂ for 24 h. L and M Indicated cells were cultured in complete media with or without CoCl₂ for 24 h. Immunoblotting (L) and quantitative real-time PCR (M) analyses were performed with the indicated antibodies and primers, respectively. The data represent the mean ± s.d. of three independent experiments (B, C, E, F, H, I, K, M). *P < 0.05; **P < 0.01; ***P < 0.001, based on the Student’s t-test