Fig. 6

AMPK-HIF-1α signaling promotes de novo serine biosynthesis, proliferation, and survival of GBM cells upon serine/glycine deprivation. A Isotopomer distributions of G6P, F6P, F1,6BP, 3-PG, 3-PS, serine, and glycine from [U-¹³C₆]-glucose in U87MG cells cultured with S/G(-) media with or without the depletion of AMPK or HIF-1α, n = 3 biologically independent samples. B and C Indicated cells with or without the depletion of AMPK or HIF-1α were cultured with S/G(-) media for 2 d (to measure glucose consumption), or 5 d (to measure intracellular serine/glycine levels). Glucose consumption (B) and intracellular serine/glycine levels (C) were measured. D Indicated cells with or without the depletion of AMPK or HIF-1α and with or without the reconstituted expression of SSP genes were cultured with S/G(-) media for 5 d. Intracellular NADPH and NADP⁺ levels (top panel) and ROS levels (bottom panel) were measured. E Indicated cells with or without the depletion of AMPK or HIF-1α were cultured with or without S/G(-) media for the indicated periods of time, and harvested for cell counting. NS, not significant. F U87MG cells with or without the depletion of AMPK or HIF-1α were cultured with or without S/G(-) media for 4 d, and stained with Annexin V. Representative staining (top panel) and quantification of the staining (bottom panel) are shown. Scale bar, 20 μm. G and H Indicated cells with or without the depletion of AMPK or HIF-1α, and with or without the reconstituted expression of SSP genes, were cultured with S/G(-) media for 4 d (for Annexin V staining) or 6 d (for cell counting). The data represent the mean ± s.d. of three independent experiments (A-H). *P < 0.05; **P < 0.01; ***P < 0.001, based on the Student’s t-test or one-way ANOVA with Tukey’s post hoc test