Fig. 3

Serine synthesis pathway genes are overexpressed in gliomas and required for brain tumor growth. A Microarrays of human gliomas and normal brain tissue were immunostained with the indicated antibodies. Representative images are shown (left panel). Data represent the mean ± s.d. (right panel). Scale bar, 100 μm. NS, not significant. B The mRNA and protein expression levels of PHGDH, PSAT1, and PSPH in NHA, GSCs and the indicated GBM cells were determined by quantitative real-time PCR (top panel) and immunoblotting (bottom panel) analyses with the indicated primers and antibodies, respectively. C U87MG cells with or without the depletion of SSP genes were cultured with or without S/G(-) media for the indicated periods of time, and harvested for cell counting. D U87MG cells with or without the depletion of SSP genes 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. E A total of 5 × 10⁵ control GSCs or SSP-depleted GSCs were intracranially injected into athymic nude mice. After 21 d, the mice were euthanized and examined for tumor growth. Hematoxylin-and-eosin–stained coronal brain sections show representative tumor xenografts (left panel). Tumor volumes were measured using the length (a) and width (b) and calculated using the equation V = ab²/2. Data represent the mean ± s.d. of 5 mice (right panel). Note that the scores of some samples overlap. Scale bar, 2 mm. F IHC analyses of the tumor tissues were performed with anti-PHGDH, anti-PSAT1, anti-PSPH, and anti-Ki-67 antibodies and TUNEL. Representative staining (left panel) and quantification of the staining (right panel) are shown. Scale bar, 100 μm. The data represent the mean ± s.d. of three independent experiments (A-D, F). *P < 0.05; **P < 0.01; ***P < 0.001, based on the Student’s t-test