Fig 2: PHGDH as a prognostic marker for glioma. a Expression analysis of PHGDH protein in normal human astrocytes and astrocytoma cell lines by Western blotting. b Expression analysis of PHGDH mRNA in normal human astrocytes and astrocytoma cell lines by Q-PCR. c Differential expression of PHGDH protein in 5 primary astrocytoma specimens and their paired tumor adjacent tissues were detected by Western blotting. d Differential expression of PHGDH mRNA in the same 5 paired specimens in Fig. 2c was detected by Real-Time RT-PCR. Expression was normalized to GAPDH and SD was from 3 independent experiments. e Kaplan–Meier curves with univariate analyses (log rank) for low PHGDH expression versus high PHGDH expression in WHO I-II glioma patients and WHO III-IV glioma patients

Fig 3: PHGDH interacts with and stabilize FOXM1 in glioma cells. a Expression of FOXM1 mRNA in U87 and U251 cells that express shRNAs for control or PHGDH was detected by Real-Time RT-PCR. Expression was normalized to GAPDH and SD was from 3 independent experiments (p = 0.1325). b Endogenous binding of PHGDH and FOXM1 were detected by using anti-PHGDH or anti-FOXM1 antibodies, respectively, in U87 and U251 cells. c U251 cells were plated in chambers precoated with poly-l-ornithine and fibronectin before stained with anti-FOXM1 and anti-PHGDH antibodies. Images were acquired using a scanning confocal microscope (Olympus FluoView FV1000). Scale bar for 20uM. d Flag-tagged FOXM1 fragments as indicated were transfected into U87 cells. 48 h post transfection, the co-IP were performed by using anti-FLAG antibody. e U251 cells that express shRNA for control or PHGDH were treated with cyclohexmide (50 µM) for 24 h before subjected to western blot analysis. The densitometry was acquired by Beckman Appraise, Beckman Instruments (p < 0.0001 in two way ANOVA). f Upper, control siRNA and PHGDH-2 siRNA were transfected into U87 glioma cells and 20 µM of MG132 were added as indicated 24 h post transfection. Total protein was analyzed 48 h post transfection by western blot. Lower, Flag-tagged FOXM1, HA-tagged Ubqutin and PHGDH-2 siRNA were co-transfected into U87 glioma cells as indicated. 48 h post transfection, co-IP experiments were done by using anti-Flag antibody

Fig 4: PHGDH was overexpressed in glioma samples at both the mRNA and protein levels. a Representing photes of parafin-embedded specimens of 132 primary astrocytoma specimens including WHO grade I–IV and 10 normal brain tissues stained by immunohidtochemistry using anti-PHGDH antibody. IgG were used as negative control. Amplifications are 200×. b Scoring of IHC staining samples. c Quantification PCR were used to detect the PHGDH mRNA expression of 40 glioma tissues (10 grade I astrocytomas, 10 grade II astrocytomas, 10 anaplastic astrocytomas and 10 glioblastomas) and 10 normal brain tissues. The expression level was normalized to GAPDH (log)

Fig 5: PHGDH silencing inhibited glioma tumorigenicity in nude mice. a 1*106 U87 and U251 glioma cells that express shRNA for control or PHGDH were injected intracranially into nude mice as described previously. Brains were harvested when the animal were moribund; the remaining animals were killed 60 days after glioma-cell injection. Each mouse’s brain was fixed in 4 % formaldehyde, and embedded in paraffin. Tumor formation was determined by histologic analysis of H&E-stained sections. Magnification, 100×. b Statistical analysis of tumor size and invasion in intracranial tumor assay, a two-way ANOVA analysis of tumor diameter or invasion lesion numbers with respect to cell line (U87 versus U251) and shRNA expression (GFP, si-PHGDH-1, si-PHGDH-2, p < 0.0001). c Kaplan–Meier curves with univariate analyses (log rank) of nude mice Intracranial Tumor Assay, p < 0.0001