Data Supplement from Decreased Expression of Cystathionine β-Synthase Promotes Glioma Tumorigenesis

Supplementary Figure S1. Cell proliferation of U87-MG subclones in vitro. 5,000 cells were seeded and cultured under 20% or 1% O2. Viable cell number was counted by trypan blue staining on days 2, 3, and 4, and normalized to the starting cell number (mean + SEM, n = 3). Supplementary Figure S2. Analysis of orthotopic tumors after injection of 1 x 104 cells into the caudate-putamen. (A) Tumor volume was determined by histological analysis 5 weeks after stereotactic injection of U87-MG parental or subclone cells into the brains of SCID mice (bar = mean, n = 7); *p < 0.05 vs shNT. (B) Depth of invasion was determined by histological analysis (mean + SEM, n = 5-7). (C) Anti-CD105 immunohistochemistry was performed and blood vessel density in tumor sections was determined (mean + SEM, n = 10-14). (D) Anti-Ki-67 immunohistochemistry was performed and stained cells were counted (mean + SEM, n = 5-7). (E) Immunohistochemistry was performed to detect active caspase-3 and stained cells were counted (mean + SEM, n = 5-7). Supplementary Figure S3. HIF-2α mRNA levels were determined by RT-qPCR (mean + SEM, n = 3) in parental, shScr, shCBS#07, and shCBS#61 subclones cultured at 20% O2.