Figure 2.
BCAT1 shows substrate-dependent expression in glioblastoma cell lines. (a–c) Effects of α-KG on BCAT1 expression in glioma cell lines. The numbers above the western blots indicate the fold ratios of protein expression relative to control cells after normalization to the tubulin loading control. The mRNA expression values represent the mean ± s.d. of triplicate samples. (a) Effect of cell-permeable dimethyl–α-KG (dm–α-KG) for 24 h on BCAT1 expression. (b) BCAT1 expression after lentiviral knockdown of α-KG–producing cytoplasmic IDH1 with two different shRNAs (shI and shII). nt, nontargeting. (c) BCAT1 expression after shRNA-mediated knockdown of IDH1 and additional supplementation of the culture medium with 1 mM cell-permeable dm–α-KG for 6 d. (d) Effect of branched-chain α-ketoacid substrates and 2-HG on brain BCAT1 activities. Values are the means ± s.d. of three independent experiments. (e) Western blot analysis showing BCAT1 protein expression in IHAwt and IHAmut cells. (f) Effect of lentiviral overexpression of BCAT1 in IHAmut cells on proliferation determined using the Click-iT EdU cell proliferation assay. Values in the graphs (e,f) represent the mean ± s.d. for n = 3 replicates. *P < 0.05 compared to empty vector control (Student’s t test). (g) Effect of dm–α-KG treatment for 24 h on BCAT1 expression in IHAmut cells. Values in the graph represent the mean ± s.d. for n = 3 replicates. (h) DNA methylation of CpGs (n = 19) in the BCAT1 promoter in IHAwt and IHAmut cells (Gene Expression Omnibus GSE30338). The average absolute methylation levels are shown. Box plots represent the median, the 25th and 75th percentiles (boxes) and the 5th and 95th percentiles (whiskers).