Fig 2: BCAT1 expression is correlated with GBM cell viability, migration, and invasion. (A) BCAT1 expression was inhibited by shRNA (sh-BCAT1) in GBM cells. Empty vector-transfected cells were used as a control (Vec). (B) CCK-8 assays indicated the viability of pGBM cells in the sh-BCAT1 or Vec group. (C,D) Representative images and statistical analysis of transwell assays of GBM cells in the sh-BCAT1 or Vec group. (E,F) Representative images of wound healing assays in GBM cells at 0 and 12 h in the sh-BCAT1 or Vec group. GBM cell migration significantly depended on BCAT1 expression. The results are representative of three independent experiments. (G) BCAT1 knockdown decreased cellular glutamate. Glutamate from GBM cells was analyzed with LC–MS/MS, and the relative abundance (by metabolite peak area) was shown. Relative metabolite abundances were normalized to cell numbers. (H) Total glutathione levels in GBM cells. No difference was seen between the sh-BCAT1 and Vec groups. (I) BCAT1 knockdown by shRNA decreased TxN expression in GBM cells. (J) Glutamate supplementation replenished TxN expression in BCAT1 knockdown GBM cells. GAPDH was used as a loading control. All data are shown as the mean ± SEM. Significance was assessed using Student t-tests and one-way analysis of variance. * p < 0.05, ** p < 0.01; NS, not significant. CCK-8: Cell Counting Kit-8. Scale bar, 100 μm.

Fig 3: BCAAs sustain the malignant proliferation of GBM tumors. (A) The survival percentage of nude mice orthotopically transplanted with BCAT1 wild-type and BACT1-deficient LN229 cells, respectively. There was a significant difference in the OS time between these two groups (p = 0.02). There were 16 biologically independent mice in each group. (B) Representative images of H&E staining of orthotopically transplanted tumors. (C) Quantification of tumor size of LN229 cells transplanted orthotopically in a nude mouse model. All data are shown as the mean ± SEM. Mean ± SEM, two-tailed T test, p =0.0003. (D) Representative images of IHC staining of BCAT1, GCLM, and TXNRD1 in orthotopically transplanted tumors. (E) Positive correlation between the staining density of BCAT1 and TXNRD1. (Pearson correlation: r = 0.7088, p =0.0099). (F) Representative images of BCAT1 staining in GBM samples. Negative (−), weakly positive (+), moderately positive (++), and strongly positive (+++). (G) Statistical analysis of BCAT1 IHC integral optical density in 90 GBM samples (IDH wild-type) and 14 diffuse astrocytoma samples (grade 4, IDH mutant). (H) Kaplan–Meier survival analysis showed that GBM patients with higher BCAT1 expression had shorter OS than those with lower BCAT1 expression (p = 0.045, log-rank test). (I) Representative images of GCLM staining in GBM samples. Negative (−), weakly positive (+), medium positive (++), and strong positive (+++). (J) Statistical analysis of GCLM IHC integral optical density in 90 GBM samples (IDH wild-type) and 14 diffuse astrocytoma samples (grade 4, IDH mutant). (K) Kaplan–Meier survival analysis showed that GCLM expression showed no correlation with OS in GBM samples (p = 0.3052, log-rank test). (L) Representative images of TXNRD1 staining in GBM samples. Negative (−), weakly positive (+), moderately positive (++), and strongly positive (+++). (M) Statistical analysis of TXNRD1 IHC integral optical density in 90 GBM samples (IDH wild-type) and 14 diffuse astrocytoma samples (grade 4, IDH mutant). In GBM samples, TXNRD1 was significantly higher in the high BCAT1 group than in the low BCAT1 group (p = 0.001). (N) Kaplan–Meier survival analysis showed that GBM patients with higher TXNRD1 expression had shorter OS than those with lower BCAT1 expression (p = 0.0103, log-rank test). All data are shown as the mean ± SEM. The p values were determined by a two-tailed t-test. Scale bar, 100 μm. *** p < 0.001.

Fig 4: Regulation of BCAA degradation and ubiquitin‐dependent proteolysis in hypophysectomized rats. Overview and number of differentially expressed phosphoproteins (DEPPs) among WT, PR and rhGH groups (A); Heat map of DEPPs between the rhGH and PR groups (B). The unit of the colour gradient was shown by Z‐score. The gene set enrichment of DEPPs by KEGG between the rhGH and PR groups (C). Differences in the key enzymes related to BCAA degradation in liver tissues (D), including western blotting of BCKDHA and p‐BCKDHA (E); Western blotting of BCAT1, BCAT2 and MuRF1 in skeletal muscle among the different groups (F). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Fig 5: Bcat1 epigenetically promotes inflammatory response by downregulating α-KG (A–D) rMC-1 was transfected with scr or siRNA targeting Bcat1 (#2). Cells were incubated in either normal glucose (5.5-mM) with mannitol (24.5-mM) as an osmotic control or high glucose (30-mM). A ChIP–qPCR assay was performed with H3K4me3 and H3K9me3 antibodies to determine enrichment at Gfap (A), Il6 (B), Tnf-α (C), and Cxcl10 (D). IgG was included as a negative control. (E) rMC-1 was transfected with Flag–BCAT1. Cells were treated with or without exogenous α-KG. Total RNA was extracted to determine the mRNA expression of Gfap, Il6, Tnf-α, and Cxcl10. (F) Flag–BCAT1 was expressed in rMC-1 cells. Cells treated with or without α-KG were subjected to ChIP–qPCR analysis to determine H3K4me3 enrichment. IgG was used as a negative control. All data are shown as mean ± SD from at least three independent experiments. *P < 0.05, **P < 0.01; n.s., no significant change.