Fig 2: Metabolic dysfunction of neuronal PPM1K and BCKDHA increased BCAA levels 24 h after cerebral I/R.a mRNA expression of Slc7a5, Bcat1, Bcat2, Ppm1k, Bckdk and Bckdha at 24 h post cerebral I/R (n = 4–5/group, t test). b The representative picture of immunoblots for BCKD, BCKDK, p-BCKDHA S293, BCKDHA, PPM1K and β-actin at 24 h postcerebral I/R. c Quantification analysis for the immunoblots in (b) (n = 4–6/group, t test). d Representative images of NeuN/PPM1K staining at 24 h post cerebral I/R in sham and I/R groups, including ischemic border (between the two dotted lines) and ischemic core areas. Scale bar = 100 μm. Representative magnified images are on the far right. Scale bar = 50 μm. f Quantification of NeuN-positive and PPM1K-positive cells number (n = 15–18 images from four animals/group, one-way ANOVA followed by multiple comparisons). e Immunofluorescence staining for NeuN/p-BCKDHA S293 at 24 h postcerebral I/R in sham and I/R groups, including ischemic border (between the two dotted lines) and ischemic core areas. Scale bar = 100 μm. Representative magnified images are on the far right. Scale bar = 50 μm. g Quantification of NeuN-positive and p-BCKDHA S293-positive cells (n = 12-18 images from four animals/group, one-way ANOVA followed by multiple comparisons). h BT2 decreased BCAA levels in tissue compared with the VEH group 24 h after reperfusion (n = 8–10/group, one-way ANOVA followed by multiple comparisons). i Quantification of NeuN-positive and p-BCKDHA S293-positive cells (n = 18–21 images from four animals/group, t test). j Immunofluorescence staining of NeuN/p-BCKDHA S293/DAPI in the ischemic core area 24 h postcerebral I/R in VEH and BT2 groups. Scale bar = 50μm. k Representative bands of p-BCKDHA S293 and BCKDHA. l Quantitative analysis of p-BCKDHA S293/BCKDHA protein expression (n = 6/group, t test). All data are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: Examination of genes related to BCAA and fatty acid metabolism in PDAC tissue. A–F: Data mining in GSE62165 dataset. (A) The expression level of SLC carriers (SLC7A5, SLC1A5, SLC3A2, SLC43A1 and SLC43A2) between PDAC and normal tissue. (B) GSEA analysis revealed enriched MYC and NOTCH signaling pathway in PDAC tissue. (C) The expression level of MYC and NOTCH (NOTCH1-4) between PDAC and normal tissue. (D) BCAA catabolism related genes BCAT1, BCAT2, BCKDHA, BCKDHB, BCKDK, ACADS and ACADSB were found significantly down-regulated in PDAC group. (E) The expression of lipogenesis related enzymes including FAS, ACACA and ACLY were significantly up-regulated in PDAC tissue. (F) CPT1A, the rate-limiting enzyme for fatty acid oxidation was notably upregulated in PDAC tissue. G–J: Data mining in GSE102238 dataset. (G) The expression level of SLC carriers (SLC7A5, SLC1A5, SLC3A2, SLC43A1 and SLC43A2) between PDAC and adjacent normal tissue. (H) NOTCH3 and NOTCH4 were upregulated in PDAC tissue while MYC was down-regulated. (I) Gene expression of BCAT1, BCAT2, BCKDHA, BCKDHB and ACADSB were decreased in PDAC tissue. (J) FAS and ACACA were increased in PDAC tissue.

Fig 4: BT2 and Fer-1 protected against neuronal ferroptosis, cerebral blood flow reduction, tissue damage, and promoted neurological functional recovery after cerebral I/R.a Representative images of NeuN/4-HNE staining and quantitative analysis of p-BCKDHA S293-positive and 4-HNE-positive cells in the ischemic border among VEH, BT2, and Fer-1 groups (n = 14–18images from four animals/group, one-way ANOVA followed by multiple comparisons). Scale bar = 100 μm. Representative magnified images are shown on the far right. Scale bar = 50 μm. b Representative western blots and quantitative analysis for of Acsl4 and Gpx4 protein expression, normalized to β-actin (n = 3–5/group, one-way ANOVA followed by multiple comparisons). c Quantification shows BT2 and Fer-1 significantly reduced brain tissue levels of MDA (n = 6–7/group, one-way ANOVA followed by multiple comparisons). d Laser speckle imaging 24 h after cerebral I/R and quantification of relative blood flow. (n = 6–9/group, one-way ANOVA followed by multiple comparisons). e Representative images of Nissl staining and quantification of surviving cells among sham, VEH, BT2, and Fer-1 groups. Scale bar = 1 mm in the upper panel and scale bar = 20 μm in the lower panel (n = 21–25 images from three animals/group, one-way ANOVA followed by multiple comparisons). f Representative images of TTC staining and qquantitative analysis of the percentage of infarct volume relative to total cerebral volume among sham, VEH, BT2, and Fer-1 groups 24 h after cerebral I/R (n = 6–7/group, one-way ANOVA followed by multiple comparisons). g Survival curves of sham, VEH, BT2, and Fer-1 groups during the 14-day observation period. Mortality data for the sham group was significantly differences from the other groups, *P < 0.05 as determined by Log-rank (Mantel-Cox) test. h Neurological changes were observed by Longa neurological scores (upper) and Garcia neurological scores (lower) among sham, VEH, BT2, and Fer-1 groups from 3–14 days. i A diagrammatic sketch marked with footprints and footprint intensity of the sham group. BT2 and Fer-1 significantly increased body regularity index (j), body speed (k), print area (l), mean intensity (m), and swing time (n) of the left forepaw and left hindpaw compared with the VEH group. n = 8–10/group, two-way ANOVA (Bonferroni’s multiple comparison test). LF: left forepaw, LH: left hindpaw. All data are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Immunoblots of selected BCAA metabolic enzymes. (A, B) Immunoblots of Bcat2, Dbt (A) and Bckdha (B) in liver tissue samples from control mice and tumor tissue samples from model mice. Images of full-length immunoblots are shown in Supplementary Fig. 7. (C–E) Quantification ratios of Bcat2 (C), Dbt (D) and Bckdha (E) in the above tissues with Gapdh as a loading control (C = control, T = treatment, n = 4). *P < 0.05, **P < 0.01 and ***P < 0.001.