Fig 2: Src phosphorylates and enhances the activity of BCKDK ex vivo.a Src phosphorylated BCKDK in a dose-dependent manner under EGF stimulation (EGF: 80 ng/mL, 15 min). b Src promoted the phosphorylation of BCKDK in HEK293T cells (left panel). Mutation in BCKDK at Y246 significantly downregulated the phosphorylation effect of Src on BCKDK (right panel). (EGF: 80 ng/mL, 15 min). c EGF induced time-dependent phosphorylation of BCKDK in HCT116, LoVo, and SW620 cells. (EGF: 80 ng/mL. 0, 5, 10, and 15 min). d HT29 and SW620 cells were treated with dasatinib at the indicated concentrations (0, 10, 20, and 50 nM). (EGF: 80 ng/mL, 15 min). e Src was knocked down in HT29 and SW620 cells by nontargeting (shMOCK) shRNA or Src shRNAs (#1-#7). f The effects of Src knockdown on the phosphorylation of BCKDK and its downstream targets (MEK1/2 and BCKDHA) were detected by western blot. (EGF: 80 ng/mL, 15 min). g Expression of p-BCKDK (Y246), p-MEK1/2 (S221), and p-BCKDHA (S293) was detected in Src+/+ and Src−/− cells (EGF: 20 ng/mL, 30 min).

Fig 3: 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 4: BT2, Fer-1, and PPM1K knockdown mitigated BCAA induced neuronal ferroptosis.a–e Neurons were pretreated with BT2 (80 μM) or Fer-1 (5 μM) for 4 h and then incubated with BCAA for 24 h. a CCK-8 assay of neurons showed the BT2 and Fer-1 rescued BCAA (5, 20 mM)-induced reductions in cell viability. GSH (b) and MDA (c) levels in neurons were detected by ELISA among control, BCAA (5 mM), BT2 + BCAA, and Fer-1 + BCAA groups. Staining for 4-HNE and C11-BODIPY581/591 shows that BT2 and Fer-1 decreased lipid peroxidation levels. d Representative images of 4-HNE/NeuN double staining in each group of neurons and quantification analysis of 4-HNE MFI. e Representative images of C11-Bodipy581/591 staining in each group of neurons and quantification MFI of oxidized C11-Bodipy581/591. f mRNA expression of PPM1K in neurons treated with medium, infected with Lenti-vector, or Lenti-shPPM1K. (g) Representative immunoblot and statistical analysis of PPM1K in neurons treated medium, Lenti-vector, or Lenti-shPPM1K. h–n Neurons were treated with Lenti-vector or Lenti-shPPM1K. h mRNA expression of Slc7a5, Bcat1, Bcat2, Ppm1k, Bckdk and Bckdha in neurons. i The representative picture of immunoblots and quantification analysis for p-BCKDHA S293/BCKDHA. j BCAA levels in neurons and neurons supernatant. k Neurons viability was detected by CCK-8 assay. l mRNA expression of ferroptosis marker genes Acsl4, Gpx4, xCT, Lpcat3, Ptgs2, and Ncoa4 in neurons. m Representative immunoblot and statistical analysis of ferroptosis marker proteins Acsl4, Ptgs2, xCT, and Gpx4 in neurons. n Representative images of C11-Bodipy581/591 staining and quantification MFI of oxidized C11-Bodipy581/591. Scale bar = 50 μm. All experiments of neurons were performed at least three independent times. All data are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: 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.