Fig 2: GOT1 is required for increased aspartate synthesis in SDH impaired cells upon complex I co-inhibition.(A) Western blot for GOT2 and Vinculin from WT 143B cells, GOT2 KO 143B cells, and the same GOT2 KO clone expressing GOT2 cDNA (AB). Vinculin is the loading control. (B) Proliferation rates of GOT2 KO 143B cells compared to GOT2 AB 143B cells treated with vehicle (DMSO), 5 µM AA5, or 5 µM AA5 and 50 nM rotenone in DMEM with 1 mM PYR (n=3). (C) Relative aspartate levels measured by LCMS metabolomics of GOT2 KO 143B cells compared to GOT2 AB 143B cells treated with vehicle (DMSO), 5 µM AA5, or 5 µM AA5 and 50 nM rotenone in pyruvate-free DMEM with 1 mM AKB for 6 hours (n=3). (D) Proliferation rates of GOT2 KO 143B cells treated with vehicle (DMSO), 5 µM AA5, 5 µM AA5 and 50 nM rotenone, or 5 µM AA5 and 20 mM aspartate in DMEM with 1 mM PYR (n=3). (E) Western blot for GOT1 and Vinculin from WT 143B cells, GOT1 KO 143B cells, and the same GOT1 KO clone expressing GOT1 cDNA (AB). Vinculin is the loading control. (F) Proliferation rates of GOT1 KO 143B cells compared to GOT1 AB 143B cells treated with vehicle (DMSO), 5 µM AA5, or 5 µM AA5 and 50 nM rotenone in DMEM with 1 mM pyruvate (n=3). (G) Relative aspartate levels measured by LCMS metabolomics of GOT1 KO 143B cells compared to GOT1 AB 143B cells treated with vehicle (DMSO), 5 µM AA5, or 5 µM AA5 and 50 nM rotenone in pyruvate-free DMEM with 1 mM AKB for 6 hr (n=3). (H) Proliferation rates of GOT1 KO 143B cells treated with vehicle (DMSO), 5 µM AA5, 5 µM AA5 and 50 nM rotenone, or 5 µM AA5, 50 nM rotenone and 20 mM aspartate in DMEM with 1 mM PYR (n=3). (I) Schematic showing how aspartate is synthesized via GOT1 from RCQ and PC in SDH-deficient cells when complex I is also impaired. Data are plotted as means ± standard deviation (SD) and compared with an unpaired two-tailed student’s t-test. p<0.05*, p<0.01**, p<0.001***, p<0.0001****. Figure 6—source data 1.Proliferation rates and relative metabolite levels by LCMS in Figure 6. Figure 6—source data 2.Uncropped western blots for Figure 6A and E.

Fig 3: Ammonia promotes T cell apoptosis and inhibits T cell proliferation.a–d, Got1-deficient and -sufficient donor P14 CD8+ T cells were isolated from C57BL/6 host mice infected with LCMV Armstrong 8 days earlier. A total of 0.25 × 106 cells were cultured with anti-CD3 and anti-CD28 in the presence or absence of NH4OH for 2 days. FACS plots (a–b) and line graphs (c–d) show the percentages of AnnexinV+ PI+ cells (a, c) and Ki-67+ cells (b, d). Data are combined from two experiments with three mice in total. The results are presented as mean ± s.d. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Comparisons were performed using two-way ANOVA. P values in c (from left to right): 3.6 × 10−6, 0.026, 0.0006, 0.003, 5.3 × 10−6, 9.7 × 10−9; in d: 0.0006, 2.3 × 10−7, 1.9 × 10−11, 6.3 × 10−7, 4.0 × 10−5, 4.8 × 10−7. Six-week-old female mice were used (a–d). Source data

Fig 4: NFAT1 binds to Got1 locus.a–d, P14 splenocytes were cultured with GP33–41 peptide for 3 days before ChIP-PCR analysis. PCR was performed using primers evenly spaced across a 10kb region of Got1 locus. Line curves show the relative quantification results of DNA fragments pulled down using indicated antibodies or IgG isotype control. Bar graphs show the binding of NFAT1 to Il2 (a known target gene of NFAT1) locus, the binding of TOX to Pdcd1 (a known target gene of TOX) locus, the binding of Eomes to Il2rb (a known target gene of Eomes) locus, and the binding of Blimp1 to Id3 (a known target gene of blimp1) locus. The data are presented as mean ± s.d. **P<0.01; ***P<0.001; ****P<0.0001. Comparisons were performed with two-way ANOVA (line graphs, a–d), a two-tailed Student’s t-test (bar graphs, a–d, data points were normally distributed). P values in a (from left to right): 9.1 × 10−13, <1.0 × 10−15, <1.0 × 10−15, 0.002; b: 0.0003; c: 0.0003; in d: 0.0002. Seven-week-old male mice were used (a–d). Source data

Fig 5: CD8+ T cell responses require GOT1 during acute infection.a, Experimental design illustration. b, FACS gating strategies used in c–l. c–l, Contour plots, histograms, and bar graphs show the flow cytometry staining results of Ly5.1+ donor P14 CD8+ T cells (c,d), KLRG1 and IL-7Rα (e,f), cytokines (g,h), Ki-67 (i,j), and cleaved caspase-3 and Bim (k–l) in Got1-deficient and sufficient P14 CD8+ T cells. Cells were stimulated with GP33–41 peptide before the flow cytometry staining (g,h). Data were pooled from two independent experiments (c–l) with ten C57BL/6 mice in each group receiving Got1-deficient and sufficient donor P14 CD8+ T cells. The results are presented as mean ± s.d. **P<0.01; ***P<0.001; ****P<0.0001; n.s., not significant. Comparisons were performed using a two-tailed Student’s t-test (c, d, %KLRG1−IL-7Ra+ T cells among donor T cells in e, f, g, h, i, j, k, l; data points were normally distributed) or the two-tailed Mann-Whitney test (%KLRG1+IL-7Ra− T cells among donor T cells in e; data points were not normally distributed). P values in c: 1.6 × 10−5, 0.006; in d: 0.675, 0.745; in e: 0.752, 0.397; in f: 0.127, 0.738; in g: 6.4 × 10−6; in h: 0.069; in i: 2.4 × 10−4; in j: 0.3959; in k: 1.1 × 10−5, <1.0 × 10−15; in l: 0.194, 0.088. Six-week-old female mice were used (b–l). Source data