Extended Data Fig. 5. 6h U-¹³C-glucose re-exposure following TGR.

WT CD8⁺ T cells isolated from spleens of C7Bl/6 mice were activated with anti-CD3 (5 mg/mL), anti-CD28 (0.5 mg/mL), IL-2 (100 U/mL), expanded for a total of 72 hours, and exposed to 10mM (black) or 1mM (orange) glucose. Cells were re-exposed to 10mM U-13C-glucose for 6h prior to polar metabolite extraction and analysis by LC-MS. 20h cultures were started at 1×10⁶ (control) and 1.5×10⁶ (TGR) cells per/ml to generate similar end concentrations for glucose pulse experiments. (a) Percent ¹³C label incorporation into nucleotide monophosphates was analyzed. Data are from n=3 biological replicates. Significance was calculated using 2-tailed Student’s t tests. * p<0.05. (b) Relative ¹³C label incorporation into serine was analyzed. Data are from n=3 biological replicates. Significance was calculated using 2way ANOVA. Significance is indicated for the ¹³C portion (open bars). *** p<0.001. The ¹²C portion (filled bars) was also significant. ** p<0.01. (c) The GSH/GSSG ratio was calculated from summed isotopologues for each metabolite. Data are from n=3 biological replicates. Significance was calculated using 2-tailed Student’s t tests, no significant difference was found. (d) Relative ¹³C label incorporation into lactate was analyzed. Data are from n=3 biological replicates. Significance was calculated using 2way ANOVA. Significance is indicated for the ¹³C portion (open bars), which was not significant. The ¹²C portion (filled bars) was significant. *** p<0.001. All error bars show SEM.