Extended Data Fig. 5: Inhibition of the mitochondrial tricarboxylate transporter SLC25A1 impairs itaconate delivery to the Salmonella-containing vacuole.
(a and b) HeLa cells stably expressing EGFP-tagged IRG1 were pre-treated with the SLC25A1 transporter inhibitor CTPI-2 for 3, 6, or 18 hs (as indicated), and then infected with wild-type S. Typhi (MOI = 6) encoding a luciferase-based itaconate biosensor. The levels of luciferase activity in the cell lysates were then measured 3 hs after infection. Each circle or square represents a single luciferase measurement. The mean ± SD and p values (unpaired two-tailed Student’s t test) of the indicated comparisons are shown (n = 6 for each category). (a) and (b) show results of two independent experiments. (c and d) Inhibition of the mitochondrial tricarboxylate transporter SLC25A1 does not impair IRG1 expression or overall itaconate biosynthesis. (c) HeLa cells stably expressing EGFP-tagged IRG1 were pre-treated with the SLC25A1 transporter inhibitor CTPI-2 for 3, 6, or 18 hs (as indicated), and then infected with wild-type S. Typhi (MOI = 6) encoding a luciferase-based itaconate biosensor as indicated in Extended Data Fig. 10. The levels of IRG1 3, 6 or 18 hours after CTPI-2 treatment were evaluated by western immunoblot with the indicated antibodies. (d) HeLa cells stably expressing EGFP-tagged IRG1 were pre-treated with the SLC25A1 transporter inhibitor CTPI-2 for 18 hs, and the levels of itaconate were measured as indicated in Materials and Methods. Each square represents a single measurement and the mean and SD are shown (n = 3 for each category).
