Extended Data Fig. 8: Itaconate delivery and bacterial growth in cells used for cryo-ET analysis.
(a) HeLa cells stably expressing IRG1 or BMDMs from C57BL/6 mice treated with LPS (200 ng/ml) for 3 hours were infected with S. Typhi (MOI = 10), and the number of CFU was determined 1 and 3 hs after infection. Each circle represents the CFU in an independent measurement; the mean ± SEM of all the measurements and p values of the indicated comparisons (two-sided Student’s t test) are shown. ns, not significant. ****p < 0.0001 (n = 6 for each category). (b and c) HeLa cells stably expressing IRG1 (b) or BMDMs from C57BL/6 mice treated with LPS (200 ng/ml) for 3 hs (c) were infected with S. Typhi (MOI = 10) carrying a plasmid encoding the itaconate nanoluciferase biosensor. One and three hours after infection, the levels of nanoluciferase were measured in lysates of the infected cells. Each circle represents a single luciferase measurement. The mean ± SD and p-values of the indicated comparisons (two-sided Student’s t-test) are shown. ****p < 0.0001 (n = 6 for each category). (d-g) BMDMs obtained from C57BL/6 (WT) or Hps4−/− were infected with S. Typhi (MOI = 10) carrying a plasmid encoding the itaconate nanoluciferase biosensor, and the number of CFU was determined 1 (a) or 3 (c) hs after infection. Alternatively, the levels of nanoluciferase were measured in lysates of the infected cells (b and d). Each circle represents the CFU in independent measurements (a and c) or a single luciferase measurement (b and d). Shown are the mean ± SEM of all the measurements (n = 6 for each category); p values of the indicated comparisons (two-sided Student’s t test) are shown. **p < 0.01 and ***p < 0.001, ****p < 0.0001. (h and i) Itaconate delivery and intracellular growth of S. Typhi expressing gtgE in cells used for cryo-ET analysis. (h and i) HeLa cells stably expressing IRG1 were infected with S. Typhi or S. Typhi- expressing gtgE (MOI = 10) carrying a plasmid encoding the itaconate nanoluciferase biosensor. The number of CFU (a) or the level of luciferase activity (b) was determined 1 hour or 3 hours after infection. Each circle represents a single measurement. Values are the mean ± SEM of all the measurements and p values of the indicated comparisons (two-sided Student’s t test) are shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 6 for each category).
