Fig 3. S. Typhimurium dormancy is negatively regulated by SpoT.

(A) HeLa cells were infected with SINA1.1-harboring S. Typhimurium, the abundance of Vac^-Cyt^- population in wild type and SPI-2 mutant ΔssaV infected cells were quantified with flow cytometry at 6 h pi. (n = 3) (B) Schematic diagram for the construction of SINA derivative, SINA1.9, yielded from the introduction of an arabinose-inducible hilA expression cassette into SINA1.1. SINA1.9 was used to rescue the reduced invasiveness of ΔdksA, ΔrelA and ΔrelAspoT mutant strains. (C) HeLa cells were infected with SINA1.1 or SINA1.9-harboring S. Typhimurium, the abundance of Vac^-Cyt^- population in (p)ppGpp biogenesis and regulon mutants, Δlon, ΔdksA, ΔrelA and ΔrelAspoT were quantified by flow cytometry at 6 h pi (n = 3) (D) Distribution of Vac^-Cyt^-, Vac⁺Cyt^- and Vac⁺Cyt⁺ populations in hilA-expressing wild type (Left) and ΔrelAspoT mutant (Middle) infected HeLa cells at 6 h pi quantified by flow cytometry. Overlay Timer^bac profile (Right) of Vac^-Cyt^- (red) and Vac⁺Cyt^- (blue) populations of wild type and Vac^-Cyt^- population of ΔrelAspoT mutant (grey) in infected HeLa cells quantified by flow cytometry at 6 h pi. (3 independent experiments) At least a total of 1000 events of infected cells were analyzed by flow cytometry in triplicate experiments. Statistics were performed using unpaired t test. ns: not significant (P > 0.05), **P < 0.01, ****P < 0.0001.