Fig. 2.

Shigella OspC3 and IpaH9.8 cooperate to promote the growth of intracytosolic Shigella. (A–E) WT HeLa cells unprimed or primed overnight with 10 ng/mL IFNγ were infected at an MOI of 3 with designated strains, each of which carries pBAD33-sfGFP, pBR322-AfaI, and pP_END-OspC3 or an EV. Thirty minutes postinfection (p.i.), PI, Hoechst, and arabinose were added to the medium, and cells were imaged using an automated fluorescent microscope. Time course of total GFP divided by the number of Hoechst-positive (Hoechst⁺) nuclei in (A) and percentage of PI⁺/Hoechst⁺ cells in (B). Values shown are the mean ± SEM of three experimental replicates. (C-F) Representative images of cells infected with the designated strains 3 h p.i. (C and E) with enlarged Insets are shown in (D and F), PI (red), Hoechst (blue), and sfGFP (green). Yellow arrows point to live PI⁻GFP⁺ cells, while orange arrows point to dead PI+GFP cells. (F) WT HeLa cells primed overnight with 10 mg/mL IFNγ were infected at an MOI of 3 with designated strains that contain pBAD33-sfGFP and pNG162-AfaI. Three hours p.i., the cells were lysed, and intracellular bacteria were enumerated. Three biological replicates were performed, and representative data are shown. Statistical significance when indicated was assessed by one- (E and F) or two-way (A and C) ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ****P < 0.0001, ns = nonsignificant.