Fig. 2. Human APOL3 targets and inflicts damage to cytosolic bacteria.

(A) APOL3^mnGFP targeting Stm^RFP by live imaging in HeLa cells (movie S1). Percentage of total Stm targeted by HA-tagged APOL family members (2 hours) is shown at right. (B) Stm^RFP targeting and replication in IFN-γ–primed ΔAPOL3 cells complemented with the indicated APOL3 variant. (C) Deconvolved wide-field images of APOL3^mnGFP targeting vacuole-confined Stm^RFP (Stm^{ΔinvA::pR1203}) with or without vacuole release with LLOMe; fold replication is shown at right. (D) Inner membrane (IM) integrity as measured by minD^mnGFP aggregation within Stm in HeLa cells expressing APOL3^RFP at 2 hours with or without IFN-γ. Quantification reflects aggregation in APOL3-coated versus uncoated bacteria or total bacteria in WT versus ΔAPOL3 cells via Fisher’s exact test. (E) Arabinose-induced GFP in Stm targeted by APOL3^FLAG in HeLa cells with or without IFN-γ. Maximal-intensity GFP/mCherry ratios are shown (mean ± SD, n = 50). (F) Immunofluorescence and SIM of APOL3^HA and LPS on Stm with or without IFN-γ at selected times. Mid-2D z-planes are shown. Quantification of LPS penetrance (25 bacilli, mean ± SEM, n = 3) and 3D surface rendering are shown below. Blue arrows indicate cryo-immunogold EM staining of APOL3^GFP in Stm-infected HeLa cells; OM, outer membrane. Micrographs are representative of at least three independent experiments. Data are means ± SEM [(A), (B), (C)] with significance by one-way ANOVA at 6 hours. ***P < 0.001. Scale bars, 5 μm [(A), (B), (C), and (E)], 2 μm (D), 1 μm (F).