Fig 2. SifA inhibits lysosome function.
(A) Quantitation from 3D confocal microscopy of the mean fluorescence signals of TGN-associated CD-MPR in HeLa cells infected with the indicated strains. (B) HeLa cells infected for 14 hours with sifA, sseJ double mutant Salmonella expressing SifA-HA or SifAL130D-HA were immunolabeled for HA (blue), TGN46 (green), and CD-MPR (red). (C) Magic Red-RR recovery assay of CatB activity in HeLa cells infected for 14 hours with the indicated strains. (D) HeLa cells were infected for 14 hours with the indicated Salmonella strains and exposed to Magic Red-RR, and the percentage of fluorescent product intensity of infected cells compared with uninfected cells from the same sample was measured at steady state by flow cytometry. (E) HeLa cells were infected as above and pulse chased with DQ-bovine serum albumin (DQ-BSA, Life Technologies, Paisley, UK), and the percentage of fluorescence between infected and uninfected cells from the same sample was measured by flow cytometry. (F) BALB/c mice were inoculated orally with the indicated Salmonella strains. After 48 hours, CD11b(+) splenocytes were harvested and subjected to Magic Red-RR assay. Bars represent the percentage of steady-state fluorescence of infected cells compared with uninfected cells from the same sample or of cells from uninfected mice compared with uninfected cells from infected animals. (G) HeLa cells were transfected with vectors encoding GFP-SifB, GFP-SifA, or GFP-SifAL130D, and 24 hours later cells were subjected to Magic Red-RR assay. In each case, fluorescence intensity in cells expressing similar levels of the indicated protein was normalized to that of untransfected control cells within the same sample and then to fluorescence caused by expression of SifB. Statistically significant relationships are denoted (*P < 0.05; **P < 0.01; ***P < 0.005).