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. 2021 Mar 2;12(2):e02983-20. doi: 10.1128/mBio.02983-20

FIG 4.

FIG 4

Secretion of CpnT in macrophages requires functional ESX-1, ESX-4, and ESX-5 systems. (A) Fluorescence microscopy of infected RAW 264.7 macrophages with WT, ESX-1-deficient (ESX-1–), ESX-4-deficient (ESX-4–), and ESX-5-deficient (ESX-5–) strains expressing cpnT. All strains expressed gfp from the L5 bacteriophage integration site. Macrophages were infected at an MOI of 2 and fixed at 24 h postinfection. Cells were permeabilized with 0.2% Triton X-100 and immunolabelled with the anti-HA antibody (CpnT-HA). Additional dyes were used to label the nuclei (Hoechst) and the actin filaments (Phalloidin). The scale bar represents 10 μm. Images are a representation of five biological replicates. (B) The fluorescence quantification of the anti-HA signal per infected RAW macrophage, from microscopy pictures, was calculated using automated image analysis by the software CellProfiler. Analysis was performed on one representative experiment out of three independent experiments with 30 to 46 infected cells. One-way ANOVA and multiple comparisons using Dunnet’s statistical test were performed for statistical significance. ****, P < 0.0001.