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. 2020 Jun 25;225(6):1005–1010. doi: 10.1093/infdis/jiaa367

Figure 2.

Figure 2.

Taok2 siRNA knockdown or inhibition of its kinase activity by the chemical compound SW172006 impairs efficient rupture of Listeria monocytogenes-containing vacuoles. A, Taok2 knockdown by siRNA (left) or inhibition of its kinase activity (right) results in reduced cytoplasmic access. HeLa cells were transfected with the YFP-CBD of the L. monocytogenes phage endolysin Ply118, which identifies cytosolic bacteria shortly after escape. The red line indicates the mean. Results were from 100 cells counted in 3 representative fields to estimate the number of CBD-labeled bacteria. Statistical analysis: 2-tailed unpaired t tests. **P < .01, ****P < .0001. B and C, Taok2 accumulation in a compartment resembling a L. monocytogenes-containing vacuole. B, HeLa cells infected with L. monocytogenes EGDe PrfA*ΔplcAΔplcB for 30 minutes and imaged. Taok2 (labeled with anti-Taok2 antibodies) is shown in red, DNA (labeled with Hoechst) is shown in green, extracellular bacteria (labeled with anti-L. monocytogenes antibodies) are shown in blue, and actin (labeled with phalloidin) is shown in white. Left image: overview. Right images: enlargement of 3 ROIs shown in the overview. In ROIs 1 and 2, Taok2 decorates intracellular bacteria (small arrows, revealed by the bacterial DNA labeling and by the absence of extracellular bacterial labeling), which are present in a vacuolar compartment and do not polymerize cytoplasmic actin (absence of actin labeling). In ROI 3, extracellular bacteria (large arrowhead, revealed by the presence of both DNA and extracellular L. monocytogenes labeling) are not decorated by Taok2. Scale bar, 5 μm. C, HeLa cells infected as in (B) using the strain L. monocytogenes EGDe PrfA* ΔplcAΔplcBΔhly. Left image: overview. Right images: enlargement of 3 ROIs shown in the overview. In all the ROIs showing either intracellular or extracellular L. monocytogenes Taok2 does not decorate the bacteria. Scale bar, 5 μm. Abbreviations: CBD, cell wall binding domain; DMSO, dimethyl sulfoxide; LM, Listeria monocytogenes; ROI, regions of interest; siRNA, small interfering RNA; YFP, yellow fluorescent protein.