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. 2021 Apr 21;40(10):e106188. doi: 10.15252/embj.2020106188

Figure 6. Tpl2[D270A] mutation impairs macrophage killing of S. aureus .

Figure 6

  1. BMDMs of the indicated genotypes were infected with YFP‐S. aureus (MOI 10) for 0.5 h. S. aureus CFU were assessed at 8 h after infection and normalised to CFU at 1 h (n = 12; 2 biological and 6 technical replicates per condition per experiment). As a control, WT cells were treated with bafilomycin A to block V‐ATPase acidification of phagosomes and phagosome proteolysis.
  2. Quantification of YFP‐S. aureus average fluorescence intensity per cell from Fig 5 panel (A) (n = 66–85 cells).
  3. Cell death following was assayed by measuring LDH release from cytosol of BMDMs 8 and 24 h post‐infection with S. aureus (MOI 10; n = 4 wells).
  4. Whole cell extracts from WT and Tpl2[D270A] BMDMs following S. aureus infection (MOI 10) were immunoblotted for the gasdermin D p30 cleavage product. HSP90 was used as a loading control.
  5. BMDMs of the indicated genotypes were infected with GFP‐C. rodentium (MOI 2) for 0.5 h. C. rodentium CFU were assessed at 2 h after infection and normalised to CFU at 0.5 h (n = 12; 2 biological and 6 technical replicates per condition per experiment). As a control, WT cells were treated with bafilomycin A.
  6. Representative images of GFP+ BMDMs labelled with LysoTracker Red DND‐99 dye (red) and DAPI, 1 h post‐infection with GFP‐C. rodentium (green).
  7. Quantification of LysoTracker Red co‐localisation with GFP‐C. rodentium from panel (F) (n = 54–63 cells).

Data information: One representative experiment out of three shown. Error bars represent SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001, not significant (ns). Panels (A, B, C, E, G) one‐way ANOVA.