Fig. 4: Host cells depleted of executioner caspases are less resistant to intracellular bacterial growth.

Parental HeLa or commercial caspase-3 KO (C3) cells (Abcam) were nucleofected with Cas9/guide RNA ribonucleoproteins (RNPs) targeting caspase-7 before enriching for deletion lines by limited dilutions. Knockout was confirmed by immunoblot using anti caspase-3, caspase-7 and α-tubulin antibodies (A). B caspase-deficient or parental HeLa lines were treated with staurosporine (STS) for 7 h before lysis and measuring DEVDase activity using DEVD-pNA. Indicated HeLa lines were infected with WT Listeria at MOI 0.1 for 16 h +/− TNF-α before the cells were lysed and DEVDase activity was measured (C), and CFU were calculated from bacterial growth curves (D and E), as shown in Figure S1. Averages +/− SEM of three independent experiments are shown. THP-1 cells were nucleofected with Cas9/guide RNPs targeting caspase-3 or -7 before cloning by limited dilutions and assessment by western blot (F). Cells of indicated genotype were infected with Lm at the MOI of 0.1 for 16 h +/− TNF-α, then the cells were lysed and DEVDase activity was assessed using fluorescent DEVD-fmk (G), and CFU were determined as above (H). WT and caspase-7 KO THP-1 cells were additionally infected with SL1344 at a MOI of 0.001 + /- TNF-α for 16 h before intracellular CFU were calculated from growth curves as shown in Figure S1G (I). Averages +/− SEM of three independent experiments are shown and significant differences between groups are indicated by asterisks. P-values are * < 0.05, ** < 0.01 and *** < 0.005.