FIG 3.

metJ deletion reduces pyroptosis and invasion in vitro. (A) Deletion of metJ reduces pyroptosis and invasion of 18592 LCLs. (B) Deletion of metJ reduces pyroptosis and invasion of THP-1 monocytes. (C) Deletion of metJ reduces invasion of HeLa cells. For the assays whose results are presented in panels A, B, and C, pyroptosis and invasion were measured at 3 h postinfection using a modified gentamicin protection assay across at least three independent experiments. Percent cell death represents all 7-AAD⁺ cells under each infected condition, with the baseline uninfected cell death being subtracted. See the gating in Fig. 1A. Data were normalized to the global mean, and P values, indicated at the top, were calculated either through a one-way analysis of variance with Tukey's multiple-comparison test or by a Student's t test. (D, E) Suppression of pyroptosis could not be rescued by treating bacteria with polyamines. Bacteria were treated with 300 μM putrescine, spermidine, or spermine for 2 h 40 min prior to infection to determine whether the effects on pyroptosis were due to effects on polyamine synthesis. For all experiments with LCLs or THP-1 monocytes, cells were infected at an MOI of 30. HeLa cells were infected at an MOI of 5. For the assays whose results are presented in panels D and E, data were generated from two independent experiments and normalized to the global mean, and P values were generated by a one-way analysis of variance with Dunnett's multiple-comparison test. Error bars represent the standard error of the mean.