Fig 1: HvKp and cKp strains induce pyroptotic cell death in BMDMs. (A–C) BMDMs were infected with hvKp or cKp (MOI of 10) for 16 h. Cell death was analyzed using (A) flow cytometry with propidium iodide (PI) staining and (B) LDH release in the supernatant. The number of PI+ macrophages was determined using flow cytometry and is shown by a representative value from the three independent experiments. (C) The cell morphologies were determined by bright-field microscopy. Images were magnified ×400. Scale bar, 20 μm. (D) BMDMs were infected with hvKp or cKp (MOI of 10) for the indicated times, and the mRNA levels of Il1b and Il18 were measured by qRT-PCR. (E) BMDMs were infected with hvKp or cKp at different MOI for 16 h. The IL-1β levels were determined using ELISA in cell culture medium. (F) BMDMs were infected with hvKp or cKp (MOI of 10) for 16 h. Cells were lysed and subjected to immunoblotting using antibodies against caspase-1, GSDMD and ACTB. (G) BMDMs were infected with hvKp or cKp (MOI of 10) for 16 h. Cells were stained with cleaved GSDMD (green) and DAPI (for nuclei). Representative images were analyzed using confocal microscopy. Scale bar, 20 μm (left). The quantification of cleaved GSDMD fluorescence (right). Data are expressed as the mean ± standard deviation of the three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 according to analysis of variance. Un, uninfected; Av, average.