Extended Data Fig. 3 |. SpeB triggers lytic death of primary mouse keratinocytes and A431 cells.

a, Primary mouse keratinocytes were infected with GAS isolate M1T1 strain 5448 or its isogenic mutant strains for the indicated times. Percentage of internalized GAS was shown by counting intracellular CFUs relative to the inoculum (left panel). Cell cytotoxicity of all the cells in a well was measured by LDH release assay (right panel). b–d, Primary keratinocytes infected with FITC-labeled GAS strains were washed with PBS before cells were stained and analyzed by confocal fluorescence imaging. Cell borders are outlined with dashed lines. Cells with intracellular GAS were quantified from 700 cells (n = 5, mean ± s.d.) (b). e, Levels of hyaluronic acid capsule at logarithmic and stationary growth phases. f, RT-PCR (left panel) and immunoblot analysis (right panel) of the expression of SpeB in the indicated GAS strains. g, h Primary keratinocytes infected or not with the indicated GAS strains for 2.5 h were analysed by phase-contrast microscopy (g), LDH release (h). i, j, A431 cells infected or not with GAS isolate M1T1 strain 5448 or its isogenic mutant strains for 2.5 h were analysed by phase-contrast microscopy (i) and LDH release (j). k, l, Equal amounts of recombinant of WT SpeB or protease activity-deficient mutant mSpeB were respectively electroporated into A431 cells for 1 h or directly added into cell culture medium for 2.5 h, followed by cell morphology observation by phase-contrast microscopy (k), cell viability assessment by CellTiter-Glo luminescent assay (l). g, i, Arrowheads indicate pyroptotic cells. c, d, g, i, k, scale bar: 10 μm. Graphs show mean ± s.d. of triplicate wells. h, j, One-way ANOVA; l, Two-tailed Student’s t-test. Data are representative of at least three independent experiments. For gel source data, see Supplementary Fig. 1.