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. 2023 Aug 1;14:1184588. doi: 10.3389/fphar.2023.1184588

FIGURE 5.

FIGURE 5

Z-LLSD-FMK or Z-YVAD-FMK inhibited GSDMD activation or pyroptosis induced by LPS + nigericin in BMDMs. BMDMs were primed with LPS for 4 h followed by nigericin for 30 min. Z-LLSD-FMK, Z-YVAD-FMK, and both combined were added 30 min before LPS + nigericin treatment. (A) Representative immunofluorescence images of cell death determination via PI (red) and Hoechst 33342 (blue) staining (scale bar = 75 μm). (B) The percentage of PI-positive BMDMs was calculated at five randomly selected image fields (n = 5). (C) LDH release in supernatants (n = 5). (D) IL-1β release in supernatants was analyzed by ELISA (n = 5). (E) Western blotting analysis was performed to detect protein levels of NLRP3, caspase-1, GSDMD, cleaved IL-1β, and HMGB1 in supernatants and cell lysates. (F–J) Quantitative analysis of expression of NLRP3, p10 (caspase-1), GSDMD-N, cleaved IL-1β, and HMGB1 in cell lysates (n = 5). β-actin served as the loading control. Data represent means ± SEM.