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. 2022 Apr 19;14(6):e15415. doi: 10.15252/emmm.202115415

Figure 4. Contribution of GSDMD in the effect of VHHASC on the release of IL‐1β and cell death induced by PFO or nigericin.

Figure 4

THP‐1 cells expressing a Dox‐inducible CRISPR‐Cas9 cassette targeting GSDMD were left untreated (–), or treated with 1 µg ml−1 Dox for one or two cycles of 72 h (1×, or 2× respectively).
  1. Immunoblot analysis of GSDMD expression following the indicated course of Dox treatment and PMA‐differentiation, as indicated. Data is from one representative of two independent experiments.
  2. IL‐1β concentration or percentage of LDH released into cell‐free supernatants of PMA‐differentiated THP‐1 cells that were treated with VHHASC (200 µg ml−1) or CRID3 (25 µM) for 30 min prior to stimulation with nigericin (10 µM, left panels) or PFO (30 ng ml−1, right panels) for 2 h. Data is average of experimental duplicates from three independent experiments, each represented by a different symbol. ns P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Two‐way ANOVA, multiple comparison (Tukey test). Data is displayed as floating bars with the max/min values and mean (thicker band).
  3. Live confocal imaging of PMA‐differentiated and nigericin‐treated (10 µM) THP‐1 cells expressing human ASC‐GFP (green) in the presence of AlexaFluor647‐labeled VHHASC (VHHASC‐AF647, 10 µg ml−1, cyan) in the medium. Cells were either left untreated (–) or incubated with VX‐765 (50 µM) for 1 h prior to nigericin stimulation. Nuclei were stained with Hoechst 34580 (magenta). Scale bar: 50 µm. Data is from one representative out of two independent experiments.

Source data are available online for this figure.