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. Author manuscript; available in PMC: 2022 Aug 19.
Published in final edited form as: Cell. 2021 Jul 21;184(17):4495–4511.e19. doi: 10.1016/j.cell.2021.06.028

Figure 1. Engineered macrophages model pyroptosis through expression of the GSDMD N-terminal domain.

Figure 1.

(A) Retroviral transduction workflow to generate Tet3G transactivator expressing Dox-inducible fluorescently tagged variants of GSDMD in iBMDMs from the Cas9 knock-in mouse and downstream characterization.

(B) Western blot of stable Cas9 expression in parental and progeny iBMDMs and stable expression of Tet3G transactivator in progeny iBMDM clones with β-actin loading control.

(C) Kinetic analysis of PI uptake by plate reader to measure bulk membrane permeability in populations of uninduced or Dox-induced (0.5 μg/ml) cells expressing NT-GSDMD-BFP or FL-GSDMD-BFP.

(D) Time course end point analysis of LDH release into cell free supernatants to measure cell lysis in populations of uninduced or Dox-induced (0.5 μg/ml) cells expressing NT-GSDMD-BFP or FL-GSDMD-BFP.

(E, F) Time course end point analysis by flow cytometry of the frequency of BFP+ (E) or PI+ cells (F) using uninduced or Dox-induced (0.5 μg/ml) cells expressing NT-GSDMD-BFP or FL-GSDMD-BFP.

(G) Time course live cell imaging of Dox-induced cells expressing NT-GSDMD-BFP or FL-GSDMD-BFP noting localization of BFP signal and PI uptake. Scale bar indicates 10 μm.

All quantification represents the mean and SEM of three independent experiments. Two-way ANOVA was used for analysis. See also Figure S1.