Fig. 5: Inhibition of pore formation by antagonistic GSDMD nanobodies triggers caspase-1-dependent apoptosis.

A, D–E, G–J THP-1 cell lines expressing C1C-mCherry (dox-inducible) as well as the indicated VHH-EGFP fusions (constitutively) were differentiated with PMA, treated with dox for 24 h, and stimulated with NLRC4 agonist MxiH for 1 h as described in Fig. 2C (A, D, G–J), with NLRP3 agonist LPS and Nig as described in Fig. 2D (E), or with 5 µM staurosporine (Stau) for 20 h to trigger apoptosis (G–J). Stimulation was performed in the absence or presence of VX as indicated. A Cells were recorded by live cell confocal microscopy and images representative of three independent experiments are displayed. Scale bars, 10 µm. D, E Cells were harvested and analyzed by flow cytometry to quantify C1C-mCherry specks. G–J Cells were harvested, stained with antibodies specific for cleaved caspase-3 and Alexa Fluor 647 (AF647)-coupled secondary antibodies, and the fraction of cells positive for cleaved caspase-3 was quantified by flow cytometry (G, I) Representative histograms of cell lines with the indicated treatments are presented in (H, J). B, C WT THP-1 cells (C) or THP-1 cell lines expressing the indicated HA-tagged VHHs (B) were differentiated with PMA, and stimulated with NLRC4 agonist MxiH as described in Fig. 2C, but in the presence of 100 nM SYTOX Green nucleic acid stain. Cells were recorded by live cell confocal microscopy including bright field recordings. The absolute number of cells positive for SYTOX Green as well as cells with pyroptotic and apoptotic morphology were enumerated per tile scan (675 µm × 675 µm) and average values from three independent experiments ± SEM are displayed. F Primary GM-CSF-differentiated monocyte-derived human macrophages were transduced and stimulated as described in Figs. 2H–K. Cells were recorded by live-cell confocal microscopy and images representative of three independent donors are displayed. Scale bar, 10 µm. Data represent average values (with individual data points) from three independent experiments ± SEM. *P  <  0.05, **P  <  0.01, ***P  <  0.001, and ****P  <  0.0001 (unpaired two-tailed Student’s t-test).