Fig. 2.

Mg²⁺ inhibits noncanonical pyroptosis in iBMDMs downstream of GSDMD cleavage. a−f Mouse immortalized bone marrow-derived macrophages (iBMDMs) primed overnight with Pam3CSK4 in advance were electroporated with LPS for 2 h, in the absence or presence of MgCl₂, MgSO₄, MgGluc₂, or 5 mM glycine as indicated. Cells electroporated without LPS (Ctrl) are used as controls. a Percentage of LDH release (used to measure cytotoxicity). b Representative brightfield cell images. Representative pyroptotic cells were indicated with white arrows. Bar = 20 μm. c Representative images of propidium iodide (red) uptake. Bar = 20 μm. d IL-1β release. e Immunoblots for pro-caspase-1 (Casp-1 p45) and its cleavage product (Casp-1 p20), and full-length gasdermin D (GSDMD-FL) and its N-terminal (GSDMD-NT) in whole cell lysate. α-tubulin is used as a loading control. f Immunoblots for GSDMD-NT in the cytosolic and membrane fractions. Cytosolic and membrane-bound proteins were separated by using a Plasma membrane protein isolation kit (see Methods). α-tubulin and transferrin receptor 1 (TFR1) are used as loading controls respectively. For panels (a) and (d), data are presented as mean ± SD; *P < 0.05, **P < 0.01, compared to the cells electroporated with LPS in the absence of Mg²⁺ and glycine. Each panel is a representative experiment of at least three replicates. See also Figs. S1 and S2. LPS lipopolysaccharide, LDH lactate dehydrogenase