Fig. 4: NINJ1 promotes PMR under mechanical strain during lytic cell death.

a, Representative images of THP-1 cells treated with 5 μg ml⁻¹ nigericin (Nig). Plasma membranes were labelled with CellBrite (red); DRAQ7 (yellow) marks nuclei of permeabilized cells. b, Time-course of LDH release, the percentage of DRAQ7⁺ cells and the percentage of ballooned cells. c, Images of parental and NINJ1-KO THP-1 cells 6 h after nigericin treatment. Permeabilized nuclei (DRAQ7⁺Hoechst⁺) appeared white; intact nuclei (Hoechst⁺ only) appeared blue. d, Time-course of LDH release, DRAQ7⁺ cells and ballooned cells in parental and NINJ1-KO cells. e, The workflow of the experiment testing the effect of flow on PMR. The diagram was created using BioRender. f, Shear rates in various human vascular beds as previously reported^31,32. g,h, Images (g) and quantification (h) of pyroptotic THP-1 cells after flow stimulation for 30 min. 5 μg ml⁻¹ nigericin was added to cells 6 h before. The arrows in the left and right panels indicate fully ruptured cells (DRAQ7⁺ nuclei lacking surrounding CellBrite staining). i, Images of parental and NINJ1-KO THP-1 cells treated with 5 ng ml⁻¹ nigericin for 6 h, then with flow at 2,073 s⁻¹ for 30 min. Static cultures were used as controls. j, LDH release and the full PMR rate in parental and NINJ1-KO cells after nigericin and flow treatment. k, LDH release and the full PMR rate in parental and NINJ1-KO cells after intracellular LPS and flow treatment. l,m, Quantification of dsDNA released from pyroptotic THP-1 cells treated with nigericin (l) and LPS (m) and flow for 30 min. Data are mean ± s.e.m. from three independent trials per group. Image quantification was done with 250–300 cells per trial. Statistical analysis was performed using two-sided unpaired Student’s t-tests versus the control (b) and two-way ANOVA followed by Bonferroni correction (d and j–m) versus the parental + nigericin or parental + LPS group. For a, c, g and i, scale bars, 25 μm. *P < 0.05.

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