Fig. 1: Caspase-9/GSDME trigged pyroptosis of syncytia formed by fusion of SARS-CoV-2 Spike and ACE2-expressing cells.

SARS-CoV-2-S-GFP HeLa cells were co-cultured with PLC-RFP-A549-ACE2 cells (a), or NLS-RFP-A549-ACE2 cells (b) at a 1:1 ratio. Four hours later, image was scanned by LSM780. The nucleus (blue) was stained by Hoechst; Bar, 20 μm. c The time-phase of cell-cell fusion progress, HeLa-ACE2 co-culture with HeLa-SARS-CoV-2-S cells, treatment with pan-caspase inhibitor, ZVAD (20 μM) or not; Images were obtained at 3 h, 6 h, 12 h 24 h; the nucleus(blue) was stained by Hoechst; Bar, 100 μm; then the cell-cell fusion was quantified (d); the LDH release (e), ATP level (f), the activity of caspase-3/7 (g) were detected at indicated time. Western blot analysis of the cells collected as indicated. ACE2, Flag, caspase-8 (C8), caspase-9 (C9), caspase-7 (C7), Cleaved-caspase-3 (Cleaved-C3), and GSMDE were probed. β-Actin as loading control (h, k and n). The cell-cell fusion was quantified after co-culture for 5 h as indicated (i and l). LDH release was measured after co-culture for 12 h as indicated (j and m). The model of SARS-CoV-2 Spike and ACE2 interaction induced syncytia pyroptosis (o). The data were shown as means ± SD, *P < 0.05, **P < 0.01, NS non-significance, and all the experiments were replicated more than three times.