Fig. 3

Cell fusion activates the innate immune response via the cGAS-STING pathway. a Scheme of co-culture experiment. b Representative fluorescence images of co-culture experiment. HEK293T cells were transfected with vector control (Vec) or increasing amounts of plasmids expressing spike (S), along with plasmids expressing EGFP for 24 h. Cells were then detached and mixed with HeLa-ACE2 expressing mCherry (HeLa-ACE2-mCherry). After 8 h, cells were subjected to fluorescence microscopy analysis. Scale bar, 250 μm. c Cytokine genes/ISGs expression in co-cultured cells. Cells were co-cultured as indicated in b. RNA extracted from the cells was evaluated by quantitative PCR. The data are expressed as fold change of the IFNB, ISG15, IL8, and CCL5 mRNA levels relative to the GAPDH control. Mean ± s.d., n = 4 independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed Student’s t-test. d Western blot analysis of cells from co-culture experiment as described in b using indicated antibodies. STING blots were performed under non-reducing (top) or reducing conditions. ✶STING dimer. e HEK293T cells were transfected with vector control (Vec) or plasmids expressing spike. After 24 h, cells were detached and mixed with HeLa-ACE2-mCherry cells as indicated. cGAS^KO, STING^KO, and MAVS^KO represent cells depleted of indicated genes. cGAS^RE represents cGAS-null cells re-expressed cGAS. The expression of IFNB mRNA was assayed as described in c. Mean ± s.d., n = 3 independent experiments. **P < 0.01, ****P < 0.0001, n.s. not significant. two-tailed Student’s t-test. f Western blot analysis of cells from the co-culture experiment as described in e. ✶ACE2 fragments generated during cell co-culture. S spike