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. 2021 Aug 2;12:4664. doi: 10.1038/s41467-021-25015-6

Fig. 2. IL-1β maturation and IL-6 production are induced by N protein.

Fig. 2

a HEK293T cells were co-transfected with plasmids encoding NLRP3, ASC, pro-Caspase-1, and pro-IL-1β, and transfected with plasmids encoding different concentrations of SARS-CoV-2 N for 48 h. Supernatants were analyzed (top) by ELISA for IL-1β. Cell lysates were analyzed (bottom) by immunoblotting. b–d THP-1 cells were stably infected with Lentivirus-CT or Lentivirus-N, differentiated into macrophages. Then stimulated with LPS (1 μg/ml) plus ATP (2.5 mM) or LPS (1 μg/ml) plus Nigericin (2 μM). IL-1β (b, top), IL-18 (c), and IL-6 (d) in supernatants was measured by ELISA. Cell lysates were analyzed (b, bottom) by immunoblotting. e, f GM-CSF-differentiated mice BMDMs were infected with Lentivirus-CT or Lentivirus-N and stimulated with LPS (1 μg/ml), LPS (1 μg/ml) plus ATP (2.5 mM), or LPS (1 μg/ml) plus Nigericin (2 μM). IL-1β (e) or IL-6 (f) in supernatants was measured by ELISA. g, h THP-1 cells were stably infected with Lentivirus-CT or Lentivirus-N, differentiated into macrophages. The cells were treated with MCC950 (0.01 μM) for 1 h and then stimulated with LPS (1 μg/ml) plus ATP (2.5 mM) or LPS (1 μg/ml) plus Nigericin (2 μM). IL-1β protein (g) and IL-6 protein (h) in supernatants were measured by ELISA. i, j GM-CSF-differentiated BMDMs isolated from NLRP3+/+ mice and NLRP3−/− mice were infected with Lentivirus-N and stimulated with LPS (1 μg/ml), LPS (1 μg/ml) plus ATP (2.5 mM), or LPS (1 μg/ml) plus Nigericin (2 μM). IL-1β protein (i) and IL-6 protein (j) in supernatants were measured by ELISA. Mock means untreated cells (a–d and g, h). Data are representative of three independent experiments and one representative is shown. Error bars indicate SD of technical triplicates. Values are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-tailed Student’s t-test. Source data are provided as a Source Data file.