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. 2022 Jan 8;29(6):1240–1254. doi: 10.1038/s41418-021-00916-7

Fig. 6. L37F NSP6 mutant failed to induce pyroptosis.

Fig. 6

A–B A549, Calu-3, 16HBE and BEAS2B cells were transfected with control empty vector or His-tagged wild-type (WT) or L37F NSP6-encoding plasmid for 24 h and then subject to immunoprecipitation (IP) by anti-His tag antibody or IgG (negative control). Five percent of the lysate used for the IP was loaded as input (n = 3). A Representative immunoblots of IP of WT or L37F NSP6 and ATP6AP1. B Quantification of relative protein levels is displayed as the ratio of intensity of IP:His to input. C A549, Calu-3, 16HBE and BEAS2B cells were transfected with control empty vector or His-tagged WT or L37F NSP6-encoding plasmid for 50 h. Whole-cell lysates were examined for His-tagged NSP6, inactive/full-length and active forms of ATP6AP1 (p45; Abcam; ab176609), CASP1 (p20), and IL-1β (p17). D–F A549, 16HBE and BEAS2B cells were transfected with control empty vector or His-tagged WT or L37F NSP6-encoding plasmid for 24 h. D Cells were then stained with LysoTracker Red and analyzed by flow cytometry. E Quantification of LysoTracker Red staining intensities (mean) in A549, 16HBE and BEAS2B cells. F Whole-cell lysates were examined for His-tagged WT or L37F NSP6, LC3B, SQSTM1/p62. G A549 and 16HBE cells were transfected with control empty vector or His-tagged WT or L37F NSP6-encoding plasmid for 48 h. Whole-cell lysates were examined for His-tagged NSP6, GSDMD (full-length and p30 NT fragment). C, F–G β-Actin was used as a loading control. Significance was assessed using one-way ANOVA with Tukey’s multiple comparison test. All the quantitative data are presented as means ± SD. ***p < 0.001.