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

Fig. 3. NSP6 impaired the autophagic flux and induced the activation of inflammasomes in lung epithelial cells.

Fig. 3

A, B Calu-3, A549, BEAS2B and 16HBE cells were transfected with control empty vector or NSP1-, NSP2- or NSP6-encoding plasmid for 24 h. A Whole-cell lysates were examined for LC3B and SQSTM1/p62 expression. Quantification of relative protein levels is displayed as the ratio of intensity of LC3B-II (B) or SQSTM1/p62 (C) to β-actin. D Calu-3 and A549 cells were transfected with control empty vector or NSP6-encoding plasmid for 24 h. Active caspase-1 was visualized using fluorescently-labelled inhibitor of caspase-1 (FLICA) probe (green). Nuclei were visualized with Hoechst stain (blue). Scale bar 20 μm. E A549 and 16HBE cells were transfected with control empty vector or NSP6-encoding plasmid for 16, 24 or 48 h. Cell death and membrane rupture were identified using propidium iodide (PI) staining. Nuclei were visualized with Hoechst stain (blue). Scale bar 100 μm. F 16HBE and BEAS2B cells were transfected with control empty vector or NSP6-encoding plasmid for 24 h. Representative scanning electron microscopy images of top-down views were shown. GSDMD-NT pores are indicated by red arrows. G–K Calu-3, A549, BEAS2B and 16HBE cells were transfected with control empty vector or NSP6-encoding plasmid for 24 h. G Cell death was monitored by PI staining and active caspase-1 was identified by FLICA probe. Fluorescence intensity was analyzed by flow cytometry. G’ Calu-3 cells treated with ATP (2 mM, 2 h) was used as a positive control. Quantification of caspase-1+ (H), PI+ (I) and caspase-1+PI+ (J) cells are displayed as percentages. K Quantification of IL-1β secretion in cell culture supernatant showed increased IL-1β levels upon NSP6 overexpression. β-Actin was used as a loading control. Significance was assessed using one-way ANOVA with Dunnett’s multiple comparison test (B, C) or unpaired two-tailed t-test (H–K). All quantitative data are presented as means ± SD. from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ns no significance.