Fig. 4: NSP6 induced accumulation of non-digestive autophagosomes without inhibiting autophagosome-lysosome fusion.

A, B Calu-3, A549 and BEAS2B cells were co-transfected with mCherry-GFP-LC3 plasmid and control empty vector or NSP6-encoding plasmid for 24 h. Bafilomycin A1 (Baf A1; 100 nM, 24 h) and starvation (no serum, 24 h) were used as positive and negative control, respectively. A Representative fluorescence images of mCherry-GFP-LC3 fusion protein (GFP, green; mCherry, red) and his-tagged NSP6 (magenta) expression. Nuclei were stained with DAPI (blue). Scale bar 10 μm. B Quantification of colocalization coefficient is displayed as the percentage of punctate mCherry signals that were positive for GFP signals. Twenty cells from each group were randomly selected for statistical analysis. C Calu-3 cells were transfected with control empty vector or NSP6-encoding plasmid for 24 h. Baf A1 was added to culture 1 h after transfection until cells were harvested. Whole-cell lysates were examined for his-tagged NSP6, LC3B, SQSTM1/p62, IL-18, inactive/full-length and active forms of CASP1 (p20 and p10), GSDMD (p30 NT fragment) and IL-1β (p17). D, E Calu-3, A549 and 16HBE cells were co-transfected with GFP-LC3 plasmid and control empty vector or NSP6-encoding plasmid for 24 h. Starvation and Baf A1 were used as positive and negative control, respectively. D Representative fluorescence images of GFP-LC3 (green), LAMP1 (red) and his-tagged NSP6 (magenta) expression. Nuclei were stained with DAPI (blue). Scale bar 10 μm. E Quantification of colocalization coefficient is displayed as the percentage of punctate LAMP1 signals that were positive for LC3B signals. Twenty cells from each group were randomly selected for statistical analysis. Significance was assessed using one-way ANOVA with Tukey’s multiple comparison test. All quantitative data are presented as means ± SD. ***p < 0.001; ns no significance.