Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium
Fig 6
STING expression promotes RV-C15 replication in HAE at 12hpi.
A: Western blot showing STING and VP1 expression at 12 and 48hpi in non-infected or RV-C15-infected HAE. Fold change graph represents STING protein levels normalized to the endogenous control (actin) and compared to non-infected cultures. Data shown are from two independent donors, represented by circles and triangles. Blot above is from the donor represented by triangles. B: Immunofluorescence detection of STING (green) in non-infected or RV-C15-infected (dsRNA+; red) ciliated cells (acetylated α-tubulin, magenta) at 12hpi (scale bar = 50μm). C: XZ orthogonal view (upper) and 3D view (below) of RV-C15-infected HAE shows the detection of STING (green) and viral dsRNA (red) in ciliated cells (acetylated α-tubulin, gray) at 12hpi (z-stacks at 1μm of thickness). D: Quantification of dsRNA and STING fluorescence levels (CTCF) in non-infected and RV-C15-infected (dsRNA+) HAE at 12hpi. Dots represent the total CTCF per cell and the line indicates the mean. Statistical analyses were done by Mann-Whitney test (two-tailed; 0.95% confidence interval; ****p<0.0001). E: Spearman correlation analysis (two-tailed; 0.95% confidence interval) between fluorescence levels for dsRNA and STING in RV-C15-infected HAE at 12hpi. Each dot represents the fluorescence levels of dsRNA and STING quantified in the same Z-slice. F: 2D split channel views of RV-C15-infected HAE showing the detection of STING (green), dsRNA (red), and STING/dsRNA colocalization (yellow) by immunofluorescence at 12hpi (scale bar = 5μm). G: 3D surface plot of RV-C15-infected HAE shows the detection of STING (green), dsRNA (red), and STING/dsRNA colocalization (yellow) by immunofluorescence at 12hpi. H: Spatial colocalization analysis shows the ratio of dsRNA/STING colocalization in RV-C15-infected HAE at 12hpi (bar = median). I: Western blot showing STING expression, quantified below, in HAE derived from BCi-NS1.1 cells transduced with non-targeting control (NTC), sgRNA1 (sg1), or sgRNA2 (sg2). J: Immunofluorescence detection of dsRNA (red) in HAE derived from BCi-NS1.1 cells transduced with NTC, sgRNA1 (sg1), or sgRNA2 (sg2) infected with RV-C15, RV-A16, or RV-B14 at 12hpi (scale bar = 100μm). K: qPCR to detect RV RNA in HAE derived from BCi-NS1.1 cells transduced with NTC, sgRNA1 (sg1), or sgRNA2 (sg2) and infected with RV-C15, RV-A16, or RV-B14 at 4 and 12hpi. Each bar represents the mean value obtained across n = 3 cultures/condition (individual dots); the black line represents the limit of detection (RV-C15 = 103.5; RV-A16 = 105.9(not visible on the graph); RV-B14 = 104.8); statistical analysis was done by Ordinary One-way ANOVA and Tukey’s multiple comparisons test (**p<0.01).