Fig 3.

IRE1α is required for optimal HCoV-OC43 infection assessed by immunofluorescence and titer. (A–D) HCT-8 cells were treated with IRE1α nuclease inhibitor 4μ8C, structurally similar negative control AMC, IRE1α nuclease inhibitor STF-083010 or DMSO solvent control prior to infection with HCoV-OC43. (A–C) Cells were fixed 48-hour post-infection. (A) Double-stranded RNA (dsRNA, green) was visualized by immunostaining, and nuclei were counterstained with TO-PRO-3 (red). (B) Relative total fluorescence intensity was calculated for dsRNA. (C) HCoV-OC43 viral proteins spike and nucleoprotein were visualized by immunostaining, and positive cells were quantified per field at 10× magnification. (D) Viral supernatant was harvested 48-hour post-infection, then diluted serially and plated on HCT-8 cells for an endpoint focus-forming assay. Data are means ± SD of six (C) or four (C+D) replicates and are representative of three (A+B) and two (C+D) independent experiments, respectively. *P < 0.05, **P < 0.01 by unpaired t-test.