Journal: iScience

Article Title: Experimental and natural evidence of SARS-CoV-2-infection-induced activation of type I interferon responses

doi: 10.1016/j.isci.2021.102477

Figure Lengend Snippet: SARS-CoV-2 infection does not inhibit type I IFN expression To determine if SARS-CoV-2 can modulate IFNβ gene expression and downstream stimulation of ISGs, Calu-3 cells were infected with SARS-CoV-2 for varying times, following which cells were mock transfected or transfected with poly(I:C). Mock-infected and mock-transfected cells served as controls. Transcript levels were quantified using qPCR. Protein expression was observed and quantified using immunoblot analysis. (A) Calu-3 cells were infected with SARS-CoV-2 (MOI 1) for 0, 24, 48, and 72 h. Cells were fixed and stained to visualize the nucleus and SARS-CoV-2 nucleocapsid (N) protein. Scale bar indicates 300 μm. (B) SARS-CoV-2 genome (UpE) levels in Calu-3 cells infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h and transfected with 100 ng of poly(I:C) or mock transfected for 6 h (n = 6). Primers for the UpE region were designed to quantify SARS-CoV-2 genome levels (see methods). 1/dCT values are represented after normalizing Ct values for SARS-CoV-2 genome levels at 18 hpi with Ct values observed at 0 hpi (immediately after removal of virus inoculum). Gel (below): UpE qPCR amplicons were visualized on an agarose gel. (C) IFNβ transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either transfected with 100 ng of poly(I:C) or mock transfected for 6 h. IFNβ transcript levels were normalized to GAPDH transcript levels (n = 6). (D) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either transfected with 100 ng of poly(I:C) or mock transfected for 6 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 6). (E) IRF7 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either transfected with 100 ng of poly(I:C) or mock transfected for 6 h. IRF7 transcript levels were normalized to GAPDH transcript levels (n = 6). (F) IFIT1, SARS-CoV-2 N, and ACTB protein expression in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with 1,000 ng of poly(I:C) or mock transfected for 24 h (n = 3). (G) IFNβ transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 0.1 or 1) or mock infected for 24 h. Twenty-four hpi, cells were transfected with 10 ng of poly(I:C) or mock transfected for 12 h. IFNβ transcript levels were normalized to GAPDH transcript levels (n = 3). (H) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 0.1 or 1) or mock infected for 24 h. Twenty-four hpi, cells were transfected with 10 ng of poly(I:C) or mock transfected for 12 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 3). (I) IFNβ transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with varying concentrations of poly(I:C) or mock transfected for 12 h. IFNβ transcript levels were normalized to GAPDH transcript levels (n = 3). (J) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with varying concentrations of poly(I:C) or mock transfected for 12 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 3). (K) IRF7 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with varying concentrations of poly(I:C) or mock transfected for 12 h. IRF7 transcript levels were normalized to GAPDH transcript levels (n = 3). (L) pTBK1-S172, TBK1, pIRF3-S396, IRF3, SARS-CoV-2 N, and ACTB protein expression in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with 1,000 ng of poly(I:C) or mock transfected for an additional 24 h (n = 3). (M) Calu-3 cells were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h, followed by transfection with 1,000 ng of rhodamine-labeled poly(I:C) or mock transfection for 3 h. Cells were fixed and stained to visualize SARS-CoV-2 nucleocapsid (N) protein and rhodamine-labeled poly(I:C). SARS-CoV-2 N and poly(I:C)-rhodamine containing cells are indicated by arrows. Cells that only contained detectable levels of poly(I:C)-rhodamine are indicated by arrow heads. Scale bar indicates 150 μm. Data are represented as mean ± SD, n = 3 or 6, p∗∗<0.01, ∗∗∗<0.001, and ∗∗∗∗<0.0001 (Student's t test and Tukey's multiple comparisons test). pTBK1-S172 and pIRF3-S396 protein expression levels are expressed as ratios of pTBK1-S172/TBK1 and pIRF3-S396/IRF3 levels, respectively. Blots were quantified using Image Studio (Li-COR) (n = 3). Ct, cycle threshold. See also Figure S3 .

Article Snippet: Poly(I:C)-Rhodamine , InvivoGen , Cat#tlrl-piwr.

Techniques: Infection, Expressing, Transfection, Western Blot, Staining, Virus, Agarose Gel Electrophoresis, Labeling