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. 2023 Mar 13;26(4):106395. doi: 10.1016/j.isci.2023.106395

Figure 3.

Figure 3

Effect of IFNs and endogenous IFITM expression on ccCoVs

(A) Huh7 cells were stimulated with IFN-α2, IFN-β or IFN-γ for 8h, 16h and 24h. Immunoblot was performed using whole cell lysates stained with anti-IFITM1, anti-IFITM2, anti-IFITM3 and anti-GAPDH.

(B) Huh7 were left untreated or treated with IFN-α, IFN-γ and IFN-λ for 18h and subsequently infected with 229Epp, NL63pp, SARS CoV-2pp or VSV-Gpp. Cytation for GFP+ cells was performed at 24h post-infection.

(C) Huh7 cells were treated with IFNs as described in panel B and infected with HIVpp carrying the hCoV-OC43 S protein. Luciferase activity was determined at 48h post-infection.

(D) Huh7 cells were treated with IFNs and infected with genuine hCoV-229E (MOI 0.01), hCoV-NL63 (MOI 0.05) or hCoV-OC43 (MOI 0.01). Whole cell lysates were collected at 24h post-infection for hCoV-229E and at 48h post-infection for hCoV-NL63 and hCoV-OC43. Intracellular viral RNA levels were determined by qRT-PCR.

(E and F) Huh7 cells were transfected with the indicated siRNAs, left untreated or treated with IFN-β 100U/ml) for 18h starting on the following day and subsequently infected with hCoV-229E (MOI 0.01) after 18h of IFN treatment. Supernatant was harvested 24h post-infection and hCoV-229E RNA and infectious titer quantified by qRT-PCR and TCID50 assay, respectively.

(G) Huh7 cells were treated as described for panel E, infected with Renilla hCoV-229E (MOI 0.01) and luciferase activity was measured 24h later.

(H) Huh7 cells transfected with IFITM targeting or non-targeting siRNA were treated with IFN-β (100 U/ml) or left untreated, infected with hCoV-NL63 (MOI 0.05) and viral RNA levels in the culture supernatants were determined by qRT-PCR two days later.

(I) Calu3 cells transfected with IFITM targeting or non-targeting siRNA were treated with IFN-β (500 U/ml) or left untreated, infected with hCoV-NL63 (MOI 0.05) and viral RNA levels in the culture supernatants were determined by qRT-PCR two days later.

(J) Huh7 cells were treated as described in panel H, infected with hCoV-OC43 and viral RNA levels in the culture supernatants were determined two days later. Bars in all panels represent the mean of three or four independent experiments (±SEM). ∗, p < 0.05; ∗∗, p < 0.01).