Fig 3. IFN-λ activates the ISRE promoter and induces antiviral ISGs in S3GFP and R4GFP cells.

(A) S3-GFP cells were transfected with either an ISRE-Luc reporter plasmid or a control EGFP-Luc plasmid and treated with increasing doses of IFN-α or IFN-λ. Cells were collected 24 hours post transfection and Firefly luciferase activity was measured. Values were normalized with one microgram of protein and expressed as fold increase over the control. (B) R4-GFP cells were transfected with pISRE-Luc plasmid and treated with increasing doses of IFN-α or IFN-λ. After 24 hours, firefly luciferase activity was measured. (C and D) S3-GFP and R4-GFP cells were treated with increasing doses of IFN-α for 24 hours then the expression of ISGs mRNA level was quantified by qRT-PCR. The value of each sample was normalized to GAPDH and the expression levels relative to the untreated control were calculated. (E and F) S3-GFP and R4-GFP cells were treated with IFN-λ for 24 hours; the expression of ISG mRNA was measures by real-time RT-PCR. The value of each sample was normalized to GAPDH and the expression levels relative to the untreated control were calculated (G and H). The protein levels of the ISGs were evaluated by Western blot in S3GFP and R4GFP cells treated with 100 I.U/mL of IFN-α or 10ng/mL of IFN-λ and collected every 24 hours for 72 hours.