(A–C) Cells electroporated with HCV replicon RNA (A, C) or infected with HCVcc (B) were treated with 5 μM CsA (A, B) or CypI (C) 4 hr later. After 48 hr, RNA was extracted and expression of IFN-β mRNA was evaluated by qRT-PCR. Data were normalised by GAPDH expression and are expressed as fold change compared to the DMSO vehicle-treated control. (D-E) CypI potency does not depend on IFN signalling. HCV replication in Huh7 cells, electroporated as described above, and treated with IFN-β (5 ng/mL) or CypI, in the presence or absence of the Jak/STAT inhibitor ruxolitinib (Rux). Rux treatment rescued HCV replication from IFN-β inhibition (D) but not from CypI (E). (F) CsA treatment induces expression of a subset of antiviral genes in HCV-replicating Huh7 cells. RNA expression of IFN-β, CCL2, MX1, RSAD2 IFIT2, ANKRD, CXCL2, CXCL10 and TNFα mRNA was evaluated by qRT-PCR at 48 hpe in Huh7 cells electroporated with HCV replicon RNA and treated with CsA (5 μM) at four hpe. Data were normalised by GAPDH expression and are expressed as fold change compared to the DMSO vehicle-treated control. All graphs show means ± standard deviation from at least three independent experiments each performed in triplicate. Statistical significance was evaluated by t-test using GraphPad Prism (**** p-value<0.0001; *** p-value<0.001; n.s. (not significant), p-value>0.05). Detection limits of the assay (D) or gene expression in DMSO-treated cells (set as 1) (F) are shown by the dotted grey line.