Table 1.
Oligonucleotide sequences for RT-qPCR.
Figure 1.
Inhibition of HCV replication by statins in genotype 1b replicon cells.
LucUbiNeo-ET replicon cells were incubated in the presence of fluvastatin (FLV), pravastatin (PRV), simvastatin (SMV), rosuvastatin (ROV) or atorvastatin (ATV) at the indicated concentrations for 24, 48 or 72 hours. (A) Expression of the LDL-receptor (LDLR) was measured by real time RT-qPCR after 48 h of statin incubation (B–F) Dose- and time-dependent inhibition of HCV replication caused by statins was measured by luciferase reporter assay. Statin mediated effects on cell proliferation were measured after 72 hours of incubation (G) and confirmed by Western Blot analysis (H).
Figure 2.
Inhibition of HCV in the infectious genotype 2a system.
Huh-7.5 cells were transfected with the full length HCV clone JcR2a. HCV replication was measured by luciferase reporter assay after 72 hours of FLV incubation (A). Huh-7.5 cells were infected with the HCV genotype 2a strain JC1 and incubated with FLV or PRV for 24 hours. Viral replication was visualized by immunofluorescent staining of the HCV protein E2 (B, upper pannel). Cell viability was verified by bright field (BF) microscopy (B, lower pannel). Representative images pairs are shown.
Figure 3.
Statin-induced HO-1 expression contributes to inhibition of HCV replication and endogenous interferon response.
HO-1-induction was measured by RT-qPCR in Huh-5-15 replicon cells after 6 hours of statin incubation (A). Western Blot analysis was used to visualize the HO-1 protein level was after 8 hours of incubation using 10 or 25 microM of FLV (B). LucUbiNeo-ET replicon cells stably expressing shRNA against HO-1 (shHO1) or a control gene (shGFP) were used to detect baseline or FLV-induced expression of HO-1 by RT-qPCR after 6 hours of incubation (C). ShGFP or shHO1 cells were incubated with statin for 72 hours. HCV replication was measured by luciferase reporter assay (D). LucUbiNeo-ET replicon cells were incubated in the presence of 10 microM of statins for 24 hours. Gene expression levels (interferon alpha 2; interferon alpha 17; interferon response genes) were analyzed by RT-qPCR (E). LucUbiNeo-ET cells were incubated with FLV, IFN alpha (Intron A) and telaprevir (TVR) alone or in combination for 24 hours. Single and combined effects on HCV replication were detected by luciferase assay (F).
Figure 4.
Statins promote HO-1 expression by reducing Bach1- and inducing KLF2- expression.
Bach1 (A) and KLF2 (B) expression levels were measured in Huh-5-15 cells by real time RT-qPCR after 6 hours of statin incubation. LucUbiNeo-ET replicon cells were transfected with siRNA directed against KLF2 (siKLF2) or against a control gene (GFP; siControl) at [10 nM] (C, D, E, F). 24 hours after transfection expression of KLF2 and HO-1 was measured by RT-qPCR (C). Cells transfected with siRNA for 24 hours were further incubated with FLV for 24 hours. Expression of KLF2, HO-1 (D) and HCV (E) was measured by RT-qPCR, HCV replication was measured by luciferase reporter assay (F).
Figure 5.
Matrix conditions predict anti-viral properties of statins.
LucUbiNeo-ET replicon cells growing on PAA gel supports (1–12 kPa: 1 kPa = soft; 12 kPa = stiff) were incubated with FLV for 72 hours. Effects on HCV replication were measured by luciferase assay (A). For the same settings cell viability was measured by MTT assay (B). LucUbiNeo-ET replicon cells were incubated with increasing concentrations of the Rho kinase inhibitor HA1100 alone or in combination with FLV at 5 µM for 72 hours. HCV replication was measured by luciferase reporter assay (C). Viability of LucUbiNeo-ET replicon cells was monitored for the same settings by MTT assay (D). Huh-5-15 replicon cells were incubated with FLV with or without coincubation with HA1100 for 6 hours. HCV replication (E) as well as HO-1 expression (F) was measured by RT-qPCR.