Ledipasvir

Huh-7.5 cells were obtained from Charles M. Rice (Rockefeller University, New York). The Huh-7.5 cell line was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM L-glutamine, sodium pyruvate, nonessential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum. Huh-7.5 cells were infected with JFH1-GFP chimera HCV using a protocol developed in our laboratory as previously described^{31 (link)}. Recombinant human IFN-α was purchased from Schering Plough, Kenilworth, NJ. Sofosbuvir was obtained from Acme Biosciences, Inc. (Palo Alto, CA) and ledipasvir was obtained from Selleck Chemicals (Houston, TX). Thapsigargin (TG), tauroursodeoxycholic acid (TUDCA), PERK inhibitor (GSK 2606414), α-tocopherol, and roscovitine were obtained from Sigma-Aldrich (St. Louis, CA). Antibody to pNrf2 was purchased from Abcam. Antibodies to GRP78 (BiP), PERK, eIF2α, peIF2α, CHOP, IRE1, GFP and Rb were obtained from Cell Signaling (Beverly, MA). Antibody to NS3 was purchased from Virogen Inc. Antibodies to HCV core and pIRE1 were purchased from Thermo Scientific. Antibody to GRP94, GADD34, pPERK, ATF4, XBP1, ATF6, Mdm2, β-actin and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Asunaprevir, boceprevir, dasabuvir, and mericitabine were purchased from MedChem Express (Monmouth Junction, NJ, USA). Telaprevir, ledipasvir, and daclatasvir were purchased from Selleckchem (Houston, TX, USA). Ombitasvir was purchased from Clearsynth (Lotus Business Park, Andheri West, India). Simeprevir was obtained from ApexBio (Boston, MA, USA). The PRK2 inhibitor HA1077 was purchased from Sigma-Aldrich (Saint Louis, MO, USA) or Selleckchem. The siRNAs used for depletion of PRK2, ROCK1, and ROCK2 and the scrambled control siRNA (siCtrl) were described previously^{21 (link)}. siRNAs were transfected into cells using Lipofectamine RNAiMAX (Invitrogen, CA, USA). PKH67 dye was obtained from Sigma-Aldrich and used for HCV particle labeling according to the manufacturer’s protocol. The following antibodies were used: a mouse monoclonal anti-HCV core antibody (clone C7–50) from Abcam (Cambridge, UK); rabbit polyclonal anti-PRK2 and p-PRK2(Thr816) antibodies from Cell Signaling Technology (Danvers, MA, USA); goat polyclonal anti-pPRK2(Thr816), and mouse monoclonal anti-ROCK1 (4H247) and ROCK2 (30-J) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and Alexa fluor488 goat anti-mouse IgG antibody from Invitrogen.