, 2005) (Supplementary

, 2005) (Supplementary Duvelisib Fig. 1) were cultured

at 37 °C (5% CO2) in Dulbecco’s modified Eagle medium-GlutaMAX-I (DMEM-GlutaMAX-I; Invitrogen, Carlsbad, CA, USA) containing 10% foetal bovine serum and 0.5 mg/mL G418 (Invitrogen, Carlsbad, CA, USA) (Sakamoto et al., 2005 and Watanabe et al., 2006). The JFH-1/K4 cell line, which comprises HuH-7 cells persistently infected with the HCV JFH-1 strain, was maintained in DMEM with 10% FCS (Takano et al., 2011). PYC was supplied by Horphag Research Co., Pegylated IFN-alpha-2a was obtained from Chugai Pharmaceutical Co., Japan. Cells were seeded into 96-well plates (5 × 103/well). After incubation for 24 h at 37 °C (5% CO2), the medium was removed and replaced with growth medium containing serial dilutions of PYC, IFN-alpha, RBV, telaprevir or simeprevir (Janssen Pharma Co., Tokyo, Japan). After 72 h, luciferase activity was measured using the Bright-Glo luciferase assay kit (Promega, Madison, WI). Measurements were made in triplicate using an AccuFLEX Lumi 400 luminometer (Aloka, Tokyo, Japan), and the results expressed as the average percentage of the control. Telaprevir-resistant R6FLR-N subgenomic replicon cell lines were established as described previously (Katsume et al., 2013). Briefly,

wild-type R6FLR-N replicon cells were seeded in 10-cm dishes in the presence of 0.5 mg/mL Volasertib cell line G418 and treated with telaprevir. The cells were incubated for 51 days with no-compound control or telaprevir (1.8 μM and 2.7 μM serially diluted in media). Fresh media and telaprevir were added every 3 days. Most cells incubated with 2.7 μM telaprevir died; however, after 3 weeks small colonies started to appear and were expanded for 4 weeks. Deep sequencing was performed

as described previously (Katsume et al., 2013) and revealed a mutation profile in NS3 (V36A, T54V and A156T) and NS5A (Q181H, P223S and S417P) which confer resistance to telaprevir. Resistant replicon cells were seeded at 5 × 103/well. After incubation for 24 h at 37 °C (5% CO2), culture medium was removed and replaced with growth medium containing serial dilutions of PYC or telaprevir alone or in combination. After 72 h, luciferase check details activity was determined using the Bright-Glo luciferase assay kit (Promega, Madison, WI, USA). Measurements were made in duplicate using a GloMax-Multi detection system (Promega, Madison, WI, USA). Cytotoxicity was measured using WST-8 cell counting kit (Dojindo, Kumamoto, Japan). Western blot analysis was performed, as described previously (Nishimura et al., 2009). Briefly, HCV replicon cells (2 × 105) were grown in a 60-mm cell culture dish. After 24 h, cells were treated with PYC for 72 h. Cells were collected and lysed with radioimmunoprecipitation buffer (1% sodium dodecyl sulphate, 0.5% Nonidet P-40, 150 mmol NaCl, 0.5 mmol ethylenediaminetetraacetic acid, 1 mmol dithiothreitol, and 10 mmol Tris, pH 7.4).

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