cDNA samples were used in triplicate for qRT-PCR
using iQ-SYBR Green Supermix (Bio-Rad Laboratories).17 Target gene levels are presented as ratio of levels detected in treated cells over levels detected in control cells, according to the ΔΔCt method.17 Huh7.5 cells were grown in supplemented Dulbecco’s modified Eagle medium (DMEM).5 HCV replicon-harboring Huh7.5 cells were established by transfecting in vitro transcribed Con1 replicon RNA followed by selection with 1 mg/mL G418.24 After selection, cells were propagated in DMEM with 0.75 mg/mL G418. Infectious JFH1 virus was obtained by transfection of Huh7.5 cells with in vitro transcribed RNA and harvesting of cell supernatant as described.6, 25 To generate viral stocks, clarified supernatant was used to infect naïve Huh7.5 cells, supernatants were recovered 7 days postinfection, and concentrated using Sunitinib solubility dmso BI 6727 in vivo an Amicon 100k device. Virus-containing supernatants were titered by FFU as described.25 Briefly, serial 10-fold dilutions of samples were plated in triplicate on 96-well plates containing subconfluent Huh7.5 cells. After 72 hours of incubation, cells were washed with phosphate-buffered saline (PBS) and fixed with ice-cold methanol. Infected cells were subsequently
identified by immunofluorescence using mouse α-Core antibody (C7-50; Abcam) and FFU/mL values were calculated using the mean of three separate wells per sample. Time course infections-protein analysis: Huh7.5 cells (3 × 105) were seeded onto four T-25 flasks and two of the flasks were infected the following day with HCV at a multiplicity of infection of 0.5-1.0. The virus was removed 16 hours postinfection and replaced with normal growth medium. Uninfected cells were split and grown in culture for the same time as their respective infected culture counterparts. Cells were harvested and lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS) on learn more day 2 and day 5 postinfection. The cell lysate was collected in 1.5-mL
microcentrifuge tubes and allowed to sit on ice for 15 minutes prior to centrifugation at 10,000g. The supernatant was collected as total cell lysate and amount of protein was estimated using the Bradford method. Drugs were incubated with cells prior to RNA and protein isolation. The following concentrations and incubation times were used: cyclopamine and tomatidine (5 μM for 48 hours for initial experiment; 24, 48, and 72 hours for time course), Shh (100 ng/mL for 48 hours), SAG (0.3 μM for 24 hours), interferon-α (500 U/mL for 48 hours), GDC-0449 (range 0-25 μM for 24 hours), 5E1 (Shh neutralizing antibody, 10 μg/mL, for 48 hours), mouse IgG1 isotype control (10 μg/mL, for 48 hours). If the experiment was done with the JFH1 HCV, drug was added at the time of infection. Supernatant media was collected from Huh7.5 cells infected and uninfected cells after 72 hours.