Treatment of DENV-infected cells with the Ltc 1 DihydrotestosteroneDHT order peptide To infect the HepG2 cells with DENV2, the cells were cultured in 24-well plates (1.5 × 105 cells/well) for 24 h at 37°C and selleck kinase inhibitor 5% CO2. The virus supernatant was added to the cells at a MOI of 2, followed by incubation for 1 h with gentle shaking every 15 min for optimal virus to cell contact. The cells were washed twice with fresh serum-free DMEM after removal of the
virus supernatant. Then, fresh complete DMEM containing 25 μM Ltc 1 peptide was added to the cultures and incubated for 72 h. The HepG2 cells were then collected, and the virus particles and expression level of the viral NS1 protein were examined using immunostaining and western immunoblotting. Time-of-addition assay This assay was performed to identify the mode of antiviral activity of the Ltc 1 peptide against DENV2 entry, replication and release from the infected cells. Three independent experiments were performed in triplicate for pre-, simultaneous and post-infection treatments. HepG2 cells were grown in a 24-well tissue culture plate (1.5 × 105 cells/well), incubated 24 h under optimal conditions and infected with DENV2 at an MOI of 2. For pre-treatment infection, 25 μM peptide was added to the cells
before virus inoculation Cediranib and incubated for 24 h. After removal of the old medium containing the peptide, the DENV2 supernatant was added, followed by incubation for 1 h with gentle shaking every 10 min for optimal virus to cell contact. The virus supernatant was removed and the cells were washed twice with fresh serum-free DMEM medium to remove the residual
virus. Fresh complete DMEM medium was added and the cultures were incubated for 72 h at 37°C, supplemented with 5% CO2. Identical applications were performed for the simultaneous treatment, except the peptide was mixed with the virus supernatant and incubated at 37°C for 1 h, and then inoculated onto the HepG2 cells. The post-treatment Isotretinoin infection was performed after inoculation of the HepG2 cells with DENV2, and complete DMEM medium with the Ltc 1 peptide was then added. The cultures including the peptide were incubated for 72 h at 37°C and 5% CO2, and three wells of infected cells in each experiment were maintained without treatment as controls. The cell supernatants were collected and stored at -80°C for viral load determination using a plaque formation assay. Dose-response assay This assay was performed to evaluate the 50% effective concentration (EC50) of the Ltc 1 peptide against DENV2. HepG2 cells were grown in six-well microplates (1.5 × 106 cells/well) for 24 h in quadruplicate experiments. The cell culture media were removed and the cells were washed three times with PBS. Then, fresh medium containing the virus supernatant was added at MOI of 2, followed by incubation for 1 h with gentle shaking every 15 min. The viral residues were removed by washing with PBS, and serial dilutions of the Ltc 1 peptide (0, 2.