Ren luc prl tk

The monocistronic minigenome construct expressing the firefly luciferase gene [74 (link)] was kindly provided by Dr. Bukreyev (UTMB). The plasmids pCEZ-NP, pCEZ-VP35, pCEZ-VP30, pCEZ-L, and pC-T7 were previously described [75 (link)]. 293T cells were plated (50,000 cells/well) onto 24-well plates in 5% FBS 1× DMEM for 24 h, and co-transfected with the following plasmids: EBOV minigenome (125 ng), pCEZ-VP30 (31.25 ng), pCEZ-NP (62.5 ng), pCEZ-L (500 ng), pC-T7 polymerase (125 ng), 100 ng of empty vector (pCAGGS) or pCAGGS-VP35 (WT, R225E, or R225K), and REN-Luc/pRL-TK plasmid (20 ng; Promega) expressing Renilla luciferase used as an internal control to normalize transfection efficiency. To test the compounds, 4 h post-transfection, treatments with pCEBS or SFC to 200, 100, 50, 25, and 12 μm in 1% DMSO were added, and cells were incubated at 37°C, 5% CO2 for 50 h. After, the cells were lysed to measure the luciferase signal using the Dual-Luciferase Reporter Assay System (Promega) with a Cytation 5 reader (Biotek). A portion of the lysate was boiled at 95°C for 10 min in 2× Laemmli buffer to evaluate protein expression via immunoblot.

The monocistronic minigenome construct expressing the firefly luciferase gene (72 (link)) was kindly provided by Dr. Bukreyev (UTMB). The plasmids pCEZ-NP, pCEZ-VP35, pCEZ-VP30, pCEZ-L, and pC-T7 were previously described (73 (link)). 293T cells were plated (50,000 cells/well) onto 24-well plates in 5% FBS 1X DMEM for 24 hours, and co-transfected with the following plasmids: EBOV minigenome (125 ng), pCEZ-VP30 (31.25 ng), pCEZ-NP (62.5 ng), pCEZ-L (500 ng), pC-T7 polymerase (125 ng), 100 ng of empty vector (pCAGGS) or pCAGGS-VP35 (WT, R225E, or R225K), and REN-Luc/pRL-TK plasmid (20 ng; Promega) expressing Renilla luciferase used as an internal control to normalize transfection efficiency. To test the compounds; four hours post-transfection, treatments with PCEBS or SFC to 200, 100, 50, 25, and 12μM in 1% DMSO were added, and cells were incubated at 37°C, 5% CO2 for fifty hours. After, the cells were lysed to measure the luciferase signal using the Dual-Luciferase Reporter Assay System (Promega) with a Cytation 5 reader (Biotek). A portion of the lysate was boiled at 95°C for 10 minutes in 2X Laemmli buffer to evaluate protein expression via immunoblot.