Figure 7

Effect of hnRNP C1/C2 knockdown on DENV protein translation. Huh7 cells transfected with hnRNP C1/C2-specific siRNA (SP) or irrelevant negative control siRNA (IR) were subjected to co-transfection with pGL3-DENV2-5′UTR-72ntC-Fluc-3′UTR construct (viral reporter) and pRL-SV40 construct (internal control reporter). At 12 h after reporter RNA transfection, cells were harvested and assessed for firefly and Renilla luciferase activities. In parallel, reporter RNA-transfected cells that were treated with DENV capsid-specific siRNA to knockdown viral reporter RNA template used for viral translation (RK) or with cycloheximide to inhibit overall protein translation (CHK) were set up as positive controls in this assay for inhibition of protein translation, compared with non-treated cells (NT). (A) Relative luciferase expression specifically indicated DENV protein translation. In each sample, relative luminescence units of firefly luciferase activity were normalized to that of Renilla luciferase activity. Normalized luciferase signals of control siRNA-transfected cells and non-treated cells were set to 1. Relative luciferase expression in hnRNP C1/C2-specific siRNA was compared to that in control siRNA-transfected cells whereas relative luciferase expression in viral reporter knockdown cells and cycloheximide-treated cells was compared to that in non-treated cells. Data represent mean and SEM of three independent experiments. Asterisks indicate statistically significant difference (***p < 0.0001) in relative luciferase expression between viral reporter knockdown cells and non-treated cells by unpaired t test. (B) Immunoblotting analysis was performed to confirm the expression of hnRNP C1/C2 and β-actin proteins in each sample. Results are representative of three independent experiments with similar outcome.