siRNA-mediated knockdown of hnRNP C1/C2 mRNA and proteins. (A) Huh7 cells transfected with hnRNP C1/C2-specific or control siRNA were infected with DENV at MOI 0.03. Cells were collected at 0, 12, 24, 36 and 48 h post-infection and subjected to total RNA extraction and subsequent reverse transcription and real-time PCR for determination of hnRNP C1/C2 and β-actin (internal control) mRNA expression. Relative hnRNP C1/C2 mRNA expression was obtained by normalization with β-actin mRNA expression in siRNA-transfected cells following DENV infection. Fold change of the relative hnRNP C1/C2 mRNA expression between the specific siRNA and control siRNA-transfected samples was compared. Data represent mean and SEM of three independent experiments. Asterisks indicate statistically significant differences (*p < 0.05; **p < 0.01) in hnRNP C1/C2 levels between the specific siRNA-transfected and control siRNA-transfected groups by unpaired t test. (B) Huh7 cells were transfected with hnRNP C1/C2-specific siRNA (SP) or irrelevant negative control siRNA (IR), followed by mock or DENV infection at MOI 0.03. Cells were collected at the indicated time points post-infection and subjected to immunoblotting analysis using monoclonal antibodies specific against human hnRNP C1/C2 and β-actin (internal protein control). Immunoblotting analysis of hnRNP C1/C2 and β-actin proteins in mock-infected and DENV-infected cells was shown as representative of three independent experiments (top panel). The intensity of hnRNP C1/C2 bands in both IR and SP samples was first normalized to that of β-actin in the same set of samples. The obtained relative intensity of hnRNP C1/C2 in the SP samples was then normalized to that in the IR samples. Results show fold change of relative hnRNP C1/C2 protein expression in the SP samples compared with the corresponding IR samples (bottom panel). Data represent mean and SEM of three independent experiments. Asterisks indicate statistically significant differences (*p < 0.05; **p < 0.01) in relative expression of hnRNP C1/C2 proteins between the specific siRNA-transfected and control siRNA-transfected samples by unpaired t test.