Figure 2.

Knockdown of IFITMs mediated by short hairpin RNA (shRNA) enhances CSFV replication. (A) Cell viability of stable IFITM-knockdown cell line. (B) Confirmation of recombinant IFITM shRNA lentivirus (shIFITM) transfection by detection of EGFP reporter. (a) Mock-transfected PAMs. (b) PAMs transfected with scrambled shRNA lentivirus (shN). (c) PAMs transfected with shIFITM. Scale bars, 100 μm. (C) Western blot analysis of IFITM expression in IFITM-knockdown cells using an IFITM1-specific antibody and an IFITM2/3 antibody against IFITM2 and IFITM3, respectively. β-actin served as an internal control. (D) Western blot analysis of IFITM expression using an anti-Flag antibody in shIFITM cell lines following transfection with CMV-IFITM1, CMV-IFITM2, or CMV-IFITM3 for 48 h. β-actin served as an internal control. (E) CSFV genomic RNA in shIFITM cell lines. The shN and shIFITM cell lines were infected with CSFV at an MOI of 1. CSFV genomic RNA levels were determined by RT-qPCR at 12 and 24 hpi. Data were normalized to β-actin expression. (F) Infectious progeny viral titers in culture medium from shIFITM cells. The viral titers of CSFV in supernatants were quantified and expressed as TCID₅₀/mL. Data (A, E, and F) represent the mean ± SD of three independent experiments and were measured in technical duplicate. Comparisons between groups were performed by Student’s t test. * p < 0.05 and *** p < 0.001.