(A–C) 293 (A), HeLa (B), or Vero (C) cell lines stably expressing tetracycline (Tet)-inducible IFITM1, IFITM2 or IFITM3 were cultured with or without Tet for 72 h and then transfected with AiV replicon RNA. Cell lysates were harvested at the indicated time points after transfection and then assayed for luciferase activity. The peak activity obtained for cells in the absence of Tet was taken as 100%. Tet-induced IFITM1, IFITM2, or IFITM3 protein expression in each cell line was detected by Western blotting (bottom panels of (A) and right panel of (B) or right panel of (C)). (D) HeLa cells were transfected with the control, IFITM1, IFITM2, or IFITM3 siRNA for 72 h and then transfected with replicon RNA. At the indicated time points after replicon RNA transfection, cell lysates were harvested and subjected to the luciferase assay. The maximum value obtained for cells treated with control siRNA was taken as 100%. (E) IFITM1, IFITM2, or IFITM3 knockdown was confirmed by Western blotting. (F) Cell viability was determined by the CellTiter-Glo assay. Data are the means ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.