Effect of EGFR knockdown on RSV infection.

(A) ATP1A1 and EGFR expression in A549 cells transfected with siRNAs targeting ATP1A1 or EGFR. A549 cells were transfected with ATP1A1 siRNA 2, EGFR siRNA, or Neg. siRNA 1 or 2, incubated for 48 h, and subjected to immunofluorescence staining for ATP1A1 (green) and EGFR (red) as described for Fig 4A. Images (z-stacks) were acquired on a Leica SP5 confocal microscope with 63x objective NA 1.4 and 2.0x zoom. Scale bar 10 μm. (B and C) EGFR knockdown reduces RSV infection and virion production. A549 cells were transfected with an EGFR-specific siRNA or Neg. siRNA 1 or 2, and 48 h later were infected with RSV-GFP (MOI = 1 PFU/cell). Infection was quantified by (B) GFP signal of the total well, scanned by ELISA reader at 17 h p.i., and RSV production was assessed by (C) plaque titration on Vero cells 24 h p.i. Data are derived from three independent experiments. The statistical significance of the difference was determined by one-way analysis of variance with Tukey’s multiple-comparison post-test and p-values of the significance for each comparison is indicated. (D) Cell viability. An ATP-based cell viability assay (CellTiter-Glo) was performed 72 h p.t. to evaluate the viability of the transfected cells. Cells were lysed, the ATP concentration was determined by luciferase activity relative to mock-transfected cells, as a measure of viability.