Knockdown of LAMP3 inhibits influenza A virus replication. (A) Knockdown of LAMP1 or LAMP3 by RNAi. A549 cells were transfected with the siRNA against LAMP1, LAMP3 or a non-targeting control RNA (scrambled) (50 nM). Seventy two hours later, cells were lysed and subjected to Western blot analysis using antibody against LAMP1 or LAMP3. (B) In-cell western assay for NP production. A549 cells grown in 96-well plates were transfected siRNAs as indicated in (A). After 72 h, cells were either infected or mock infected by influenza virus, and were subjected to in-cell western assay at 24 h p.i.. NP was visualized via labeling with NP specific antibody, and endogenous lamin A was labeled as a control. (C) Quantitative analysis of in-cell western using Odyssey Imaging System. * Student's t test, p = 0.031.(D) A549 cells were transfected with LAMP3, LAMP1 or control siRNAs, cells were then infected with A/PR/8/34 virus at a MOI of 0.1 at 72 h post-transfection. Cell supernatants were harvested and subjected to TCID50 assay 24 h p. i.(E) A549 cells were left untransfected, or were transfected with an IFN-β reporter plasmid, along with the scrambled siRNA, siRNA against LAMP3 (SiLAMP3), or poly (I:C) at 50 ng/ml (a positive control for IFN promoter activation), and analyzed 48 h later for luciferase assay.