Cell culture and virus infection
The human lung carcinoma cell line A549, human cervical cancer cell line HeLa, Madin Darby Canine kidney cell line MDCK and African green monkey kidney cell line Vero were purchased from American Type Culture Collection (ATCC; Manassas, VA) and were maintained in the Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C. A influenza A virus strain A/PR/8/34 (H1N1) was used in this study. For virus infection, A549 cells or MDCK cells were inoculated with virus at the indicated multiplicities of infection (MOI). Two hours later, the inoculum was removed, and cells were washed twice with Hank's solution prior to the addition of serum-free DMEM containing 5% BSA and 1 μg/ml TPCK-trypsin (Sigma-Aldrich, St. Louis, MO).
Reverse transcription PCR
To determine the mRNA expression level of various LAMPs during influenza A virus infection, cells were infected with A/PR/8/34 virus at a MOI of 0.5. At different time points post infection, total cellular RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA samples were treated with DNase I (Pierce, Rockford, IL), and reversely transcribed using a Superscript cDNA synthesis kit (Invitrogen) following the manufacturer's protocols. cDNA samples were subjected to PCR amplification and electrophoresis analysis. The primer sequences were as follows:
LAMP1, 5'-GCGAGCTCCAAAGAAATCAA-3' (forward), 5'-TGGACCTGGGTGCCACTAA-3' (reverse), LAMP2, 5'-CTCTGCGGGGTCATGGTG-3' (forward), 5'-CGCACAGCTCCCAGGACT-3' (reverse) LAMP3, 5'-GCGTCCCTGGCCGTAATT-3' (forward) 5'-TGCTTAGCTGGTTGCTGGA-3' (reverse).
Western blot analysis
Cells were lysed in RIPA buffer containing 150 mM NaCl, 25 mM Tris (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 1 mM EGTA, and 1 mM EDTA with proteinase inhibitor cocktail (Roche, Indianapolis, IN), and total protein concentration was determined by the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL)before separation by SDS-PAGE. Expression of LAMP1 or LAMP3 protein was detected by the rabbit polyclonal anti-LAMP1 antibody (Sigma, L1418) or the rabbit polyclonal anti-LAMP3 antibody (AVIVA, ARP41598_P050), respectively.
siRNA against LAMP1 (M-013481-01), LAMP3 (M-004716-01), along with the non-targeting control (D-001206-13-05) were purchased from Dharmacon (Boulder, CO). A549 cells were transfected with the siRNAs (50 nM) using Dharmafect1 (Dharmacon) according to the manufacturer's instructions.
A549 cells were seeded in 24-well plates at a cell density of 1.5 × 105 cells per well. The next day, cells were left untransfected, or were transfected with pGL3-IFN-β-Luc and pRL-SV40, along with the scrambled siRNA, siRNA against LAMP3, or poly (I:C) using Lipofectamine 2000 (Invitrogen). At 48 h post transfection, cells were harvested, and cell lysates were subjected to luciferase activity assays (Promega, Madison, WI).
Indirect immunofluorescence assay
A549 cells either infected or mock infected by influenza A virus were fixed in 4% paraformaldehyde and permeabilized with PBS containing 0.5% Triton X-100 (pH 7.4). Then, the cells were blocked with PBST (PBS containing 0.1% Tween-20; pH 7.4) containing 5% BSA for 50 min at room temperature. Further, the cells were incubated with mouse polyclonal anti-influenza A virus NP antibody (1:500; Millipore, Temecula, CA) at 4°C overnight. After three washes with PBST, cells were incubated with anti-mouse IgG/DyLight594-conjugated antibody (1:500; Zhongshanjinqiao Biotech, Beijing, China) for 1 h, and cell nuclei were labeled with Hoechst dye 33258 (Beyotime, Nantong, China) according to the manufacturer's instructions. For localizing LAMP3 and influenza virus, Hela cells were transfected with LAMP3-GFP (Origene, Rockville, MD), and were stained with NP antibody 18 h post-transfection. After extensive washes with PBST, cells were incubated with anti-mouse IgG/DyLight594-conjugated antibody as indicated above. Fluorescence images were obtained by using a Leica TCS SP5 laser scanning confocal microscope.
In-cell western assay
The cells infected with influenza A virus or mock infected were immunostained with influenza A virus NP antibody together with a rabbit IgG antibody against Lamin A (1:1000; Sigma-Aldrich) as described above. Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody (1:500; LI-COR, Lincoln, NE) and goat anti-rabbit IgG IRDye 680 antibody (1:500; LI-COR). Protein expression was quantified using the Odyssey Imaging System. For statistical analysis, integrated intensities of fluorescence in wells were determined using software provided with the imager station (LI-COR). The relative amount of NP protein was obtained by normalizing to the endogenous lamin A.