Reagents and antibody
Luteolin was purchased from J&K Scientific (CAS:491-70-3, Beijing, China), and oseltamivir carboxylate (OC) was from Medchem Express (CAS: 187227-45-8, NJ, USA). Luteolin and OC were dissolved in DMSO (2 mM stock). Golgicide A was purchased from Selleck (CAS:1139889-93-2, Shanghai, China). Pitstop2-100 was obtained from Abcam Biochemicals (Cambridge, MA, USA). The following antibodies were used for Western blot: β-actin (1:5000) (Cell Signaling Technology), β-COP (1:1000) (abcam), and IAV nonstructural protein (NS1, 1:400) (Santa Cruz), respectively.
Cells and virus
Madin-Darby canine kidney (MDCK) cells and Vero cells (African green monkey kidney) were grown at 37 °C in minimum essential medium (MEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (10,000 U/mL). Extra 1% MEM nonessential amino acids solution was added to MDCK cells.
Influenza virus A/Fort Monmouth/1/1947 (H1N1) was purchased from the America Type Culture Collection (ATCC, VR-97™). Influenza virus A/Jiangxi/312/2006 (H3N2) was kindly donated by Professor Yuelong Shu from the Institute for Viral Disease Control and Prevention, China Centers for Disease Control and Prevention, Beijing, China.
Cell viability test
To measure luteolin cytotoxicity, MDCK (2.5 × 104), Vero (3 × 104) and Calu-3 (2 × 104) cells were grown in 96-well plates. After being cultured for overnight, cells were incubated with various concentrations of luteolin that ranged from 3.75 to 240 μM for an additional 48 h. Cell viability was measured using a CCK-8 assay kit (TransGen, Beijing, China) following the manufacturer's instructions. The absorbance of each well was recorded at 450 nm using a Multiskan MK3 microplate reader.
Cytopathic effect (CPE) assays
For CPE assays, MDCK cells in 96-well plates were infected with influenza virus at an MOI of 0.001 for 2 h, and then incubated with a maintenance medium supplemented with or without luteolin. After 48 h, the CPE was recorded. Then, we calculated the 50% CPE inhibition concentration (IC50) values of luteolin. For the antiviral assay, MDCK and Vero cells were infected with two IAV strains. After 24 h, we collected cells and supernatants by freezing and thawing thrice, and then determined virus titer by TCID50 assay for 48 h.
A time-of-addition experiment was conducted as previously described with some modifications . Briefly, MDCK cells were inoculated with 0.01 MOI of virus at 37 °C for 2 h. The media with or without luteolin 15 µM were added during the periods of – 2 to 8 h, 0–8 h, 2–8 h, 4–8 h and 6–8 h. After each incubation period, the collected cells were washed with PBS, and then determined the viral mRNA through qRT-PCR.
Hemagglutination inhibition (HI) assay
The effect of luteolin on the agglutination of chicken RBCs by influenza virus was determined by an HI assay . Briefly, 50 µL of twofold dilutions of the compound in normal saline were prepared and mixed with 50 µL influenza virus solution (equivalent to 4 HA units) in a U-bottom 96-well plate. The plate was kept at 4 °C for 30 min, and then 100 µL 1.2% chicken erythrocyte suspension was added to each well. Lastly, cell agglutination in the well was checked visually after incubation for 40 min at room temperature.
Indirect immunofluorescence assay
MDCK cells were washed twice with PBS and incubated for 2 h with influenza virus (0.001 MOI) at 37 °C in 5% CO2 atmosphere for adsorption. After 2 h, inoculum was decanted and infected cells were supplemented with maintenance medium with or without luteolin. After 18 h, MDCK cells were fixed for 10 min with 4% paraformaldehyde at room temperature, and washed three times with PBS to remove the fixation buffer. The cells were permeabilized in 0.5% Triton X-100 in PBS for 15 min. The samples were then incubated with 3% BSA for 1 h at room temperature and further incubated with the mouse anti-influenza virus M2 antibody overnight at 4 °C. After washing three times with TBST, cells were incubated with Alexa Fluor 488 anti-mouse IgG (TransGen) for 1 h. The nucleus was stained with Hoechst 33342 (Beyotime) for 5 min. Photos were taken with an Olympus TH4-200 microscope.
For co-localization studies with β-COP and GM130, fixed Vero cells were permeabilized in 0.5% Triton X-100 for 15 min and blocked with 3% BSA for 1 h at room temperature. The cells were incubated with rabbit anti-β-COP and mouse anti-GM130 antibody overnight at 4°C, rinsed with TBST, stained with Alexa Fluor 488 anti-rabbit IgG (TransGen) and Alexa Fluor 594 anti-mouse IgG (Cell Signaling Technology) for 1 h. The nuclei were stained with DAPI (ZSBIO, Beijing). Images were acquired with a Zeiss LSM 710 microscope.
Protein extracts (10 µg) were subjected to electrophoresis with 10% polyacrylamide gel and were blotted onto PVDF membranes (Millipore Corp.). The membrane was incubated with primary antibody overnight at 4 °C, followed by incubation with HRP-conjugated rabbit anti-mouse or goat anti-rabbit IgG. The signal was detected with an ECL kit.
Quantitative real-time RT-PCR
For intracellular viral RNA quantification, total RNA was extracted from the cells using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. QRT-PCR analysis was performed as described as follows. Each qRT-PCR reaction mixture (15 μL) contained 7.5 μL of 2xTransStart Tip Green qPCR SuperMix, 0.3 μL of forward and reverse primers (10 μmol/L), 2 μL RNA Template, 0.3 μL of one step enzyme Mix, 0.3 μL of passive reference dye and 4.3 μL RNase-free Water (TransGen, Beijing, China). Oligonucleotide primer pairs were (1) forward 5′-GACCRATCCTGTCACCTCTGAC-3′ and reverse 5′-GGGCATTYTGGACAAAKCGTCTACG-3′ for influenza virus M2 (2) forward 5′-AGTCAAGGCTGAGAACGGGAAACT-3′ and reverse 5′-TCCACAACATACTCAGCACCAGCA-3′ for dog glyceraldehyd-3-phosphate dehydrogenase (GAPDH). Triplicate qRT-PCRs were prepared for each sample. The CT method was applied using the GAPDH cycle threshold for normalization to evaluate influenza virus M2 mRNA expression.
All data were analyzed by the SPSS 19.0 software using one-way ANOVA, and the two independent groups were analyzed via Student’s t test. Values and error bars represent the average and standard deviations from three independent experiments. Statistical differences were considered significant at *p < 0.05; and **p < 0.01.