Flua np 4f1

MDCK cells were seeded on a 24-well plate at a density of 1 × 10⁵ cells/well. 2–5 (12.5 μM), 1 (12.5 μM, positive control), or DMSO (0.125%, negative control) were mixed with MOI 0.1 of A/PR/8/34 virus and incubated for 30 min prior to the cells being added. At 4, 8, 12, or 24 h post-infection, the cells were lysed in a buffer containing 125 mM Tris-HCl, pH 6.8, 5% sodium dodecyl sulfate, 25% glycerol, 0.1% bromophenol blue, and 10% β-mercaptoethanol and boiled for 5 min. The cell lysates were separated on a 10% polyacrylamide gel. The proteins were transferred to a polyvinylidene fluoride microporous membrane (Millipore, MA, USA). FluA-NP 4F1 (SouthernBiotech) or a goat anti-influenza A viral NS1 antibody (vC-20; Santa Cruz Biotechnology, CA, USA) were used as primary antibodies to detect their respective proteins. A rabbit anti-β-ACTIN antibody (13E5; Cell Signaling, MA, USA) was used as an internal control. The secondary antibodies, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (SouthernBiotech) or donkey anti-goat IgG (sc-2020; Santa Cruz Biotechnology), were used as appropriate. The signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). Signal intensities were measured using ImageJ software, and the protein levels of NP and NS1 were normalized to that of β-actin.

MDCK cells were seeded in a 96-well plate (1 × 10⁴ cells/well). H₂O, EtOAc, Hex, or CHCl₃ extracts from the stems of J. multifida (3.1-25 μg/mL) or (+)-(S)-bakuchiol (3.1-25 μM) were mixed with influenza A virus at a MOI of 0.1 in the infection medium and incubated for 30 min at 37 °C in the presence of 5% CO₂. DMSO (0.031-0.25%) was used as the negative control. Each mixture was added to the cells and incubated for 24 h at 37 °C in the presence of 5% CO₂. The cells were fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C and then permeabilized by the addition of 0.3% Triton X-100 for 20 min at 25 °C. A mouse antibody for the detection of the NP of A/PR/8/34 (FluA-NP 4 F1; SouthernBiotech, AL, USA) was used as the primary antibody. Alexa Fluor488-conjugated goat anti-mouse IgG (H + L) antibody (Life Technologies, CA, USA) was used as the secondary antibody. Cell nuclei were then stained using diamidino-2-phenylindole (DAPI; Life Technologies). The wells were photographed using a fluorescence microscope (BIOREVO BZ-9000, Keyence, Osaka, Japan), and the percentage of influenza A NP-positive cells per DAPI-positive cells were calculated based on measurements recorded with BZ-H1C software (Keyence).

MDCK cells were seeded in a 96-well plate (1 × 10⁴ cells/well). (+)-(S)-Bakuchiol, (−)-(R)-bakuchiol, or ME was mixed at a concentration of 12.5–50 μm with influenza A virus (A/PR/8/34, A/CA/7/09, or A/Aichi/2/68) at an MOI of 0.1 in the infection medium and incubated for 30 min at 37 °C under 5% CO₂. DMSO (0.125–0.5%) was used as the negative control. Each mixture was added to the cells and incubated for 24 h at 37 °C under 5% CO₂. The cells were then fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C before permeabilization with 0.3% Triton X-100 for 20 min at room temperature. Mouse antibodies detecting the NP of A/PR/8/34 and A/Aichi/2/68 (FluA-NP 4F1, SouthernBiotech) or the NP of A/CA/7/09 (AA5H, AbD Serotec) were used as primary antibodies, as appropriate (26 (link)). Horseradish peroxidase-conjugated goat anti-mouse IgG (SouthernBiotech) was used as the secondary antibody. To visualize the infected cells, TrueBlue peroxidase substrate (KPL) was added and incubated for 15 min; color development was terminated by washing with H₂O. The wells were photographed under a microscope, and the stained cells were counted. Each half-maximal (50%) inhibitory concentration (IC₅₀) value was then calculated based on the cell numbers.