Apigenin (#A106676), hesperetin (#H107700), kaempferol (#K107145), naringin (#N107344), naringenin (#N164488), quercetin (#Q111274) and PD98059 (#P126620, ERK inhibitor) were purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD (Shanghai, China). 6-Methylflavanone (6Met, #M1403, TAS2R inhibitor) was obtained from TCI Development Co., Ltd (Shanghai, China). U73122 (#662035, PLCβ inhibitor) was obtained from Calbiochem (San Diego, CA). Dulbecco’s modified Eagle’s medium (DMEM), fatal bovine serum (FBS), penicillin, streptomycin and trypsin were purchased from Gibco (#27250018, Grand Island, NY). Cell culture flasks and plates were purchased from Corning Incorporated (Corning, NY).
Cell Cultures And Drug Treatment
The human BEC 16HBE14o- cells, one kind of basal cell featured with functional tight junction and well-developed barrier properties , were obtained as a gift from Prof. Gruenert of University of California (San Francisco) . They were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, L-glutamine and antibiotics at 37 °C in a 5% CO2 humidified incubator. Cells were passaged at 90% confluence using 0.05% trypsin in 1:3 or 1:4 proportions. The healthy cells with cubic-like morphology were used for the following experiments.
Stock solutions (1 M) of apigenin, hesperetin, kaempferol, naringin, naringenin, and quercetin were prepared in DMSO. The stock solution (1 M) of denatonium was dissolved in phosphonate buffer solution (PBS). All those stock solutions were kept frozen at -20 °C. The cells cultured in basal DMEM medium without FBS were exposed to different concentrations of bitter tastants, with or without inhibitors (6Met, U73122, and PD) for TAS2R signaling. For each experiment, an appropriate vehicle control was run in parallel, with the final concentration of DMSO at 0.1% (v/v) to avoid the cytotoxicity of DMSO.
Cell Morphological Assessment
To directly present the effect of six bitter flavonoids on cell morphology, the cells were treated with different concentrations of bitter flavonoids for 48 h and then imaged by phase contrast microscopy using an inverted optical microscope with a 20x objective (Primovert, Carl Zeiss, Jena, Germany). The view-field for imaging was chosen randomly to prevent subjective bias.
Cell Proliferation Assay
The cell proliferation was evaluated by using trypan blue exclusion cell counting method. To this end, 16HBE14o- cells were seeded at the density of 2 × 104 cells/cm2 into 6-well plates for 24 h. Then the medium was replaced with fresh serum free DMEM containing bitter tastant at different concentrations. After the cells were incubated for 48 h with/without inhibitors for TAS2Rs signaling or ERK activity, cell number was counted by using cytometer after staining with 0.4% trypan blue for living cells. The cell proliferation is presented as a ratio of cell number in experimental groups to that of the vehicle group (% of control).
Cell Viability Assay
To verify the cell proliferation analysis, the cell viability of 16HBE14o- cells were tested by using ATP luminescence assay, which can efficiently measure metabolic activity in the cells . Briefly, subconfluent, exponentially growing 16HBE14o- cells at the density of 1 × 104 cells/cm2 were seeded in 96-well plates and cultured for 24 h. Then the medium was replaced with fresh serum free DMEM containing bitter tastant at different concentrations. After the cells were incubated in this medium for 48 h, the medium was replaced again with fresh serum free DMEM so that the ATP secreted from the cells into the culture medium during culture was excluded from the cell viability measurement. Then the cell viability was determined using ATP luminescent cell viability assay (#C0065M, Beyotime Biotechnology, China), which directly presents the metabolic activity of cells based on the quantitation of the amount of ATP in cells. The cell viability is expressed as the percentage of luminescence (RLU) in experimental groups to that of the vehicle group (% of control).
Cell Cycle Analysis
Cells were seeded in 6-well plate at a density of 2 × 104 cells/cm2 and then exposed to 0.1, 0.5 and 1 mM naringin with/without inhibitors (6Met, U73122, and PD) for 48 h. Cells were collected and fixed with 70% ethanol. Following incubation on ice for 30 min, samples were washed with ice-cold phosphate-buffered saline (PBS) and then centrifuged at 1200 rpm for 5 min. Pellets were resuspended in PBS with RNase A (#ST578, Beyotime) (100 µg/mL) and propidium iodide (#ST511, Beyotime) (40 µg/mL). Samples were kept at 37 °C for 30 min in the dark. The cell cycle profile was analyzed using a flow cytometer (BD Accuri C6, BD Biosciences, San Jose, CA) and analyzed with ModFit software (Verity Software House, Topsham, ME).
Assessment Of Intracellular [Ca2+] In Cultured BECs
Intracellular calcium signals were visualized as described previously, using the membrane permeable [Ca2+]isensitive fluorescent dye Fluo4 acetoxymethyl ester (Fluo4/AM) (Sigma, St. Louis, MO). Briefly, cultured BECs were inoculated (~ 104 cells per dish) into confocal Petri dishes with a glass bottom. Next, the cells were incubated with 5 µM Fluo4/AM for 45 min at 37˚C in a 5% CO2 incubator. The cells were then washed with Tyrode's solution and incubated for 20 min to allow complete deesterification of the cytosolic dye. The fluorescence intensity of the sample (Fluo4/AM labeled BECs) was measured by laser scanning confocal microscopy (LSM710, Carl Zeiss) using an excitation wavelength of 488 nm and an emission wavelength of 505 nm, which represented the influx of the [Ca2+]i. Subsequent image processing and analysis were performed off-line using Image J software. Baseline Fluo-4 fluorescence (F0) was determined by averaging the first 10 frames of each experiment. [Ca2+]i was represented as F/F0, with F calculated by integrating Fluo-4 over entire cells for global [Ca2+]i.
Real-time qPCR Assessment Of Cyclin Protein Expression
Cyclin proteins, including Cyclin A, B, D and E are molecules that regulate cell proliferation and cell cycle. In order to determine whether naringin influences these molecules in bronchial epithelial cells, we used real-time qPCR to measure the mRNA expression level of Cyclin A to E in 16BEC14o- cells with/out exposure to naringin at 01 or 1 mM for 24 h. Total RNA from cultured BECs was extracted using the TRI Reagent RNA Isolation Reagent (#T9424, Sigma). 500 ng total RNA was used to generate 1st strand cDNA using the Revert Aid First Strand cDNA Synthesis Kit (#K1622, Thermo, MA, USA). Primers for Cyclin A, Cyclin B, Cyclin D, and Cyclin E as target genes and GADPH as reference gene were purchased from General Biosystems (Anhui, China). PowerUp SYBR Green Master Mix (#A25742, Applied Biosystems, CA, USA) was used. The reaction was run in the Real-time PCR system (StepOnePlus, Applied Biosystems) using 1 µl of the cDNA in a 10 µl reaction according to the manufacturer’s instructions in triplicate. Calibrations and normalizations were done using the 2−∆∆CT method, where ∆∆CT = CT (target gene) -CT (reference gene). Fold changes in mRNA expression were calculated from the resulting CT values from three independent experiments.
The FRET biosensor ERK, EKAREV, was prepared as reported previously . Cells were seeded in 6-well plates and grown to reach 40% of confluency. Cells were then transfected with 2.5 µg of DNA per well using Lipofectamine 3000 transfection kit (#L3000-008, Thermo). After 12 h, the medium was replaced with 10% FBS DMEM without antibiotics overnight. After transfection for 24 h, cells were seeded on 10 µg/ml fibronectin-coated glass-bottom dishes in 10% FBS DMEM for 12 h. Before imaging on microscope, cells were starved in DMEM without FBS overnight. Live cell imaging was performed as previously reported [19, 20]. By using a live cell image system (Cell Observer 1A, Carl Zeiss), cells were excited at a 436 nm wavelength, and the fluorescence of the CFP (emission at 480) channel and YFP channel (emission at 535) were recorded, respectively. FRET/CFP, was used to represent the level of FRET ON state.
Images were acquired every 2 min. After 10 min, drugs were added to the cell medium by gentle pipetting. The ECFP and FRET images were processed and quantified by Fluocell, a MATLAB-based Open Source image analysis software package(http://github.com/lu6007/fluocell [21, 22]).
Statistical analysis was performed by using GraphPad Prism (Graph Pad Software, La Jolla, CA). Unless stated otherwise, data were reported as means ± s.e.m, and n represents the number of samples. One-way analysis of variance (ANOVA) followed by Post Hoc test was carried out for multiple comparisons. The significance level was set at p < 0.05 (*p < 0.05; **p < 0.01).