Human IL-5 and IL-4 Enzyme-linked immunosorbent assay (ELISA) kits were supplied by Ray Biotech. Recombinant human IL-13 and R130Q were purchased from PeproTech (London, UK). Cell counting kit-8 and Fluo-3/AM were obtained from Beyotime (Jiangsu, China).
Culture of primary hBSMCs
Primary hBSMCs and cell culture agents were supplied by ScienCell (ScienCell, Carlsbad, CA, USA) and cultured at 37°C in a humidified atmosphere with 5% CO2 using a smooth muscle cell medium (SMCM; Science Cell) according to the supplier’s instruction. The medium was supplemented with 2% fetal bovine serum, 0.5 ng/mL human epidermal growth factor, 5 μg/mL insulin, 2 μg/mL human fibroblast growth factor-basic, 50 μg/mL gentamicin, and 50 ng/mL amphotericin B. Medium change was performed every 2 days. The hBSMCs were passaged when they reached 90-95% confluence. Cells between the third and sixth passage with 80-85% confluence were used for further experiments.
Treatment with IL-13
For the treatment experiments, hBSMCs were first cultured in a serum-free medium for 24 h. The serum-deprived cells were then stimulated with increasing concentrations (1 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml) of recombinant human WT IL-13 or IL-13 R130Q for 24 h. The specific concentration and incubation times were chosen based on previous reports conducted in this area [21, 22] .
Cell proliferation assay
For the cell proliferation assay, hBSMCs were incubated in 96-well plates at a density of 1x104 cells per well with SMCM and treated with IL-13 R130Q or WT IL-13 for 24 h. Then, 10 µl of cell counting kit-8 (CCK-8) solution (Beyotime, Jiangsu, China) was added to each well. The cells were incubated with the solution at 37°C for 2 h. The proliferation activity of each clone was quantified by measuring the absorbance at 450 nm with an enzyme immunoassay analyzer (Bio-Rad, Hercules, CA, USA).
Cell migration assay
Migration of hBSMCs was analyzed using a modified Boyden chamber  separated by an 8 mm polycarbonate membrane (Costar, Corning, USA) coated with 0.01% collagen type-I in 0.01N HCl solution (Sigma). The free-floating hBSMCs that detached from the plate with trypsin-EDTA (Invitrogen Canada Inc, Burlington, ON) following 24 h of serum deprivation were washed and resuspended in SMCM at a density of 5*104 cells/ml. The cell suspension was added to the upper chamber of the modified Boyden chamber apparatus. The lower chambers contained IL-13 R130Q or WT IL-13 (1, 10, 50, 100 ng/ml) in the culture medium. After incubation for 24 h at 37°C in a humidified atmosphere with 5% CO2, hBSMCs that have migrated to the lower side were counted under a phase-contrast microscope (OPTIPHOT, Nikon, Japan). The magnification was 20X and four-five random fields were counted for each chamber.
Intracellular calcium concentration
After pretreatment with WT IL-13 and IL-13R130Q for 24h, cells were incubated at 37°C in a medium containing 5 mM Fluo-3AM (a Ca2+- sensitive fluorescent probe) for 1 h followed by 30 min in extracellular saline. After that, the relative median fluorescent activity was measured with flow cytometry as an indicator of intracellular calcium (Ca2+) concentration.
ELISA for IL-4 and IL-5
Before measurement, hBSMCs were incubated in 6-well cell culture plates at a density of 1x105 cells/cm until 80-85% confluence was achieved and then treated with IL-13 R130Q or WT IL-13 for 24 h after serum deprivation. The concentrations of IL-4 and IL-5 (expressed as picograms per milliliter) in the collected cell culture supernatant were determined by ELISA kits following the manufacturer’s instructions without any prior dilution.
All values were expressed as mean ± standard deviation (SD). Continuous variables were compared with one-way analysis of variance. A P value of < 0.05 was considered statistically significant.