Hbsmcs

Normal human BSM cells (hBSMCs; a male donor: purchased from Cambrex Bio Science Walkersville, Inc., Walkersville, MD, USA) were maintained in SmBM medium (Cambrex Bio Science Walkersville, Inc., Walkersville, MD, USA) supplemented with 5% fetal bovine serum, 0.5 ng/mL human epidermal growth factor (hEGF), 5 µg/mL insulin, 2 ng/mL human fibroblast growth factor-basic (hFGF-b), 50 µg/mL gentamicin, and 50 ng/mL amphotericin B. Cells were maintained at 37 °C in a humidified atmosphere (5% CO₂), fed every 48–72 h, and passaged when cells reached 90–95% confluence. Then the hBSMCs (passages 5–7) were seeded in 24-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) and were cultured without serum. Twenty-four hours after the starvation period, the cells were loaded with Fluo-8/AM (2.5 M: AAT Bioquest, Inc., Sunnyvale, CA, USA) in serum-free SmBM medium for 90 min at 37 °C. The cells were washed with PBS and maintained in Krebs–Henseleit solution described above. The intracellular Fluo-8 fluorescence was monitored using fluorescence microscope (Keyence, Osaka, Japan) with BZ-X filter GFP (470/40, 535/50 nm). Images were pictured using time-lapse imaging (Keyence), and analyzed with BZ-X analyzer (Keyence). Change in the cytosolic Ca²⁺ level was calculated as ratio to the basal fluorescence intensity.

Normal human bronchial smooth muscle cells (hBSMCs; Cambrex Bio Science Walkersville, Inc., Walkersville, MD, USA) were maintained in SmBM medium (Cambrex) supplemented with 5% fetal bovine serum, 0.5 ng/mL human epidermal growth factor (hEGF), 5 µg/mL insulin, 2 ng/mL human fibroblast growth factor-basic (hFGF-b), 50 µg/mL gentamicin, and 50 ng/mL amphotericin B. Cells were maintained at 37 °C in a humidified atmosphere (5% CO₂), fed every 48-72 h, and passaged when cells reached 90–95% confluence. Then, the hBSMCs (passages five through seven) were seeded in 6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) at a density of 3500 cells/cm² and, when 80–85% confluence was observed, cells were cultured without serum for 24 h before addition of recombinant human IL-13 (100 ng/mL; PeproTech EC, Ltd., London, UK). At the indicated time after the IL-13 treatment, cells were washed with phosphate-buffered saline, immediately collected and disrupted with 1x SDS sample buffer (150 µL/well), and used for Western blot analyses. Total RNAs containing miRNAs were extracted using a Vantage^TM total RNA purification kit (OriGene) according to the manufacturer’s instructions.