Amphotericin

Normal human lung fibroblasts (NHLFs) from the American Type Culture Collection (Manassas, VA, U.S.A.) were used throughout this study. They were cultured in complete FBM medium with Fibroblast Growth Medium-2 SingleQuots supplemented with 2% fetal bovine serum (FBS), 0.1% insulin, 0.1% human basic fibroblast growth factor (bFGF), 30 μg mL^{− 1} gentamicin, and 15 ng mL ⁻¹ amphotericin (Lonza Walkersville, Inc., Walkersville, MD, U.S.A.). The cells were maintained under a subconfluence condition at 37 °C with 5% CO₂. Early passages of NHLFs (passages 2−5) were used in this study. For the generation of stable SOX2 knockdown cells, 2 × 10⁵ cells were seeded in each well of 6-well plates with a total volume of 1 mL of media and incubated overnight. SOX2 knockdown experiments were conducted using lentivirus-mediated gene transfection. The cells were washed, and the culture medium was replaced with 2.5 mL per well of fresh medium containing 2 μg mL⁻¹ hexadimethrine bromide. The cells were then infected with either control or SOX2 shRNA lentivirus (Applied Biological Materials Inc., Richmond, BC, Canada) and incubated for 24 h. Next, the cell culture medium was replaced with 2 mL of fresh medium, and cells were further cultured for 48 h and allowed to recover for 24 h prior to experiments.

Rat vascular SMCs (Lonza) were maintained in DMEM/F12 supplemented with 20% fetal bovine serum and 0.1% GA-1000, containing 30 μg/ml Gentamicin and 15 ng/ml Amphotericin (Lonza). For immunostaining, cells were grown on 4-well chamber slide (Thermofisher) and the plasmid DNA encoding human Slingshot-1L (WT or CS, a phosphatase mutant) fused with YFP (generous gift from Dr. Kensaku Mizuno), were transfected into cells using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Forty eight to 72 hrs after transfection, cells were fixed in 100% Methanol at −20°C for 20 minutes, blocked with 2% BSA fraction V (Wako Chemical, Japan) containing 0.1% Tween-20 for 1 hour, and incubated with phospho-cofilin antibodies (Santa Cruz, 1:500) overnight at 4°C. Alexa Fluor 568-conjugated secondary antibodies (Invitrogen) were used at a dilution of 1:200 for 1 hour at 37°C. Slides were covered with Vectarshield containing DAPI (Vector Laboratories). Fluorescence images were obtained using a LSM700 (ZEISS).