T g ha vsmc

The T/G HA-VSMC (vascular smooth muscle cell) line (CRL-1999) was purchased from ATCC (American Type Culture Collection, Virginia, USA). This line of VSMC is a normal human cell line established from the normal aorta of an 11-month old child and one of the best-characterized cellular models for the analysis of vascular smooth muscle cell biology. Cells were cultured in F-12K Medium (ATCC, No. 30–2004) supplemented with 0.05 mg/ml ascorbic acid, 0.01 mg/ml bovine insulin, 0.01 mg/ml human transferrin, 10 ng/ml sodium selenite, 0.03 mg/ml Endothelial Cell Growth Supplement, 10 mM HEPES, 10 mM TES, and 10% fetal bovine serum under a 5% CO₂ in air-ventilated incubator. To knock-down Csk, VSMCs were transiently transfected with control or Csk siRNA using Lipofectamine 2000 and treated for 24 hours with DMSO or PP2 after 24 hours transfection and then proteins were extracted for Western blotting.

Human ovarian cancer cell lines (TOV112D, TOV-21G, SKOV3, MDAH2774), lung fibroblast cells (HFL1), and normal aorta smooth muscle cells (T/G HA-VSMC) were obtained from the American Type Culture Collection (Manassas, VA, USA). TOV112D, TOV-21G, SKOV3, MDAH2774, and HFL1 were cultured in DMEM/F12 with 10% fetal bovine serum and appropriate amounts of penicillin and streptomycin at 37°C with 5% CO2. T/G HA-VSMC cells were cultured in DMEM/F12 with 10% fetal bovine serum, 0.05% mg/ml ascorbic acid, 0.01 mg/ml insulin, 0.01 mg/ml transferrin, 10 ng/ml sodium selenite, and 0.03 mg/ml endothelial cell growth supplement. For apoptosis analysis, cells transfected with BAIAP2L1 siRNA were either irradiated with UV (100 J/M²) or treated with 10 μM cisplatin (Fresenius Kabi, NC, USA) for 24 hrs before analysis.

The human aortic vascular smooth muscle cell line (T/G HA-VSMC, Cat. CRL-1999) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Primary cell types including human aortic smooth muscle cells (HASMCs, Cat. #6110) and rat aortic smooth muscle cells (RASMCs, Cat. #R6110) were purchased from ScienCell (San Diego, CA, USA). Culture medium used in this study includes Dulbecco Modified Eagle Medium (DMEM, Hyclone) and Smooth Muscle Cell Medium (SMCM, ScienCell) while the other cell-culture reagents were purchased from Life Technologies (Thermo Fisher Scientific Inc. Waltham, MA USA). Note that supplemented culture medium represents culture medium supplemented with 10% FBS and 1% penicillin and streptomycin.
Chemokines used in this study to induce migration of VSMCs were PDGF-BB (Peprotech, Cat. #AF-100-14B) and TNF-α (Peprotech, Cat. #AF-300-01A). The materials used during device fabrication were SU-8 photoresist (MicroChem Corp., Newton, MA, USA) and 184 silicone elastomer (Dow Corning Corp., Midland, MI, USA). Fibronectin (Sigma) and collagen type I (Sigma) were used for channel surface coating.

Human vascular smooth muscle cell line T/G HA VSMC (ATCC® number: CRL-1999™) was purchased from American Type Culture Collection. Cells were cultured in Ham's F12K medium with 2 mM L-glutamine supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified incubator with 5% CO₂. Then, cells were trypsinized, transferred to 10 cm tissue culture dishes, and cultured to subconfluence. Cells at passages 4–8 were used. Cells overexpressing PAK4 were obtained by transfection with PAK4 ORF expression clone vector, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. For further experiments, cells were cultured in 6-well or 96-well plates with serum-free medium for 24 h.

The human aortic vascular smooth muscle cell line (T/G HA-VSMC), which was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), was a gift by Dr. Chen Jian’s Lab. The human umbilical vein endothelial cell line (PUMC-HUVEC-T1) and human monocytic cell line (THP-1) were purchased from National Infrastructure of Cell Line Resource of China (Beijing, China). T/G HA-VSMC and PUMC-HUVEC-T1 cell lines were maintained in high-glucose DMEM (Life Technologies, Gibco) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Gibco), MEM Non-essential amino acids (MEM NEAA 100×, Life Technologies, Gibco), penicillin (100 U mL⁻¹, Gibco, USA) and streptomycin (100 μg mL⁻¹, Gibco, USA). THP-1 cells were maintained in RPMI 1640 Medium (Caisson Labs) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U mL⁻¹) and streptomycin (100 μg mL⁻¹). The cell lines were cultured in cell incubator (37 °C, 5% CO₂) (Thermo Fisher Scientific). Human DiI-Acetylated LDL and LDL were purchased from Yeasen (Shanghai, China).