Tenascin c

Non-tissue culture-treated 96-well plates were coated with 100 μl of the following matrices in triplicate at given concentrations: Fibronectin at 1 μg/ml, rat tail Collagen-I (BD Biosciences, Franklin Lakes, NJ, USA, 354236) at 0.5 μg/ml, Matrigel at 0.5 μg/ml, Tenascin-C (EMD Millipore, CC 065) at 3 μg/ml, Laminin-I at 10 μg/ml, Collagen-IV at 10 μg/ml and latency-associated peptide (LAP) (Sigma-Aldrich, L3408) at 0.5 μg/ml. Three wells of each plate were coated with 0.1% bovine serum albumin (BSA, PAA Laboratories, K45-001) in phosphate-buffered saline (PBS) (w/v) as a control. Coated plates were incubated for 1 hour at 37 °C after which wells were washed twice with 100 μl PBS per well.
Modified β6-1089 or N-1089 cells were serum starved for 24 hours prior to the experiment. 3 × 10³ cells were seeded in 100 μl of serum-free Ham’s F12 per well and allowed to adhere for 1 hour at 37 °C. Then 0.25 μl of Calcein AM (Invitrogen, C1430) cell tracker was added and incubated for 15 minutes at 37 °C after which the plate was washed twice with 100 μl PBS per well, and 100 μl of SFM was added. The plate was read at 490/520 nm on a fluorescent reader (BMG Labtech, Aylesbury, UK, FLUOstar Optima). The adhesion was calculated as the fluorescence value of adherent cells and normalised to that of control cells.

Collagen I (rat tail; Sigma Aldrich) and Collagen IV (from human placenta; Sigma Aldrich) were diluted to 20ug/mL in 0.5M acetic acid, tenascin C (human purified; Millipore) was diluted to 5ug/mL in PBS, fibronectin (from human plasma; Millipore), laminin (mouse purified; Millipore) and hyaluronan (high molecular weight; R&D Systems) were diluted to 20ug/mL in PBS. Diluted coatings were applied to cover wells or flasks for 2h at room temperature, then removed and washed with PBS. PBS was removed and cells were seeded in their appropriate media.
For assays using pairwise ECM combinations, Collagen I and Collagen IV were diluted to 40ug/mL in 0.5M acetic acid, tenascin C was diluted to 10ug/mL in PBS, and other ECM components were diluted to 40ug/mL in PBS. These ECM dilutions were mixed 1:1 for each pairwise combination prior to plating. Additional concentration-matched single ECM controls were generated by further diluting single ECM components 1:1 in PBS.

Tissue culture plates were incubated overnight with a 10 µg/ml solution of collagen-I (Sigma-Aldrich, #C7774), collagen-IV (Sigma-Aldrich, #C6746), fibronectin (Sigma-Aldrich, #F0895), laminin (Sigma-Aldrich, #L6274) or tenascin-C (#CC065, Millipore). Wells were washed with PBS, blocked with 1% BSA/PBS and again washed with PBS as previously described [14] (link). For cell derived ECM preparations, ASM cells from control and asthma derived cells were cultured in 12-well culture plates for 14 days, media aspirated and cells removed using ammonium hydroxide (50 mM) and Triton X-100 (0.05%). Following incubation with DNase I (20 U/ml) plates were washed with PBS and stored overnight (4°C). This preparation results in a cell free bioactive ECM coating [29] (link). Plates were allowed to warm to room temperature prior to seeding serum starved ASM cells, and cell culture supernatants were harvested after 24 hours.

Transfection complexes were formed combining 30 nM single siRNA or 15 nM each of combined siRNAs with 10 μl Dharmafect 1 or 2 and OPTI-MEM in a total volume of 400 μl. After a 20-minute incubation, 1.6 ml antibiotic-free media containing 10% FBS was mixed in and the total volume added to cells at 50% confluence on 60 mm plates. After 5 hours of incubation at 37°C/5% carbon dioxide, an additional 2 ml complete media were added to each plate. Next day, the cells were trypsinized and cells were plated (8,000 cells for MDA-MB-231 and SUM-149; 10,000 cells for MCF-7L^TamR) into four wells per transfection condition in Primaria 96-well plates coated with 0.33 mg/ml Matrigel (354234; BD Biosciences), 0.2 μg/ml tenascin-C (CC065; Millipore Billerica, MA, USA) and 0.1 μg/ml fibronectin (F-0895; Sigma). Ninety-six hours following transfection initiation, CellTiter 96® Aqueous One Assays were performed according to the manufacturer’s instructions (Madison, WI, USA). Absorbance values for media alone were subtracted from the experimental values. The data shown represent the results of five experiments for MDA-MB-231, of four experiments for SUM-149, and of two experiments for MCF-7^TamR.