Soft agar

Soft agar, Bacto-agar powder and Type 1 collagen (COL1) were purchased from BD Biosciences (Mississauga, On, Canada). TRIZOL reagent was purchased from Invitrogen (Burlington, On, Canada). Quantitect reverse transcription kit, Taq DNA polymerase kit and SYBRGreen kit were purchased from QIAGEN (Toronto, On, Canada). Developer and fixation solution kits were purchased from Kodak (Rochester, NY, USA). Unless stated otherwise, all other products were from Sigma-Aldrich (Oakville, On, Canada).

To assess anchorage-independent growth, soft-agar colony forming assay was performed using 8000 cells per well. A layer of agar containing 3.0 ml of 0.6% soft agar (BD Biosciences, USA) in (basement membrane extract) BME was poured into wells of a 6-well cell culture dish and allowed to set at room temperature for 30 min. A second layer containing 1 ml 0.35% soft agar in BME containing cells (800 cells/ml) was placed on the top of the first layer and allowed to set at room temperature for 30 min. Cells were incubated in an incubator at 37 °C for 14 days, and the number of colonies were counted, and the images were captured using an Olympus microscope.

The cells were seeded into 6 cm culture dishes at the density of 2 × 10⁵ cells/dish. The transfections were mediated by lipofectamine 2000 according to manufacturer’s instructions (Invitrogen, Carlsbad, CA). For cell counting, the cells were treated with trypsin (0.25% trypsin plus EDTA, Life Technology, Carlsbad, CA), collected, re-suspended into 10 ml regular medium, and the single cell suspension was analyzed on hemocytometer for cell counting with trypan blue (Sigma-Aldrich, St. Louis, MO). For cell plating efficiency assay, the colonies were fixed with 4% paraformaldehyde, stained with crystal violet (0.5% w/v), and counted under inverted microscope and evaluated for their survivability. For colony formation assay, each 6-well plate was loaded with 2 ml 0.5% agar as base layer of soft agar (BD Biosciences, San Jose, CA) first, and 2 ml 0.3% agar top layer mixed with the transfected prostate cancer (LNCaP or PC3) cells (0.5 × 10⁴ cells and 5 × 10⁴ cells/ml). The colonies formed in soft agar were counted under inverted microscope after 10 days of transfections. The cellular colonies composed of more than 10 cells were counted as positive colonies.