Biocoat control insert

LNCaP cells grown in phenol red-deficient RPMI 1640 medium containing 10% FBS for 72 hrs were dissociated using Accutase (Invitrogen) and counted with a hemocytometer. 75,000 cells were seeded per well in 24-well plates with phenol red-deficient RPMI medium containing 1% charcoal-stripped FBS. The top and bottom of Biocoat control inserts (BD Biosciences, Palo Alto, CA), an 8 μm membrane pore size, were coated with 5 μg/ml of fibronectin in DPBS for 2 hrs at 37°C and subsequently washed with 1 DPBS and dried at 25°C. Cells were seeded into the top chamber, and the bottom chamber was filled with phenol red-deficient RPMI medium containing 1% charcoal-stripped FBS plus androgen (0, 0.1, 1, or 10 nM R1881). Migration was allowed to proceed at 37°C under 5% CO₂ for 18–24 hrs. The cells were then fixed with −20°C methanol for 10 min at 25°C, and inserts were stained with 0.5% crystal violet (Sigma) in 25% methanol for 10 min at 25°C. Inserts were washed with ddH₂O for 5 mins at 25°C and visualized under a light microscope to count cells. The means and standard deviations for counted cells were calculated, and ANOVA was used to determine statistical significance (*p ≤ 0.05, n = 3) between vehicle (ethanol) and androgen-treated cells.

To assess cell migration, 4 × 10⁴ cells in 150 μL DMEM with 1% FBS and indicated concentrations of EP (0, 10, 20 and 30 mM) were seeded in the upper chamber of transwell migration chambers (8 μm BioCoat Control Inserts, Becton Dickinson Labware, Bedford, MA, USA). A total of 600 μL DMEM containing 10% FBS was added into the lower chamber. After 24 h, the cells in the upper chamber were fixed by 4% paraformaldehyde for 20 min at room temperature, stained with 1% crystal violet for 15 min, and photographed in three independent 20× fields for each well. Transwell invasion assay was similarly performed with transwell upper chamber pre-coated with Matrigel. All experiments were performed in triplicate.

Transwell cell migration assays were performed on 12 well plates with 8-µm BioCoat control inserts (Becton-Dickinson, Franklin Lakes, NJ, USA). In total, 1–2×10⁵ U251 or U87 cells transfected with IF1 or NT shRNA were suspended in 500 µl serum-free DMEM and then seeded in the upper well of a Transwell chamber. DMEM (750 µl) supplemented with 10% FBS was added to the lower well. Subsequent to completion, the membranes were removed, the cells on the side facing the upper well were wiped with a cotton swab, and the adherent cells on the undersurface of the insert were stained using crystal violet. At least six representative images of each well were captured and the number of cells were counted using ImageJ software. The BioCoat Matrigel invasion chamber (Becton Dickinson Labware) was used for Transwell cell invasion assays and the following protocols were the same as the Transwell cell migration assays. The experiments were performed in triplicate (15 (link)).

To assess cell migration in vitro, MCF-7 cells (1 × 10⁵ cells in 100 μl DMEM supplemented with 1% FBS) were placed in the top chamber of transwell migration chambers (8 μm BioCoat Control Inserts, Becton Dickinson Labware, Bedford, MA). The lower chamber was filled with 600 μl DMEM supplemented with 10% FBS. After 24 h, unmigrated cells were removed from the upper surface of the transwell membrane with a cotton swab, and migrated cells on the lower membrane surface were fixed, stained, photographed, and counted under high-power magnification.