Transwell permeable polyester membrane inserts

Before plating, all transwell chambers were incubated at 37 ^oC with 5% CO₂ for 30 minutes with unsupplemented medium to acclimate. Cells were then trypsinized, washed, and switched to serum-free medium. For the migration assays, 5×10⁴ cells DU145 (shControl and PLEC-KD) and C4–2 (shControl, PLEC-KD) were plated in unsupplemented RPMI medium in the upper chambers of 24-well transwell inserts (Transwell™ Permeable Polyester Membrane Inserts from Corning Inc.) with 8 μm-pore size membranes. For invasion assays, 1×10⁵ cells were plated in unsupplemented RPMI medium in 24-well Matrigel-coated Boyden chamber transwell inserts (Corning Inc.) with 8 μm-pore size membranes. FBS-supplemented medium was added to the bottom of each transwell. For migration and invasion, chambers were incubated for 20 hours, fixed with ice cold 100% methanol for 20 minutes, and then stained with 0.1% Crystal Violet for 20 minutes at room temperature. Wells were then washed in a room temperature water bath for 10 minutes and air dried overnight. Wells were imaged on a stereomicroscope at 80X, three images per well and two wells per condition. Cell number represents the average of three well images in duplicate ± SD. Experiments were performed in triplicate. Representative images are presented.

48 hours prior seeding into 24-well transwell inserts, pore size 8.0 um, 6.5 mm diameter (Transwell^™ Permeable Polyester Membrane Inserts, Corning Inc, Corning, New York), cells were treated with erastin (1.25 μM), (1S,3R)-RSL3 (0.125 μM) or an equal volume of DMSO as a control. 5×10⁴ cells were then seeded in serum-free media in 24-well transwell inserts. The inserts were incubated in 10% FBS supplemented media in 24-well plates for 20 hours. The bottom chamber was filled with 10% FBS supplemented media containing erastin (1.25 μM), (1S,3R)-RSL3 (0.125 μM) or an equal volume of DMSO as a vehicle control. Cells that passed through the membrane were fixed and stained with 0.01% crystal violet solution. For migration assays with higher concentrations of compounds, cells were seeded in serum-free medium including erastin (5 μM), or (1S,3R)-RSL3 (0.5 μM) in 24-well transwell inserts. The inserts were then incubated in 10% FBS supplemented medium including erastin (5 μM), or (1S,3R)-RSL3 (0.5 μM) in 24-well plates for 20 hours.