Qcapture 64

For wound healing assays, cells were grown on a 12‐well plate until 95% confluence in the regular medium. Prior to the experiment cells were serum starved for 3 h along with the addition of 1 µg/mL of mitomycin C to inhibit cell proliferation. All cells were then incubated in their respective media with 10% FBS and the monolayer was scratched using a 200 µL pipette tip. Finally, images were taken using an Olympus CKX41 microscope with QCapture ×64 software (Surrey, Canada) immediately after the scratch, marked as 0 h and after 24 h. For transwell migration assays, 25 000 DU145 cells were suspended in serum free media along with treatments indicated in figures and placed in transwell inserts with 8 µM pore membranes (Corning, New York, NY). In all experiments, lower wells contained MEM + 10% FBS and cells were allowed to migrate for 6 h. For pre‐treatment experiments, cells were pretreated with shTRPM7 for 48 h and/or TGFβ for 24 h. Cells on the inside of the transwell membrane were removed with a cotton swab and cells on the lower side of membrane were fixed with methanol and stained with hematoxylin. Four random fields at 10× were counted indicating total cells migrated.