Xm10 digital camera system

Cells were seeded in 6 well plate at a density of 7.5 × 10⁵ cell/well and allowed reach confluence at 37 °C, 5% CO₂. Then, a “wound” was made by manual scratching with a 200 µL pipette tip and cells were gently washed with 1× PBS. The “wounded” areas were photographed in specific wound sites (two sites for each wound) at 40× magnification using an Olympus IX51 inverted microscope equipped with an Olympus XM10 Digital Camera System every 24 h until wound closure. The relative migration distance (5 measures by wound) was calculated with the following formula: relative migration distance (%) = (A–B)/C × 100, where A is the width of cell wound at 0 h incubation, B is the width of cell wound after specific h of incubation, and C is the width mean of cell wound for 0 h of incubation. For relative migration distance, the results were analyzed using the beWound-Cell Migration Tool (Version 1.5) [50 ]. At least three independent experiments were performed.

For migration capacities, cells were seeded (6‐well plate) in complete DMEM at an optimal density to obtain at least 95% of confluence in the next 24 h and incubated at 37 °C, 5% CO₂. The procedure and the calculation formula were performed as previously described [34 ].
The wound areas were photographed in two specific sites at 40× magnification using an Olympus IX51 inverted microscope equipped with an Olympus XM10 Digital Camera System (Olympus, Tokyo, Japan). At least three independent experiments were performed.

UMUC3^KD and UMUC3^CTRL cells were seeded in 6-well plate at a density of 7.5x10⁵ cell/well and allowed reach confluence at 37ºC, 5% CO₂. Then, a “wound” was made by manual scratching with a 200 µL pipette tip and cells were gently washed with 1X PBS. The ''wounded'' areas were photographed in specific wound sites (two sites for each wound) at 40x magnification using an Olympus IX51 inverted microscope equipped with an Olympus XM10 Digital Camera System every 12h until wound closure. The relative migration distance (5 measures by wound) was calculated with the following formula: relative migration distance (%) = (A-B)/C x100, where A is the width of cell wound at 0h incubation, B is the width of cell wound after specific hours of incubation, and C is the width mean of cell wound for 0h of incubation. For relative migration distance, the results were analyzed using the beWound-Cell Migration Tool (Version 1.5) (developed by A.H.J. Moreira, S. Queirós and J.L. Vilaça, Biomedical Engineering Solutions Research Group, Life and Health Sciences Research Institute- University of Minho; available at http://www. besurg.com/sites/default/files/beWoundApp.zip). At least three independent experiments were performed.