24 well insert

For the transwell migration assay, 2 × 10⁴/200μl H446 or H2227 cells were plated in the top chamber with the non-coated membrane (24-well insert, pore size: 8 μm, BD Biosciences). For the invasion assay, 5 × 10⁴/200μlH446or H2227 cells were plated in the top chamber with extracellular matrix gel(Sigma, E1270)-coated membranes (24-well insert, pore size: 8 μm, BD Biosciences). In both assays, the cells were plated in medium without serum, and medium with 15% serum was used as a chemoattractant in the lower chamber. The cells were incubated at 37°C for 24 hours, and cells that did not migrate through or invade the pores were removed with a cotton swab. Cells on the lower surface of the membrane were fixed with methanol and stained with0.1% crystal violet (Sigma). Cells in 9 random fields of view at100xmagnification were counted and expressed as the average number of cells per field of view. Pictures were taken with an inverted phase-contrast microscope at 100x magnification (Olympus,CKX41).

The migration assay was performed on MDA-MB-231 cells when they reached greater than 90% confluence. Cells were incubated for 24 h with freshly prepared Leibovitz’s L-15 medium containing either vehicle alone or ODZ10117, followed by scratching with pipette tips and washing with PBS. The images were obtained using the LAS microscope software.
The invasion assay was performed using a Boyden chamber system containing growth factor reduced Matrigel diluted with serum-free media at a ratio of 1:3. Diluted Matrigel was transferred into a 24-transwell support (BD 24-well insert, 8 μm pore transparent PET filter) and incubated at 37 °C for 4–5 h for gelling. MDA-MB-231 cells in 100 μL Leibovitz’s L-15 medium containing 1% FBS were seeded in the upper chamber and incubated for 24 h in the presence of either vehicle alone or ODZ10117. The lower chamber was filled with 500 μL of 10% Leibovitz’s L-15 medium containing 5 μg/mL fibronectin. The Matrigel-containing upper chamber was rinsed with PBS, fixed, stained with Diff-Quik solution, and subsequently rinsed with distilled water. The migrated cells were captured using the LAS microscope software.

To conduct wound healing assay, cells were seeded into 12-well plates and then incubated over 90% confluence. The plate was scratched with pipette tips and washed with PBS. Cells were incubated for 24 h with fresh DMEM medium containing either vehicle alone or ODZ10117. Digital images were obtained using the Leica Application Suite (LAS) Microscope Software (Leica Microsystems).
Invasion assay was performed using a Boyden chamber system (Neuro Probe, Gaithersburg, MD, USA). Growth factor reduced Matrigel (354230, BD Matrigel™, BD Biosciences) was diluted with serum free media with ratio of 1:3. Diluted Matrigel was transferred into 24-transwell (BD 24-well insert, 8 μm pore transparent PET filter) and incubated at least for 4 to 5 h for gelling at 37 °C. Cells in 100 μL DMEM containing 1% FBS were seeded in the upper chamber and incubated for 24 h in the presence of either vehicle alone or ODZ10117. The lower chamber was filled with 500 μL of 10% DMEM containing fibronectin (5 μg/mL, ECM001, Sigma-Aldrich). Matrigel containing upper chamber was rinsed with PBS, fixed, stained with Diff-Quik solution (Sysmex Corporation, Kobe, Japan), and subsequently rinsed with distilled water. The migrated cells were captured using the LAS Microscope Software (Leica Microsystems).