Qcm ecmatrix cell invasion assay

Twenty-four hours after infection, cells were harvested and subjected to the following assays. For migration assays, infected cells (6 × 104) were plated in triplicate in the top chamber of Transwells (Millicell Hanging Cell Culture Inserts, PIEP12R48, Millipore Corporation) with a membrane containing 8-μm diameter pores in 300 μl serum-free RPMI1640. The inserts were then placed into the bottom chamber wells of a 24-well plate containing RPMI1640 with 20 % FBS as a chemo-attractant. After 24 h of incubation, cells remaining on the insert top layer were removed by cotton swab scrubbing, and cells on the lower surface of the membrane were fixed in 100 % methanol for 15 min, followed by staining with Giemsa solution. Cell numbers in five random fields (400×) were counted for each chamber and the average values were calculated.
For invasion assays, infected cells (1.5 × 105) were plated in the top chamber with Matrigel-coated membrane (QCM ECMatrix Cell Invasion Assay, Millipore Corporation), whereas the bottom chambers were filled with conditioned medium. After 48 h incubation, migrated cells (lower side of the membrane) were counted as described above.

For migration assays, infected GC cells (BGC-823 and SGC-7901; 1 × 10⁵) were plated in triplicate in the top chamber of Transwell plates (Millicell Hanging Cell Culture Inserts, PIEP12R48, Millipore Corporation, Burlington, MA, USA) with a membrane containing 8 μm diameter pores in 250 μl of serum-free RPMI1640. The inserts were then placed into the bottom chamber wells of a 24-well plate containing RPMI1640 with 20% FBS as a chemoattractant. After 24 h incubation, cells remaining on the insert top were removed with a cotton swab, while cells on the lower surface of the membrane were fixed in 100% methanol for 15 min, followed by staining with Giemsa solution. Cell numbers in five random fields (200x) were counted for each chamber to calculate average values.
For invasion assays, infected cells (1 × 10⁵) were plated in the top chamber of Matrigel-coated membranes (QCM ECMatrix Cell Invasion Assay, Millipore Corporation), whereas the bottom chambers were filled with conditioned medium. After 24 h incubation, migrated cells (lower side of the membrane) were counted as described above.

For wound-healing assay (Scratch assay), SkBr3 and MDA-MB-453 cells expressing the empty vector, MTSS1-GFP, SCAMP1-HA or both were cultured to 80% confluence and serum starved for 24 h, after which a scratch was made in the middle of each well using a 10 μl pipette tip. Images from triplicate experiments at 0, 24 and 48 h were taken and the distances between the edges of the scratch were measured at 3 different points using Carl Zeiss AxioVision software^{35 (link)} (Buczek et al., 2016). The measurements were expressed as percentages of gap closure. For the well-cell invasion assay, we used the colorimetric QCM ECMatrix Cell Invasion Assay (ECM550, Millipore, Ltd.) and the cell count of invasive cancer cells were performed following the manufacturer’s recommendations. For the proliferation assay, cells were cultured (10000 cells per/well) and then transfected for 24 h and 48 h. Media were removed and the proliferation was assessed using the CyQUANT® NF Cell Proliferation Assay Kit (C35007, Life Technologies, Ltd.) and following the manufacturer’s recommendations. Cell–cell adhesions were assessed following transfections (48 h) and using Vybrant™ Cell Adhesion Assay Kit (V13181, Fisher Scientific), and the manufacturer’s recommendations.