Coolpix digital camera

Confluent MCF-7 cell monolayers were serum-starved overnight, scratched with a p-200 micropipette tip, and treated with 100 ng/ml epidermal growth factor (EGF) for 16 hours. For live imaging, phase micrographs were taken with a Nikon TMS phase Microscope equipped with a Nikon Coolpix digital camera (Nikon, Mississauga, ON, Canada) to capture the migratory infilling of the scratch/wound. Wound areas were quantified using CellSens Software (Olympus, Richmond Hill, ON, Canada).
In some experiments the scratched monolayers were treated with either dimethylsulfoxide (DMSO; vehicle control) or the ezrin inhibitor NSC668394 (Millipore, Etobicoke, ON, Canada [39 (link)]) diluted to 10 μM in DMSO.

Human breast cancer cell lines, MDA-MB-231, SUM149, SUM159, BT-474, T-47D, and MCF-7, were obtained from the American Type Culture Collection (ATCC, MD, USA). Cells were stably transfected with a construct containing cDNA of tdTomato as outlined in the Supplementary Materials and Methods under ‘Cell lines’.^{27 (link)} Human mammary fibroblasts were a kind gift from Dr Gary Luker at the University of Michigan. All cells were incubated at 37 °C with 5% CO₂ in a humidified incubator.
Descriptions of cell-specific culture media are given in Supplementary Materials and Methods under ‘Cell type specific media used for breast cancer cell lines’. tdTomato protein expression was detected by fluorescence microscopy using a ×20 objective attached to a Nikon inverted microscope, equipped with a filter set for 528 to 553 nm excitation and 600 to 660 nm emission and a Nikon COOLPIX digital camera (Nikon Instruments, Inc, Melville, NY, USA).^{27 (link)}

A collagen gel contraction assay was performed as described previously41 (link) with minor modifications. We used this assay to examine the inhibitory effect of Avagacestat on the contractile activity of LX2 cells. A collagen suspension (5 ml) containing 3.0 ml Collagen G1 (5 mg/ml, Matrix biosciences, Morlenbach, Germany), 0.5 ml 10x M199 medium (Sigma), 85 ul 1N NaOH (Sigma) and sterile water was mixed with 1.0 ml (2 × 10⁶ cells) human LX2 cells. Collagen gel-cells suspension (0.6 ml/well) was plated in a 24‐well culture plate and allowed to polymerize for 1 h at 37 °C. Once polymerized, 1 ml of 0.5% serum containing medium was added with or without TGFβ (5 ng/ml) together with 10 μM Avagacestat or PBS followed by detachment of the gels from the culture wells. Digital images were made at 0, 24, 48 and 72 h using a Nikon Coolpix digital camera (Nikon, Mississauga, ON, Canada). Measurement of collagen gel diameter at the indicated time points was performed using Image J imaging software (NIH, Bethesda, MD). Gels were measured by tracing around the edges of the gel disk and normalized with their respective well size in each image. Gel contraction experiments were performed in duplicates in three independent experiments.

Matrigel precoated Millicell culture inserts (8.0 µm pores, 10 mm diameter) contained in 24-well plates were used to assess chemotaxis. Before the experiment, inserts were washed twice with DMEM and rehydrated for 30 min in DMEM. An aliquot of Matrigel (1 : 10 in DMEM without serum) was added to the inserts. The insert with Matrigel was incubated at 37°C for 1 h to enable polymerization. A suspension of 5 × 10⁴ MDA-MB-231 cells was homogenously added in the upper chamber whereas the lower chamber was filled with DMEM containing 10% FBS. Cells were incubated for 48 h in the presence of AVME (10 and 20 μM) or DMSO (control) to allow them to migrate. Afterward, the upper surface of the transwell membrane was wiped gently with a cotton swab to remove nonmigrating cells and fixed with 5% glutaraldehyde followed by staining with 0.1% crystal violet solution for 10 min. Images were captured with a Nikon COOLPIX digital camera. A minimum of five fields per insert was counted by image editor software (ImageJ®). Each experiment was performed twice, and the average of cells/field was calculated. The number of untreated cells that invaded was considered to be 100%, and the percentage of cells treated with AVME was calculated based on the number of cells in the control group.

Human kidney cancer cell line HEK293T was obtained from the American Type Culture Collection (ATCC). These cells were grown in DMEM supplemented with 10% v/v fetal bovine serum (FBS). HEK293T cells were seeded into 6-well, 24-well plates or 35mm-dishes coated with fibronectin, collagen and bovine serum albumin (respectively 1 μg/ml, 30 μg/ml and 10 μg/ml in coating medium) [41 (link),42 (link),43 (link)]. Primary human pulmonary arterial endothelial cells (HPAEC) were purchased from Clonetics (San Diego, CA). These were seeded into T-25, T-75 or 6-well plates coated with fibronectin, collagen and bovine serum albumin (respectively 1 μg/ml, 30 μg/ml and 10 μg/ml in coating medium) [41 (link),42 (link),43 (link)]. HPAECs were grown in Medium 200 supplemented with low serum growth supplement LSGS (Cascade Biologics, Carlsbad, CA) and were used between passages 4 and 10 [44 (link)]. The human endothelial cell line EA.hy926 was a gift from Dr. Yang-Ming Yang (Dept. Cell Biology & Anatomy, New York Medical College) and grown in DMEM supplemented with 10% v/v fetal bovine serum and 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine [31 (link)]. Phase contrast microscopy was carried out daily and at the conclusion of each experiment using a Nikon Diaphot Microscope and a Nikon Coolpix digital camera.