Matrigel

The transwell cell migration assay measures the capability of cells to migrate through the transwell membrane, whereas the transwell cell invasion assay measures the invasion of cells through the extracellular matrix and transwell membrane. Cells marked with the cell-permeant dye calcein-AM (Sigma-Aldrich, St. Louis, MO, USA) were diluted in serum-free culture medium and plated on top of the filter membrane in a transwell with 8 µm pore size (FluoroBlock™, Corning, Corning, NY, USA) placed in a 24-well plate. Culture medium containing 10% FBS was added to the bottom chamber and plates were incubated at 37 °C and 5% CO₂ to allow the cells to settle down. For invasion assays, Matrigel^® (R&D Systems, Minneapolis, MN, USA) was loaded on top of the transwell membrane and cells were added on top of the Matrigel coating to simulate invasion through the extracellular matrix. The fluorescence was measured at the bottom of the plate at 8 h (migration assays), and at 24 h for (invasion assays), and then normalized against the control condition.

The migration and invasion of tumor cells were analyzed in 24-well Boyden chambers with 8-μm pore size polycarbonate membranes (Corning, NY, USA). For invasion assays, the membranes were coated with Matrigel (3432-005-01, R&D Systems) to form matrix barriers. Briefly, SK-HEP-1 or SNU-449 cells in serum-free DMEM or RPMI were placed into the upper chamber of 24-well Boyden chamber coated without or with Matrigel (R&D Systems), while the lower chamber was filled with 600 μl 10% FBS-containing DMEM/RPMI. After 10 h of incubation, cells were fixed and stained with crystal violet. All the migrated/invaded cells were counted.

Vascular mimicry assay was completed as described previously [32 (link)]. Briefly, 100 µl chilled Matrigel (Corning or R&D) was added to a chilled 48-well plate and incubated for at least 30 min at 37 °C to allow Matrigel polymerization. 30,000 melanoma cells in 300 µL were seeded dropwise to each well in low serum media apart from the YUMM lines being seeded in full media. In experiments including YAP/TAZ inhibitors, drugs were added to the mixture of cells and media prior to adding dropwise to the well. Vascular mimicry was evaluated by microscopy at 6 h. Multiple images per experimental group were taken and each experiment was repeated a minimum of three times. The number of complete meshes were manually quantified, and the number of junctions and tube length were quantified using the WimTube application from Wimasis [52 ].

Ninety-six-well plates were coated with 50 μl Matrigel (R&D Systems, catalog number 3432-005-01) and incubated at 37 °C for 30 min. Control (1 × 10⁴) and CRISPR/Cas9-edited HUVECs in 100 μl EGM medium were seeded in each well, respectively. After 4 h, images were captured using the Leica epi-fluorescence microscope. Branches number and length were quantified using ImageJ software.

Transwell inserts (8 μm pore size; Corning) coated with Matrigel (R&D Systems, Minneapolis, MN, USA) were used for measuring cell invasion. The lower chamber of the transwell plates were filled with 750 μL medium with 20% FBS and the upper chamber was filled with 200 μL cells suspended with medium without FBS. The cells on the lower surface of Transwell inserts were fixed and then stained by crystal violet. Image pro‐plus (Media Cybernetics, Inc., Bethesda, MD, USA) was used for counting the cell numbers. Cells in three different wells were averaged.

SD rat embryonic (E) 18-day-old rat spinal cord tissue (thoracolumbar spinal cord segment) was collected for spinal cord slice culture. After cutting the spinal cord tissue into thin slices with microscissors, tissues were placed on a coverslip precoated with Matrigel (R&D, USA). The coverslips were placed into a 24-well plate, 200 μl of culture medium (DF12+10% FBS) was then added, and the plate was placed in an incubator to culture for 4 hours; subsequently, 300 μl of medium (DF12+10% FBS) was added, and the cells were cultured for another 7 days, with the medium being changed every other day. After 7 days of culture, the growth of neurites was observed. If the growth of the neurites was good, the neurites were removed along the spinal cord slices with a blade under a microscope, and the in vitro culture was continued for 7-10 days to observe the regeneration of neurites after in vitro neuronal injury. The measurement of neurites was performed using 3 slices per group, and 10 fields of view (200x magnification) were observed under a microscope (Olympus, Japan) for each slice. The lengths of neurites in all images were measured and averaged. Comparisons between groups were performed. In the spinal cord slices, the lengths of all neurites were measured and compared between groups.