3.5 cm petri dish

We collected cells (coaA/flamindo2, coaA/pinkflamindo, or the 4-color strain) from their HL5 growth media, washed and resuspended them in PDF buffer (1.5 g KCl, 1.6 g K₂HPO₄, 1.8 g KH₂PO₄ in 1 l water; pH 6.4; autoclaved, then added 1 ml 1 M CaCl₂ and 2.5 ml 1 M MgSO₄), and counted them using a hemocytometer. We placed 5 × 10⁶ cells in PDF onto a surface of KK2 (2.2 g KH₂PO₄ and 0.7 g K₂HPO₄ per 1 l) + 2% Noble agar (Difco, Franklin Lakes, NJ, USA) in a 3.5 cm Petri dish (Corning, NY, USA). We allowed the cells to settle on the agar surface for >10 min, then wicked away the PDF solution. We used a razor blade to cut out a ∼1 cm × ∼1 cm square of agar and flipped it onto a large circular glass coverslip (MatTek, Ashland, MA, USA) to sandwich the cells between the agar and glass as a near monolayer to visualize the cells with minimal vertical cell stacking.

A375 melanoma cells were seeded in six well plates or 3.5 cm petri dish (Corning) at a density of 125,000 cells/well. The following day, the cells were transfected with siRNA; after 48 h 100,000 A375 cells were added to each chamber of an ibidi wound insert in a 3.5 cm petri dish (Ibidi). The outside of the insert was filled with 1.5 ml of DMEM 10 % FBS. In parallel, cells were also seeded in duplicate to assess knockdown efficiency by immunobloting. The next day, the insert was removed to generate the wound and the plate was gently washed with 10 % FBS DMEM to remove any floating cells. Wound closure was monitored for 48 h by live imaging on a Zeiss Axiovert 200 microscope (10x objective, phase 1) in a controlled environment (5 % CO2, 37 °C). The percent wound closure was calculated by measuring the distance of the gap at three points using Northern Eclipse Software (NES, Empix Imaging, Mississauga, Ontario, Canada).

C2C12 (Mouse myoblasts) cells were incubated with BMP-2 peptides, BMP-2 protein (positive control) peptides with BMP-2 protein, and cells without any treatment were taken as a control for six days in a 3.5 cm Petri dish (Corning, Oneont, NY, USA) with cell number 3 × 10⁵ cells. The medium was changed every three days with BMP-2 protein and peptides. The cells were harvested, re-suspended in lysis buffer containing protease inhibitors (ab65621, abcam, Cambridge, UK), and sonicated to obtain cell lysate. For Western blot experiments, 20 micrograms of each treated and untreated cell lysates were resolved on SDS-PAGE. RUNX2 (runt-related transcription factor) was detected by the antibodies from Abcam (ab23981, abcam, Cambridge, UK). RUNX2 was performed 3 times.