Cell shape change in U2OS cells

U2OS control and SPECC1L-kd cells were cultured in standard DMEM high glucose media supplemented with 10% fetal bovine serum (Life technologies, Carlsbad, CA). For AJ change, 2 × 10⁵ cells were plated on 0.1% porcine gelatin treated glass (Sigma-Aldrich, St. Louis, MO), and observed for cell shape change. Cells were collected at various indicated timepoints: 4 hours post plating (t = 1), 24 hours post plating (t = 2), confluency without cell shape change (t = 3), cell shape change (t = 4), 24 hours following cell shape change (t = 5), and 48 hours following cell shape change (t = 6) (Figs. 1, ​,2,2, ​,3).3). To modulate the PI3K-AKT pathway, cells were cultured in described concentrations of PI3K-AKT inhibitor, Wortmannin (TOCRIS Biosciences, Minneapolis, MN), or activator, SC-79 (TOCRIS Biosciences, Minneapolis, MN). Media with chemicals was replaced daily.