Cell migration and invasion assays were done using Boyden chambers as described previously.41 (link) In brief, 2.5 × 104 cells were suspended in base medium devoid of growth factors and placed in each insert coated with or without Matrigel matrix (BD Biosciences). Cells were allowed to move toward full media filled in the bottom well for 24 hours and stained with 4 μg/ml Calcein AM dye (Invitrogen). All Boyden chamber assays were performed at least three times in triplicate.