The PI3K p110α isoform regulates endothelial adherens junctions via Pyk2 and Rac1

C. TNF-alpha stimulated p110alpha or control siRNA transfected HUVECs previously labelled with celltracker orange dye were grown on collagen matrices in transwell chambers. Celltracker green-labelled THP-1 cells were added to HUVECs, and TEM allowed to proceed toward an MCP-1 gradient in the lower chamber, for either 10 or 60 minutes before fixation. Fixed samples were stained with AlexaFluor 633- conjugated phalloidin to visualize F-actin (grey) and z-stacks collected. Threedimensional reconstructions were produced using Volocity software. A small gamma correction was applied to the red channel in order to visualise weak cell tracker staining at the edges of cell, which are difficult to distinguish upon 3D rendering