An image-based genome-wide screen to unbiasedly identify Sema3E-PlexinD1 downstream signaling molecules. (A) Schematic illustration of the screen strategy. HUVECs endogenously express PlexinD1 and undergo cell collapse after Sema3E treatment. The RNAi screen identified genes that, when knocked down, block the Sema3E-induced cell collapse. (B) Schematic illustration of the automated screen procedure. Cells were transfected with smart pools of siRNA (four different sequence targets for each gene) for each well of the 384-well plates, and after 48 h Sema3E was added to the culture media for 25 min. Cells were fixed, stained, and imaged, and the cell collapse phenotype was quantified using the combination of CellProfiler and our custom-developed image analysis algorithm. (C) Automated imaging of a positive control well (PlexinD1 siRNA-transfected) and a negative control well (nontargeting siRNA-transfected) for each 384-well screening plate (images were generated from the DiI channel). HUVECs were transfected with nontargeting siRNA or PlexinD1 siRNA and treated with control ligand or Sema3E. Sema3E caused cell collapse resulting in decreased surface area and the appearance of protrusions (arrows). These cytoskeletal changes were completely blocked by PlexinD1 siRNA. Bar, 100 µm. (D) Quantification of cell collapse. Unlike cells treated with the control ligand, cells stimulated with Sema3E underwent cytoskeletal collapse, which was significantly blocked when PlexinD1 siRNA was used. Error bars indicate SD. *, P < 0.01. Control nontargeting siRNA did not alter the cellular response elicited by Sema3E.