Image-based siRNA screen of Rho GEFs and GAPs

A list of all human proteins containing either a Dbl homology or DOCK homology domain (GEFs), as well as those containing a RhoGAP domain (GAPs), was generated by bioinformatic analysis of the UniProt database (http://www.uniprot.org). The Stealth siRNA library consisted of four separate oligonucleotides with nonoverlapping sequences per gene (Thermo Fisher Scientific and Technology Development Studio of the Max Planck Institute of Molecular Cell Biology and Genetics; see Table S1). Unique oligos were arrayed in individual wells of 96-well plates, each plate also including 4 scramble oligo wells, 3 ephrinB2-targeting wells, and 2 wells targeting both ephrinB1 and 2. The final concentration of oligonucleotides in each well was 14 nM. Each plate was assayed twice. The trans-endocytosis assay in SKN-H2B-RFP cells was performed as described in theTrans-endocytosis assay section and analysis with CellProfiler software is detailed in Fig. 2 A. A cutoff (X ≥ vesicles) set to contain 40% of cells from the mean scramble value for each individual plate was used to determine z-scores (see Fig. S5, A and B). Z-scores for each well were calculated at that same cutoff using the formula: (observed value − scramble mean)/scramble SD. The mean z-score per oligo was calculated and hits determined when either below −1.25 or above 1.75. Candidate genes were ranked by mean z-score for all four oligos.