Comprehensive GEF screen. (A) Flowchart of screen. WB, Western blot. (B) Histogram of knockdown efficiency of all hairpins used in the screen. Freq., frequency. Only hairpins with depletion greater than 50% were considered for the screen (red). N (total number of hairpins) = 125. (C) Histogram of wound-healing rate. Mean ± SD = 42.2 ± 8.8 µm h−1. (D) Boxplots of z-scores for off-target controls (green, knockdown efficiency = 0%, n = 24), positive controls (Pos Cntl; red, CDC42, RAC1, and β-PIX, n = 21), and the screened GEFs (blue). Box represents 25%–75%, whiskers 5%–95% of the data, assuming a normal distribution. Z-scores were calculated relative to the off-target control. (E) Summary of GEFs identified as hits in the screen and their phenotypes, PC3 in speed or directionality signifies long-range communication. β-PIX was a GEF with known effects on collective migration and used as a positive control (cyan). Magenta labels hits that were not followed up. Black cross indicates that these phenotypes were excluded (see Fig. S2 for details). (F and G) Visualization of measures that were altered by the hits identified in the screen and that were validated. Shown are z-scores for each well. Positive controls (red): CDC42 (circle), RAC1 (cross), and β-PIX (diamond), off-target controls (knockdown = 0, green), GEFs that were screened (blue) but not identified (circle) or validated (cross) as hits, and validated hits. (F) Wound-healing rate. (G) PC1 (top) and PC3 (bottom).
