Characterization of transient effects induced by Shp2 activation. (A and B) Relative Protrusive (A) and Retractive (B) area changes for RapR-Shp2 (gray) and FRB (brown) expressing cells binned over 10 min intervals, mean activity at each 2 min timepoint overlayed as a solid line. Error bars show 90% CI. Analysis of statistical significance at the 0.05 level was determined using one-way repeated measures ANOVA with Dunnett’s correction post hoc. (A) RapR-Shp2 expressing cells had significant differences in protrusive activity means (F[23,1] = 6.63, P < 0.001). There was significant difference in means for time points 20–170 min relative to the 0 min time point. FRB expressing cells did not show significant changes in protrusive activity at the 0.05 level (F[23,1] = 1.27, P = 0.184). (B) RapR-Shp2 expressing cells had significant differences in retraction activity means (F[23,1] = 9.40, P < 0.001). There was significant difference in means for time points 40–130 and 150–180 min relative to the 0 min time point. FRB expressing cells retraction activity was not significant at the 0.05 level (F[23,1] = 1.10, P = 0.337). (C and D) Analysis of morphological changes induced by CA-Shp2. HeLa cells transiently expressing CA-Shp2-mVenus were stained with CellMask Deep Red membrane marker, imaged live and analyzed as in Fig. 4. N = 30 cells, three independent biological replicates. Significance was determined via two-tailed Student’s t test with α = 0.05, n.s. is nonsignificant. (C) Cell area analysis binned over 1 h intervals. (D) Changes in cell migration determined by path length over 1 h intervals. Box and whisker plots: Box indicates first through third quartile with median line, whiskers are 10–90%, open square represents mean. (E) Representative images of RapR-Shp2-mVenus and mCherry-ipep-FRB after rapamycin addition or CA-Shp2-mVenus localization in HeLa cells.