Figure 5.
Transient cell spreading and migration of HeLa cells induced by RapR-Shp2 activation. HeLa cells transiently expressing mCherry-ipep-FRB alone (control, N = 22 cells, two separate biological replicates) or coexpressed with RapR-Shp2-mVenus-flag (N = 26 cells, six separate biological replicates) were stained with CellMask Deep Red membrane marker and imaged live. Images of CellMask were used to assess morphological changes. Rapamycin was added at 0 min (gray line in A, B, and D). (A and B) Analysis of changes in cell area (A) and Protrusive Activity (B) for cells expressing RapR-Shp2 and FRB (black line) or FRB alone (brown line). Shaded area represents 90% CI. (C) Change in cell area induced by activation of Shp2 (gray) compared to FRB control (brown) binned over 1 h intervals. RapR-Shp2 caused significant change in spreading (F[3,1] = 12.69, P < 0.001) and significant induction over FRB control (F = 8.80, P < 0.001). FRB control cells exhibited no significant change in cell area over time (F[3,1] = 1.19, P = 0.32). (D) Analysis of retraction activity for cells expressing RapR-Shp2 and FRB (black line) or FRB alone (brown line). Shaded area represents 90% CI. (E) Change in cell migration induced by activation of RapR-Shp2 (gray) compared to FRB alone control (brown) (shown as path length over 1 h intervals). RapR-Shp2 caused significant change in migration relative to basal at all time points (F[3,1] = 7.50, P < 0.001) and significant induction over FRB control (F = 28.03, P < 0.001). FRB control cells exhibited no significant change in cell migration over time (F[3,1] = 0.08, P = 0.97). (C and E) Box and whisker plots: Box indicates first through third quartile with median line, whiskers are 10–90%, open square represents mean. Analysis of statistical significance at the 0.05 level was determined using one-way repeated measures ANOVA with Dunnett’s correction relative to the basal (−60 to 0) time-frame post hoc. To evaluate the effect of RapR-Shp2 versus FRB control two-way repeated measures ANOVA with Holm-Bonferroni correction was performed for pairwise analysis of each time point for RapR-Shp2 and FRB control. *P < 0.05, **P < 0.01, ***P < 0.001.

Transient cell spreading and migration of HeLa cells induced by RapR-Shp2 activation. HeLa cells transiently expressing mCherry-ipep-FRB alone (control, N = 22 cells, two separate biological replicates) or coexpressed with RapR-Shp2-mVenus-flag (N = 26 cells, six separate biological replicates) were stained with CellMask Deep Red membrane marker and imaged live. Images of CellMask were used to assess morphological changes. Rapamycin was added at 0 min (gray line in A, B, and D). (A and B) Analysis of changes in cell area (A) and Protrusive Activity (B) for cells expressing RapR-Shp2 and FRB (black line) or FRB alone (brown line). Shaded area represents 90% CI. (C) Change in cell area induced by activation of Shp2 (gray) compared to FRB control (brown) binned over 1 h intervals. RapR-Shp2 caused significant change in spreading (F[3,1] = 12.69, P < 0.001) and significant induction over FRB control (F = 8.80, P < 0.001). FRB control cells exhibited no significant change in cell area over time (F[3,1] = 1.19, P = 0.32). (D) Analysis of retraction activity for cells expressing RapR-Shp2 and FRB (black line) or FRB alone (brown line). Shaded area represents 90% CI. (E) Change in cell migration induced by activation of RapR-Shp2 (gray) compared to FRB alone control (brown) (shown as path length over 1 h intervals). RapR-Shp2 caused significant change in migration relative to basal at all time points (F[3,1] = 7.50, P < 0.001) and significant induction over FRB control (F = 28.03, P < 0.001). FRB control cells exhibited no significant change in cell migration over time (F[3,1] = 0.08, P = 0.97). (C and E) Box and whisker plots: Box indicates first through third quartile with median line, whiskers are 10–90%, open square represents mean. Analysis of statistical significance at the 0.05 level was determined using one-way repeated measures ANOVA with Dunnett’s correction relative to the basal (−60 to 0) time-frame post hoc. To evaluate the effect of RapR-Shp2 versus FRB control two-way repeated measures ANOVA with Holm-Bonferroni correction was performed for pairwise analysis of each time point for RapR-Shp2 and FRB control. *P < 0.05, **P < 0.01, ***P < 0.001.

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