Figure 6.
Role of ERK activation in mediating RapR-Shp2-induced changes in cell morphology. HeLa cells transiently coexpressing RapR-Shp2-mVenus-flag and mCherry-ipep-FRB were stained with CellMask Deep Red membrane marker and treated with 10 nM Trametinib (N = 39 cells, four separate biological replicates) or 15 μM FR180204 ERK1/2 inhibitor (N = 24 cells, three independent biological replicates) for 1 h prior to imaging. Rapamycin was added at 0 min (gray line in A and D). CellMask Deep Red images were used for analysis. Data for RapR-Shp2 from Fig. 4 included for comparison (gray). (A and B) Analysis of changes in cell area for trametinib (mustard) or FR180204 (mint) treated cells. (A) Shaded area represents 90% CI. (B) Box and whisker plots of relative cell area change binned over 1 h intervals for FR180204-treated cells (mint, F[3,1] = 0.48, P = 0.700) and for Trametinib-treated cells (mustard, F[3,1] = 1.88, P = 0.138). Both inhibitors cause significant reduction in cell spreading versus RapR-Shp2: Trametinib (F[3,1] = 10.79, P < 0.001), FR180204 (F[3,1] = 8.30, P < 0.001). (C) Changes in cell migration (determined as path length over 1 h intervals) for FR180204-treated cells (mint, F[3,1] = 10.68, P < 0.001) and for Trametinib-treated cells (mustard, F[3,1] = 6.05, P < 0.001). (D–F) Analysis of the protrusive and retraction activity (D) for trametinib (mustard), FR180204 (mint), and nontreated cells (data from Fig. 4, B and D in gray). Shaded area represents 90% CI. (E and F) Bar graph of Protrusive (E) and Retraction (F) activity binned over 10 min intervals. Error bars show 90% CI. (E) There were significant differences in protrusive activity means for FR180204-treated cells (mint, F[23,1] = 9.53, P < 0.001) at time points 10–180 min and for Trametinib-treated cells (mustard, F[23,1] = 10.91, P < 0.001) at time points 20–180 min. (F) There were significant differences in retraction activity means for FR180204-treated cells (mint, F[23,1] = 12.39, P < 0.001) at time points 30–180 min and for Trametinib-treated cells (mustard, F[23,1] = 14.33, P < 0.001) at time points 40–180 min. (B, C, E, and F) 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 min, −1 h, or 0 min) time-frame post hoc. (B and C) Box and whisker plots: Box indicates first through third quartile with median line, whiskers are 10–90%, open square represents mean. To evaluate the effect of RapR-Shp2 versus each inhibitor two-way repeated measures ANOVA with Holm-Bonferroni correction was performed for pairwise analysis of each time point for RapR-Shp2 and each inhibitor. *P < 0.05, **P < 0.01, ***P < 0.001.

Role of ERK activation in mediating RapR-Shp2-induced changes in cell morphology. HeLa cells transiently coexpressing RapR-Shp2-mVenus-flag and mCherry-ipep-FRB were stained with CellMask Deep Red membrane marker and treated with 10 nM Trametinib (N = 39 cells, four separate biological replicates) or 15 μM FR180204 ERK1/2 inhibitor (N = 24 cells, three independent biological replicates) for 1 h prior to imaging. Rapamycin was added at 0 min (gray line in A and D). CellMask Deep Red images were used for analysis. Data for RapR-Shp2 from Fig. 4 included for comparison (gray). (A and B) Analysis of changes in cell area for trametinib (mustard) or FR180204 (mint) treated cells. (A) Shaded area represents 90% CI. (B) Box and whisker plots of relative cell area change binned over 1 h intervals for FR180204-treated cells (mint, F[3,1] = 0.48, P = 0.700) and for Trametinib-treated cells (mustard, F[3,1] = 1.88, P = 0.138). Both inhibitors cause significant reduction in cell spreading versus RapR-Shp2: Trametinib (F[3,1] = 10.79, P < 0.001), FR180204 (F[3,1] = 8.30, P < 0.001). (C) Changes in cell migration (determined as path length over 1 h intervals) for FR180204-treated cells (mint, F[3,1] = 10.68, P < 0.001) and for Trametinib-treated cells (mustard, F[3,1] = 6.05, P < 0.001). (D–F) Analysis of the protrusive and retraction activity (D) for trametinib (mustard), FR180204 (mint), and nontreated cells (data from Fig. 4, B and D in gray). Shaded area represents 90% CI. (E and F) Bar graph of Protrusive (E) and Retraction (F) activity binned over 10 min intervals. Error bars show 90% CI. (E) There were significant differences in protrusive activity means for FR180204-treated cells (mint, F[23,1] = 9.53, P < 0.001) at time points 10–180 min and for Trametinib-treated cells (mustard, F[23,1] = 10.91, P < 0.001) at time points 20–180 min. (F) There were significant differences in retraction activity means for FR180204-treated cells (mint, F[23,1] = 12.39, P < 0.001) at time points 30–180 min and for Trametinib-treated cells (mustard, F[23,1] = 14.33, P < 0.001) at time points 40–180 min. (B, C, E, and F) 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 min, −1 h, or 0 min) time-frame post hoc. (B and C) Box and whisker plots: Box indicates first through third quartile with median line, whiskers are 10–90%, open square represents mean. To evaluate the effect of RapR-Shp2 versus each inhibitor two-way repeated measures ANOVA with Holm-Bonferroni correction was performed for pairwise analysis of each time point for RapR-Shp2 and each inhibitor. *P < 0.05, **P < 0.01, ***P < 0.001.

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