Figure S3.
Role of SH2 domains of Shp2 in mediating ERK activation and ROCK II-dependent cell migration. (A) Analysis of Western blot intensity for ERK activation following RapR-Shp2 stimulation (Fig. 7 A). All phospho-ERK (pT202/pY204) signals are normalized to total ERK blot intensity and to the signal for nontransfected cells not treated with rapamycin. Statistical significance was determined using two-tailed Student’s t test comparing independent SH2 domain mutants with and without rapamycin. (B–E) HeLa cells transiently expressing R32L or R138L RapR-Shp2 and mCherry-FRB were stained with CellMask Deep Red membrane marker and imaged live, data from Fig 7. CellMask images were used for analysis of morphological changes. (B–D) Protrusive (B) and Retraction (C) activity of HeLa cells expressing R32L RapR-Shp2 (rose), R138L RapR-Shp2 (cyan), or (D) R138L RapR-Shp2 treated with 10 µM Y-27632 (teal). Bar graph shows activity binned over 10 min intervals. (B and D) There were significant differences in protrusive activity means for R32L RapR-Shp2 expressing cells (rose, F[23,1] = 3.74, P < 0.001), R138L RapR-Shp2 expressing cells (cyan, F[23,1] = 17.67, P < 0.001), and R138L RapR-Shp2 pretreated with Y-27632 (teal, F[23,1] = 6.75, P < 0.001). (C and D) There were significant differences in retraction activity means for R32L RapR-Shp2 expressing cells (rose, F[23,1] = 3.04, P < 0.001), R138L RapR-Shp2 expressing cells (cyan, F[23,1] = 21.81, P < 0.001), and R138L RapR-Shp2 pretreated with Y-27632 (teal, F[23,1] = 12.24, P < 0.001). (E) Changes in cell migration induced by activation of R138L RapR-Shp2 in the absence (cyan, data from Fig. 7 D) and presence (pink, F[3,1] = 19.92, P < 0.001) of 10 nM trametinib. Box plot indicates path length over 1 h. (F) Cell area change in response to R138L RapR-Shp2 activation following treatment with trametinib (F[3,1] = 1.27, P = 0.295), binned over 1 h intervals. (G) Representative immunoblot of HeLa cells pretreated with 10 nM Trametinib or 10 µM Y-27632 for 1 h and activated with EGF for 5 min. Immunoblot of ERK 1/2 (pT202/pY204), total ERK 1/2, Myosin Light Chain 2 (pT18/pS19), and total Myosin Light Chain 2. (right) Quantification of signal intensities for phospho-ERK normalized to total ERK (F = 22.66, P = 0.002) and phospho-Myosin Light Chain 2 normalized to GAPDH (F = 2.77, P = 0.14). N = 3 independent biological replicates. Analysis of statistical significance at the 0.05 level was determined using one-way ANOVA with Holm-Bonferroni correction for pairwise comparisons. Error bars in A–D and G show 90% CI. (B–F) Analysis of statistical significance at the 0.05 level was determined using one-way repeated measures ANOVA with Dunnett’s correction post hoc relative to basal (−1 h, −60 to 0 min, 0 min). *P < 0.05, **P < 0.01, ***P < 0.001. Box and whisker plots in E and F: Box indicates first through third quartile with median line, whiskers are 10–90%, and open square represents mean.

Role of SH2 domains of Shp2 in mediating ERK activation and ROCK II-dependent cell migration. (A) Analysis of Western blot intensity for ERK activation following RapR-Shp2 stimulation (Fig. 7 A). All phospho-ERK (pT202/pY204) signals are normalized to total ERK blot intensity and to the signal for nontransfected cells not treated with rapamycin. Statistical significance was determined using two-tailed Student’s t test comparing independent SH2 domain mutants with and without rapamycin. (B–E) HeLa cells transiently expressing R32L or R138L RapR-Shp2 and mCherry-FRB were stained with CellMask Deep Red membrane marker and imaged live, data from Fig 7. CellMask images were used for analysis of morphological changes. (B–D) Protrusive (B) and Retraction (C) activity of HeLa cells expressing R32L RapR-Shp2 (rose), R138L RapR-Shp2 (cyan), or (D) R138L RapR-Shp2 treated with 10 µM Y-27632 (teal). Bar graph shows activity binned over 10 min intervals. (B and D) There were significant differences in protrusive activity means for R32L RapR-Shp2 expressing cells (rose, F[23,1] = 3.74, P < 0.001), R138L RapR-Shp2 expressing cells (cyan, F[23,1] = 17.67, P < 0.001), and R138L RapR-Shp2 pretreated with Y-27632 (teal, F[23,1] = 6.75, P < 0.001). (C and D) There were significant differences in retraction activity means for R32L RapR-Shp2 expressing cells (rose, F[23,1] = 3.04, P < 0.001), R138L RapR-Shp2 expressing cells (cyan, F[23,1] = 21.81, P < 0.001), and R138L RapR-Shp2 pretreated with Y-27632 (teal, F[23,1] = 12.24, P < 0.001). (E) Changes in cell migration induced by activation of R138L RapR-Shp2 in the absence (cyan, data from Fig. 7 D) and presence (pink, F[3,1] = 19.92, P < 0.001) of 10 nM trametinib. Box plot indicates path length over 1 h. (F) Cell area change in response to R138L RapR-Shp2 activation following treatment with trametinib (F[3,1] = 1.27, P = 0.295), binned over 1 h intervals. (G) Representative immunoblot of HeLa cells pretreated with 10 nM Trametinib or 10 µM Y-27632 for 1 h and activated with EGF for 5 min. Immunoblot of ERK 1/2 (pT202/pY204), total ERK 1/2, Myosin Light Chain 2 (pT18/pS19), and total Myosin Light Chain 2. (right) Quantification of signal intensities for phospho-ERK normalized to total ERK (F = 22.66, P = 0.002) and phospho-Myosin Light Chain 2 normalized to GAPDH (F = 2.77, P = 0.14). N = 3 independent biological replicates. Analysis of statistical significance at the 0.05 level was determined using one-way ANOVA with Holm-Bonferroni correction for pairwise comparisons. Error bars in A–D and G show 90% CI. (B–F) Analysis of statistical significance at the 0.05 level was determined using one-way repeated measures ANOVA with Dunnett’s correction post hoc relative to basal (−1 h, −60 to 0 min, 0 min). *P < 0.05, **P < 0.01, ***P < 0.001. Box and whisker plots in E and F: Box indicates first through third quartile with median line, whiskers are 10–90%, and open square represents mean.

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