Insulin-Like Growth Factor-I E-Peptide Activity Is Dependent on the IGF-I Receptor
Figure 6
EB increases in myoblast proliferation and migration are MAPK and IGF-IR signaling dependent.
A. C2C12 cells were plated in 96 well plates, starved for 6 hours, and treated with synthetic E-peptides. A BrdU plate assay was used to quantify proliferating cells, where increased absorbance is correlated with increased proliferation. Bars represent means ± s.e.m. of N = 10 wells. B. A similar BrdU assay was used to visualize the proliferating cells on slides. Cells were treated as above (EA and Scr 100 nM, EB and IGF 10 nM), fixed, and stained with BrdU and DAPI. Total cells and proliferating (BrdU positive) cells were counted from 3 10× fields for each slide, and bars represent means ± s.e.m. of N = 5 slides. For A and B, *, p<0.05; ***, p<0.001 for comparisons to NoTx via 1-way ANOVA followed by a Tukey post-hoc test. C. C2C12 cells were tested as in A, except an inhibitor of MEK, PD 098059 (PD, 50 µM) or IGF-IR (NVP, 100 nM) was included in the cell media. Bars represent means ± s.e.m. of N = 18 wells for No Inhibitor (No Inh) and N = 8 for with inhibitors. D. C2C12 cells were plated in the upper chamber of 24-well plate trans-well migration inserts in 0% serum media. Cells were allowed to migrate for 5 hours and stained with DAPI, imaged and counted. Synthetic E-peptides (100 nM) were added to upper and lower chambers with or without inhibitors (PD 50 µM, NVP 100 nM). Images were taken as in B, and bars represent means ± s.e.m. of N = 4 slides. For C and D, *, p<0.05; ***, p<0.001 for comparisons to NoTx via 2-way ANOVA followed by a Bonferroni post-test. †, p<0.05 for comparisons to No Inh via 2-way ANOVA followed by a Bonferroni post-test.
