Fig. 5: IGF-1 c.258 A > G synonymous mutation promotes the proliferation, migration, and mineralization of H2O2-induced CNCC cells and MC3T3-E1 cells.
From: IGF-1 c.258 A > G synonymous mutation ameliorates senile osteoporosis
a SA-β-gal staining of CNCC-IGF-1-A and CNCC-IGF-1-G cells stimulated with H2O2. Scale bar 100 μm. b Quantification of SA-β-gal positive CNCC-IGF-1-A and CNCC-IGF-1-G cells. c The expression level of senescence-associated genes (p16, p21, and p53) in H2O2-induced CNCC-IGF-1-A and CNCC-IGF-1-G cells using qPCR. d, e The expression level of IGF-1 and IGF-1R detected by western blot. f CCK-8 assay of the proliferation of H2O2-induced cells of different genotypes at 0, 1, 2, 3, and 4 days. g The expression level of cell proliferation-related genes (CyclinD1, PNCA, and CDK4) in H2O2-induced different genotypes cells by qRT-PCR. h EdU incorporation in aged different genotypes cells. Scale bar 200 μm. i Statistical results of EdU staining, CNCC-IGF-1-G cells increased the number of EdU cells. j, k Scratch assay of H2O2-induced different genotypes cells and quantitative analysis. White dashed lines indicate the start (0 h), half (9 h), and end (18 h) positions of different genotypes cells after scraping. Scale bar 50 μm. l Representative images of ALP and ARS staining of H2O2-induced different genotypes cells. m Quantitative analysis of the ALP activity in H2O2-induced different genotypes cells. n Quantitative analysis of the mineralization in H2O2-induced different genotypes cells using 10% cetylpyridinium chloride. o Quantitative qRT-PCR results of mRNA expression of ALP, OCN, OPN, COL-1, OSX, and RUNX2 in aged different genotypes cells. A–O Results in MC3T3-E1 cells corresponding to (a–o). P Binding affinity detection of IGFBP-3 or IGFBP-5 with the IGF-1 of two genotypes in the MC3T3-E1 cells. The results are presented as mean ± SD (n = 5). *P < 0.05; **P < 0.01; ***P < 0.001.
