Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation
Fig 1
ECM stiffness regulates the cellular phenotype of human mesenchymal stem cells (hMSCs) and the localization and transcriptional activity of TAZ.
(A) In indicated hydrogels, cell adhesion and morphology were visualized by light microscopy. Bright field images were taken 24hr after seeding. (B) ECM stiffness controls focal adhesion complex formation and TAZ localization. hMSCs were immunostained with an anti-vinculin antibody to detect focal adhesions (green fluorescence signal) 24 h after seeding. TAZ localization was visualized as a red fluorescence signal. DAPI was used to stain the cell nucleus. (C) The expression of TAZ target genes, including CTGF and CYR61, were analyzed by qRT-PCR using the cells in panel (A). Target gene expression was normalized to the GAPDH expression. Data is shown as fold induction. Asterisks indicate statistical significance (***p < 0.005, t-test). (D) The luciferase reporter gene CTGF-luc was introduced into hMSCs. After 16 h, the transfected cells were plated on 1.37 or 4.47 kPa hydrogels. After 24 h, luciferase reporter gene activity was analyzed. The pGL3-basic luciferase reporter gene, which has no promoter for transcription, was used as a negative control. A Renilla luciferase-expressing vector was used as a transfection control. Luciferase activity was normalized to Renilla luciferase activity and is expressed as relative fold induction. (***p < 0.005, t-test). (E) hMSCs were seeded on a 1.37 or 4.47 kPa hydrogel, and twenty four hours after seeding, total RNAs were isolated and qRT-PCR analysis was assessed to see the expression of TAZ gene. Gene expression was normalized to GAPDH.