Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation
Fig 5
Inhibition of the ERK or JNK signaling pathway induces TAZ cytoplasmic localization on stiff hydrogels.
(A) hMSCs on a 4.47 kPa hydrogel were treated with 10 μM U0126 or 10 μM SP600125. After 12 h of treatment, cells were subjected to immunostaining with an anti-TAZ antibody. The red fluorescence signal shows the location of TAZ, and DAPI was used to stain the nuclei. The results show that even in cells on a stiff matrix, TAZ is localized evenly to the cytoplasm and nucleus following ERK or JNK inhibition. (B) Approximately 100 cells in panel (A) were counted, and TAZ localization was analyzed in these cells. The counting procedure was done using the Image J program. The number of cells that showed an even cytoplasmic-nuclear or cytoplasm-dominant TAZ localization was higher in the presence of an ERK or JNK inhibitor than in the absence of either inhibitor. (C) Cell lysates in panel (A) were prepared, and the activity of the Hippo signaling pathway components LATS and MST kinase was analyzed by immunoblotting. The phosphorylation status of LATS and MST kinase was analyzed with p-LATS1 and p-MST1/2 antibodies, respectively. Total LATS1 and MST2 levels were detected as a loading control.