Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation
Fig 3
A stiff ECM activates the ERK and JNK signaling pathway.
(A) hMSCs were plated on a 1.37 or 4.47 kPa hydrogel, and after 12 h, cell lysates were prepared and analyzed by immunoblotting. To assess the activity of ERK and JNK, phosphorylated ERK (p-ERK) and phosphorylated JNK (p-JNK) antibodies were used, respectively. As a control, total ERK and JNK protein was analyzed. The levels of phosphorylated ERK and JNK were increased in hMSCs cultured on the stiff matrix. GAPDH was used as a loading control. (B) Immunocytochemistry of p-ERK and p-JNK in hMSCs on a 1.37 or 4.47 kPa hydrogel. Cells in panel (A) were fixed and subjected to immunocytochemical staining with a p-ERK or p-JNK antibody. The signals for p-ERK or p-JNK were green fluorescence. DAPI was used for nuclear staining. (C) Fluorescence signals in panel (B) was quantified by image J software and corrected total cell fluorescence was calculated by fluorescence signal with elimination of background signal. AU is arbitrary unit.