Dopamine Suppresses Osteogenic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells via AKT/GSK-3β/β-Catenin Signaling Pathway

Figure 5

LiCl rescues the attenuation of DA on rBMSC osteogenic differentiation via the AKT/GSK-3β/β-catenin pathway. rBMSCs were pretreated with 5 mM LiCl for 2 h and then cultured in osteogenic medium containing 10^-5 M DA. (a) ALP staining of rBMSCs after 7 days of differentiation. Optical photos (upper photo) and microscopic images (lower photo). (b) ALP activity of rBMSCs measured after 7 days of differentiation. (c) Calcium nodules assessed by Alizarin Red staining after 14 days of differentiation. Optical photos (upper photo) and microscopic images (lower photo). (d) Relative quantification of Alizarin Red staining. (e) Expressions of Col1a1, Alp, Runx2, Opn, and Ocn measured by qRT-PCR assay after 7 days of differentiation. (f, g) Expression of COL1A1, ALP, RUNX2, OPN, and OCN measured by western blot assay after 7 days of differentiation. (h) Expression of GSK-3β and p-GSK-3β measured by western blot assay after 24 h of differentiation. (i) Quantification of p-GSK-3β/GSK-3β ratio. (j, k) The expression levels of nuclear and cytoplasmic β-CATENIN analyzed by western blot. (j) and quantified (k) in the indicated groups after 24 h of differentiation. (l) The translocation of β-CATENIN in the nucleus assessed by immunofluorescence assay after 24 h of differentiation. β-CATENIN was stained as red and nuclei were stained by DAPI showing blue. Scale bar, 100 μm. and compared to OM group; ^# and ^## compared to DA group; ns: no significance.

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