FIGURE 4.

The effects of r‐irisin on osteoblast and osteoclast differentiation in BMSCs and BMMs. (a) Real‐timePCR analysis of the mRNA expression level of osteogenic related gene markers on Day 5 (n = 3). (b) Total protein level of β‐catenin in BMSCs during the osteoblast‐induced cell culture with or without r‐irisin. (upper lane). β‐Actin was used as a loading control (lower lane; n = 3). The β‐catenin protein level in cytoplasmic (c) and nuclear (d) fractions and the relative expression calculated using β‐actin or lamin B1 as loading controls for cytoplasmic and nuclear expression, respectively (n = 3). (e) Alizarin red staining for bone nodules of osteoblast‐induced BMSCs culture with and without irisin for 3 weeks. (f) The mRNA expression level of osteoclastogenesis gene expression on Day 5 (n = 3). (g) Nfatc1 protein expression on Day 5. (h) Western blot analyses with antibodies recognizing p‐Akt1, Akt1, p‐p38, p38, and calcineurin in osteoclast‐induced BMMs treated with and without irisin. β‐Actin was used as a loading control. (i) TRAP staining and quantitative analysis of the TRAP⁺ MNCs. All values are shown as mean ± SD. AA, amino acid; Alp, alkaline phosphatase; BMM, bone marrow–derived macrophage; BMSC, bone marrow–derived mesenchymal cell; BSP, bone sialoprotein; Col1, collagen 1; CON, control; F/I KO, Osx‐Cre:FNDC5/irisin KO mice; KO, knockout; MNC, multinucleated cell; mRNA, messenger RNA; Nfatc1, nuclear factor of activated T cells c1; PCR, polymerase chain reaction; RANKL, receptor activator of nuclear factor‐κΒ ligand; Runx2, Runt‐related transcription factor 2; TRAP, tartrate‐resistant acid phosphatase. *p < .05 versus control