FIGURE 1.

Construction and identification of the F/I KO mice, and its implication in osteogenesis, browning effect and circulating irisin level in vivo. (a) The targeting strategy of FNDC5^flox/flox mice. Truncated protein from deletion of exon2: MPPGPCAWPPRAALRLWLGCVCFALVQ AEEGCADAPVHSGGEHHHPVLRSLGPGGGHRIYRPCAGHLHPGTEPSQ* (28 aa N‐terminal sequence of irisin and 47 aa frame‐shifted nonsense aa). (b) Southern blot (upper) and standard PCR (lower) were performed to identify the FNDC5^flox/flox ES cells and F/I KO mice. The FNDC5 mRNA expression level of bone, muscle and inguinal fat (c) and irisin protein level of bone and skeletal muscle (d) in F/I KO mice and control mice (n = 3). β‐Actin was used as a loading control in western blot analysis. Relative mRNA level expression of osteogenic markers Col1, BSP, and OSX (e) and cathepsin K, Mmp9, and Trap (f) in bone tissue (femur and tibia) of F/I KO and control mice were calculated and then normalized to GAPDH (n = 3). (g) The expression level of the genes related to white adipose tissue browning Cidea, Prdm16, and Ucp‐1 in white adipose tissue (inguinal fat) of F/I KO and control mice (n = 3). (h) The circulating irisin level of F/I KO and control mice (n = 6). Values are shown as means ± SD. BSP, bone sialoprotein; Col1, collagen 1; CON, control; F/I KO, Osx‐Cre:FNDC5/Irisin KO mice; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; KO, knockout; Mmp9, matrix metalloproteinase 9; mRNA, messenger RNA; OSX, osterix; PCR, polymerase chain reaction; Prdm16, PR domain‐containing 16; Trap, tartrate‐resistant acid phosphatase; Ucp‐1, uncoupling protein 1; WAT, white adipose tissue. *p < .05, ***p < .001, ****p < .0001 versus control