Figure 5.

WNT5B suppresses osteoblast differentiation and promotes adipogenesis

(A–C) mOBs were isolated from calvaria then treated with various doses of rWNT5B at day 0 (D0), day 7 after differentiation (D7) or day 14 after differentiation (D14) for 24 h. The levels of (A) Sp7, (B) Alpl, and (C) Bglap were detected by qPCR.

(D) mOBs were treated with or without 50 ng/mL rWNT5B and stained for alkaline phosphatase (ALP) and Alizarin Red.

(E) The levels of Alizarin Red in Ctrl and WNT5B groups were quantified at O.D. 570 nm and shown as % of the control.

(F) mOBs were infected with lentivirus containing sh-Ctrl or sh-Wnt5b, before collecting mRNA or protein. The levels of WNT5B mRNA and protein were normalized with β-actin and quantified by ImageJ.

(G and H) Cells were differentiated for 7 days in the presence or absence of 50 ng/mL rWNT5B, then (G) expression of Bglap was detected by qPCR and (H) alkaline phosphatase (ALP) activity was detected at O.D. 405 nm then was normalized with total protein.

(I) Bone marrow-derived MSCs were treated with or without 50 ng/mL rWNT5B in osteogenic differentiation media for 12 days and stained with Alizarin Red. CFU-OB was reported as the number of colonies.

(J) Bone marrow-derived MSCs were treated with or without 50 ng/mL rWNT5B in adipogenic differentiation medium for 14 days and stained with Oil Red O.

(K) The levels of Oil Red O between Ctrl and WNT5B groups were quantified as % Area by ImageJ.

(L and M) Expression of (L) Pparg and (M) Adipoq in MSC-derived adipocytes treated with or without rWNT5B for 24 h were detected by qPCR. Three different biological replicates were obtained and a representative image or graph from one replicate is presented. n = 3, Student’s t test: ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05.