Fig. 4.

Conditional miR-140 knockout and conditional miR-140 over-expression regulate bone deterioration and SSPC cell fate in vivo.

(A-F) Adeno-associated-virus-F4/80-cre-ZsGreen (AAV-F4/80-Cre-ZsGreen) and adeno-associated-virus-NC-ZsGreen (AAV-NC-ZsGreen) were injected into intra bone marrow of obese miRNA-140^flox/flox littermate to generate BMM conditional miRNA-140 knockout mice and negative controls. Two months after injection, femur specimens were collected. n = 5 per group. (A) Timeline diagram of obese BMM conditional miRNA-140 knockout mice generation. (B–C) Representative μCT images (B) and quantitative μCT analysis (C) of trabecular bone. (D) Representative images of osteocalcin staining (D, top). Red arrows mark osteoblasts. Scale bar: 50 μm; representative images of H&E staining (D, bottom). Scale bar: 100 μm; (E-F) Quantification of the number of osteoblasts (E) and number of adipocytes (F). (G–N) Cross miRNA-140TG^flox/flox mice with Prrx1-cre mice to generate SSPC conditional miR-140 over-expression mice (miRNA-140^SSPC-OE). (G) Diagram of miR-140TG^flox/flox mice construct and miRNA-140^SSPC-OE mice generation. An exogenous donor sequence containing terminators and miR-140 was inserted into chromosome 11 in wild-type mice. Loxp sites are depicted by yellow arrows, miR-140^SSPC-OE represents Prrx1-cre; miR-140TG^flox/+ mice. (H-I) Representative μCT images (H) and quantitative μCT analysis of trabecular bone (I) from miRNA-140^SSPC-OE and miRNA-140 TG^flox/flox mice. n = 4 per group. (J–K) Representative images of osteocalcin staining (J) and quantification of number of osteoblasts (K). n = 4 per group. Red arrows mark osteoblasts. Scale bar: 50 μm. (L-N) Representative images of H&E staining (L) and number and area of adipocytes (M-N). Scale bar: 100 μm. Data is shown as mean ± SD. *P < 0.05, **P < 0.05, ns, no significant. (Student t-test).