Fig 2: Cell culture and treatments. (A) 3T3-L1 cells were treated with miR-34a mimics or inhibitor (siRNA) and then were induced by adipocyte differentiation medium. (B) C2C12 cells were pre-treated by miR-34a mimics or inhibitor (siRNA) and treated with oleate or conditioned medium from the differentiation progress of 3T3-L1-derived adipocyte medium. The expression of adipogenic markers (i.e., Pparg, Cebpa, Fabp4, and Plin1) and the lipid droplet deposition in C2C12 cells were detected. (C) 3T3-L1 and C2C12 cells were co-cultured and treated with miR-34a mimics or inhibitor (siRNA) to detect the expression of adipogenic markers and lipid droplet deposition.

Fig 3: miR-34a abolished the suppressive function of overexpressed Lef1 on fat deposition during 3T3-L1 differentiation. The expression of Lef1 (A), Pparg (B), Cebpa (C), Fabp4 (D), and perilipin1 (E) after cells transfected with pcDNA3.1-Lef1, siLef1 and their corresponding control during cell differentiation (n = 6). The expression of Lef1 (F), Pparg (H), Cebpa (I), Fabp4 (J), and perilipin1 (K) after cells transfected with miR-34a mimics NC + pcDNA3.1 or mimics + pcDNA3.1-Lef1 or with miR-34a inhibitor NC + siNC or inhibitor + siLef1 during cell differentiation (n = 3). The protein expression of Lef1 during cell differentiation in the rescue experiment (n = 3) (G). * p < 0.05, ** p < 0.01, *** p < 0.001 versus NC.

Fig 4: Silencing miR-34a suppresses adipogenesis. miR-34a inhibitor (siRNA) could efficiently decrease the expression of miR-34a in 3T3-L1 (n = 6) (A). The expression of Pparg (B), Cebpa (C), Fabp4 (D), Plin1 (E), and LEF1 (F) on day 0 and day 8 during 3T3-L1-derived preadipocyte differentiation after miR-34a silencing (n = 6). The protein expression level of Ppar? (G), C/ebpa (H), ß-catenin (I), and Lef1 (J) in NC and miR-34a inhibitor groups along with differentiation via Western blot (n = 3) (K). Lipid droplet accumulation in 3T3-L1 cells on day 0 and 8 of differentiation detected using BODIPY staining (L) and Oil Red O staining (M) in NC and miR-34a inhibitor groups. * p < 0.05, ** p < 0.01, *** p < 0.001 versus NC.

Fig 5: BMMSCs from ovariectomized rats (OVX-BMMSCs) exhibited impaired osteogenic potential and enhanced adipogenic differentiation ability (A) BMMSCs from Sham and OVX rats exhibited similar cell morphologies. Scale bar, 100 ?µm. Magnificantion, 100X. (B) Proliferation of Sham- and OVX-BMMSCs (C) Gene expression of Sirt1. (D–E) Protein expression of SIRT1, RUNX2 and PPAR? (F) Representative images and quantitative analysis of ARS staining that was conducted to evaluate matrix mineralization. Scale bar, 100 ?µm. Magnificantion, 100X. (G) Osteoblast-related gene expression: Runx2, Sp7 and Bglap (H) BMMSCs were induced towards adipogenesis for 14 days and stained by oil red O. Representative images and quantitative analysis were used to examine lipid formation in mature adipocytes. Scale bar, 100 ?µm. Magnificantion, 100X. (I) Adipocyte-specific gene expression: Lpl, Pparg and Fabp4. Values are the mean ?± ?S.E.M of six independent experiments (n ?= ?6) in cell proliferation assays, four independent experiments (n ?= ?4) in RT-PCR experiments, ARS assays, and Oil Red O staining assays, and three independent experiments (n ?= ?3) in Western blot assays. Statistically significant differences are indicated by * where p ?< ?0.05 or ** where p ?< ?0.01 between the indicated groups.Abbreviations: BMMSCs, bone marrow mesenchyml stem cells; OVX, ovariectomized; SIRT1, Silent information regulator type 1; RUNX2, runt-related transcription factor 2; Sp7, osterix; Bglap, bone gamma carboxyglutamate protein; PPAR?, peroxisome proliferator-activated receptor ?; Fabp4, fatty acid binding protein 4; LPL, lipoprotein lipase.