Figure legends:
Figure 1. Identification of rat BMSCs and cell viability assay.(A) P3 rat BMSCs morphology was assessed by the phase‑contrast microscopy. (B) Effects of CORM-3 (100, 200 or 400 µM) on rat BMSCs viability. (C and D) Identification of osteogenic differentiation. Alizarin red staining of rat BMSCs cultured in the osteogenic (C) or control (D) medium for 21 days. (E and F) Adipogenic differentiation identification. Oil red O staining of rat BMSCs cultured in the adipogenic (E) or control (F) medium for 21 days. (G and H) Clonogenesis experiment. Rat BMSCs were seeded at a density of 3 cells/cm2 and cultured in control medium. After 10 days, crystal violet staining of rat BMSCs was observed by visual (G) and the phase‑contrast microscopy (H). Magnification, x100; scale bar, 100 µm. The experiment was repeated in triplicate. Data were presented as the mean ± standard deviation (n=3). * P < 0.05 as indicated.
Figure 2. Expressions of miR-195-5p and Wnt3a during osteogenic differentiation of rat BMSCs. Rat BMSCs in the CORM-3 or osteogenic group were cultured in the osteogenic medium with or without 200 μΜ CORM-3, respectively. Rat BMSCs in the control group were cultured in the control medium. (A) RT-qPCR analysis of miR-195-5p on 24 hours, normalized to U6. (B) Representative western blot images of three independent experiments for Wnt3a protein expression at 24, 48 and 72 hours and quantitative results of western blot images, using ImageJ software, normalized to Tubulin. The experiment was repeated in triplicate. Data were presented as the mean ± standard deviation (n=3). as indicated.
Figure 3. Effects of miR-195-5p on the CORM-3-induced osteogenic differentiation of rat BMSCs. (A) The rat BMSCs were transfected with miR-195-5p mimics or mimics NC, miR-195-5p inhibitor or inhibitor NC. After 48 hours, the expressions of miR-195-5p were determined by RT-qPCR, normalized to U6. (B and C) The rat BMSCs were transfected with miR-195-5p mimics or mimics NC (B), miR-195-5p inhibitor or inhibitor NC (C) for 24 hours and then cultured in the osteogenic medium containing 200 μΜ CORM-3. Meanwhile, cells in the osteogenic, CORM-3 or control group were cultured in the osteogenic medium, osteogenic medium containing 200 μΜ CORM-3 or control medium respectively. After 3 and 7 days, the mRNA expressions of Runx2 and OPN were determined by RT-qPCR, normalized to β-actin. (D and E) The rat BMSCs were cultured as (B and C) described above. After 3 and 7 days, the protein expressions of Runx2 and OPN were determined by Western blot, then analysed using ImageJ software, normalized to GAPDH. (F) The rat BMSCs were cultured in different mediums as described above. After 14 days, the mineralization was determined by alizarin red staining and semi quantitative analysis. The experiment was repeated in triplicate. Data were presented as the mean ± standard deviation (n=3). ^ P < 0.05 vs. control; * P < 0.05 as indicated.
Figure 4. miR-195-5p directly targets Wnt3a. (A) The design of luciferase reporters with Wnt3a 3′UTR-wt or Wnt3a 3′UTR-mut. (B and C) Western blot images and analysis for Wnt3a protein expression in rat BMSCs after 48 hours transfection with miR-195-5p mimics or mimics NC (B), miR-195-5p inhibitor or inhibitor NC (C), using ImageJ software, normalized to Tubulin. (D) Effect of miR-195-5p mimics on luciferase activity in 293T cells transfected with either the 3′UTR-wt reporter or the 3′UTR-mut reporter for Wnt3a. The experiment was repeated in triplicate. Data were presented as the mean ± standard deviation (n=3). as indicated.
Figure 5. Effects of Wnt3a on the CORM-3-induced osteogenic differentiation of rat BMSCs. (A) Western blot images and analysis for Wnt3a protein expression in rat BMSCs after 48 hours transfection with pcDNA3.1-Wnt3a, Wnt3a siRNA or their corresponding controls. (B and C) The rat BMSCs were transfected with pcDNA3.1-Wnt3a or NC (B), Wnt3a siRNA or NC (C) for 24 hours and then cultured in the osteogenic medium containing 200 μΜ CORM-3. Meanwhile, cells in the CORM-3 or control group were cultured in the osteogenic medium containing 200 μΜ CORM-3 or control medium respectively. After 3 and 7 days, the mRNA expressions of Runx2 and OPN were determined by RT-qPCR, normalized to β-actin. (D and E) The rat BMSCs were cultured as (B and C) described above. After 3 and 7 days, the protein expressions of Runx2 and OPN were determined by Western blot, then analysed using ImageJ software, normalized to GAPDH. (F) The rat BMSCs were cultured in different mediums as described above. After 14 days, the mineralization was determined by alizarin red staining and semi quantitative analysis. The experiment was repeated in triplicate. Data were presented as the mean ± standard deviation (n=3). as indicated.
Figure 6. miR-195-5p affected CORM-3-induced osteogenic differentiation of rat BMSCs by targeting Wnt3a. (A) The rat BMSCs were co-transfected with miR-195-5p mimics and pcDNA3.1-Wnt3a, miR-195-5p mimics and NC for 24 hours and then cultured in the osteogenic medium containing 200 μΜ CORM-3. Meanwhile, cells in the CORM-3 or control group were cultured in the osteogenic medium containing 200 μΜ CORM-3 or control medium respectively. After 3 and 7 days, the mRNA expressions of Runx2 and OPN were determined by RT-qPCR, normalized to β-actin. (B) The rat BMSCs were cultured in different mediums as described above. After 3 and 7 days, the protein expressions of Runx2 and OPN were determined by Western blot, then analysed using ImageJ software, normalized to GAPDH. (C) The rat BMSCs were cultured in different mediums as described above. After 14 days, the mineralization was determined by alizarin red staining and semi quantitative analysis.
The experiment was repeated in triplicate. Data were presented as the mean ± standard deviation (n=3). ^ P < 0.05 vs. control; * P < 0.05 as indicated.