Journal: International Journal of Molecular Sciences

Article Title: Mettl3 Regulates Osteogenic Differentiation and Alternative Splicing of Vegfa in Bone Marrow Mesenchymal Stem Cells

doi: 10.3390/ijms20030551

Figure Lengend Snippet: Osteogenic differentiation of BMSCs and expression of m 6 A methyltransferase and demethylases. ( A , B ) The formation of mineralized nodules was analyzed in BMSCs undergoing osteogenic differentiation on Days 7, 14, and 21. The “white” scale bars represent 100 μm (original magnification ×100). ( C ) ALP activity assays. ALP activity was significantly higher in the cells cultured in osteogenic differentiation medium for seven days. ( D ) The expression of Runx2 , Alp and Ocn was assessed using qRT-PCR on Days 7 and 14 of culture in osteogenic induction medium. Gapdh was used as an internal control. ( E ) The expression of Runx2 and Osterix was examined using Western blotting. Vinculin was used as an internal control. The band intensities were analyzed using ImageJ software. ( F , G ) RNA methylation modification-related enzymes were detected using qRT-PCR and Western blotting in cells cultured in osteogenic induction medium for 7 and 14 days. Gapdh was used as an internal control for qRT-PCR. Vinculin was used as an internal control in Western blotting. All of the results represent the mean ± standard deviation of three independent experiments ( n = 3). Significant difference compared with the control (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The membrane was blocked in TBST containing 5% nonfat milk for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4 °C: Mettl3 (1:1000; Proteintech, Chicago, IL, USA), Fto (1:1000; Abcam, Cambridge, UK), Alkbh5 (1:1000; Proteintech, Chicago, IL, USA), Runx2 (1:1000; Abcam, Cambridge, UK), Osterix (1:1000; Abcam, Cambridge, UK), Akt and P-Akt (1:1000; Cell Signaling Technology, Boston, MA, USA), Gapdh (1:1000; Abcam, Cambridge, UK), and Vinculin (1:1000; Cell Signaling Technology, Boston, MA, USA).

Techniques: Expressing, Activity Assay, Cell Culture, Quantitative RT-PCR, Western Blot, Software, Methylation, Modification, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Mettl3 Regulates Osteogenic Differentiation and Alternative Splicing of Vegfa in Bone Marrow Mesenchymal Stem Cells

doi: 10.3390/ijms20030551

Figure Lengend Snippet: Effect of Mettl3 knockdown on the osteogenic differentiation potential of BMSCs. ( A ) A green fluorescence protein marker was used to determine the transfer efficiency of Mettl3 knockdown in BMSCs. After transfection for 72 h, the cells were observed under a microscope (on the left). The right image is an immunofluorescence image taken at the same time. The “black” scale bars represent 100 μm (original magnification ×100). ( B ) The expression level of Mettl3 was determined using Western blotting in the Mettl3 -shRNA and Mettl3 -shCtrl groups. Vinculin was used as an internal control. The band intensities were analyzed using ImageJ software. ( C ) The mRNA expression levels of Alp and Ocn in the Mettl3 -shRNA and Mettl3 -shCtrl groups were assessed using qRT-PCR after 7 and 14 days of osteogenic induction. Gapdh was used as an internal control. ( D ) The protein levels of Runx2 and Osterix in the Mettl3 -shRNA and Mettl3 -shCtrl groups were assessed using Western blotting after 7 and 14 days of osteogenic induction. Vinculin was used as an internal control. The band intensities were analyzed using ImageJ software. ( E ) ALP activity was determined in Mettl3 -shRNA and Mettl3 -shCtrl cells cultured in osteogenic differentiation medium for seven days. ( F ) The formation of mineralized nodules was analyzed in the Mettl3 -shCtrl and Mettl3 -shRNA groups undergoing osteogenic induction on Days 7, 14, and 21. Mineralization was analyzed using Alizarin Red S staining. All of the results represent the mean ± standard deviation of three independent experiments ( n = 3). Significant difference compared with the control (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The membrane was blocked in TBST containing 5% nonfat milk for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4 °C: Mettl3 (1:1000; Proteintech, Chicago, IL, USA), Fto (1:1000; Abcam, Cambridge, UK), Alkbh5 (1:1000; Proteintech, Chicago, IL, USA), Runx2 (1:1000; Abcam, Cambridge, UK), Osterix (1:1000; Abcam, Cambridge, UK), Akt and P-Akt (1:1000; Cell Signaling Technology, Boston, MA, USA), Gapdh (1:1000; Abcam, Cambridge, UK), and Vinculin (1:1000; Cell Signaling Technology, Boston, MA, USA).

Techniques: Fluorescence, Marker, Transfection, Microscopy, Immunofluorescence, Expressing, Western Blot, shRNA, Software, Quantitative RT-PCR, Activity Assay, Cell Culture, Staining, Standard Deviation