Facsdiva ver 6

In brief, C2C12 cells were seeded in 96-well plates, and transfected with control, miR-145-5p mimic, miR-145-5p inhibitor, LY294002, or LY294002+ miR-145-5p inhibitor, after which cell proliferation was determined at 12, 24, 48 and 72 h using a colorimetric MTT assay was performed to determine cell viability according to the manufacturer's protocol. For 5-ethynyl-2′-deoxyuridine (EdU) proliferation analysis, C2C12 cells were treated with 10 µM EdU (Guangzhou RiboBio Co., Ltd.) after 24 h posttransfection. EdU staining was performed according to the manufacturer's protocol. Cell nuclei were stained with DAPI (Beyotime Institute of Biotechnology). Images (magni cation, ×100) of the entire well were captured using a uorescent microscope (DMi8; Leica, German). FCM Cells were stained with Annexin V-FITC and propidium iodide (PI) using Annexin V-APC/7AAD apoptosis detection kit (Catalog No. AP105-100-KIT, Multisciences (Lianke) Biotech, Hangzhou, China). Apoptotic cells analysis performed ow cytometry (BD FACSCanto II ow cytometer and FACSDiva ver. 6.1; BD Biosciences), according to the manufacturer's protocol.