Giemsa stain kit

EdU incorporation was detcted employing the EdU assay kit (C10310-1, RiboBio, Guangzhou, China) according to the requirements of the instructions. Brie y, PCa cells PC-3 and LNCaP were seeded to 24well-plates (1 Î 10 5 cells/well) and cultured overnight. Then, cells were transfected with pcDNA3.1-LINC00893 overexpression plasmids or pcDNA3.1-vector using lipofectamine 2000 (Invitrogen). 48h post transfection, cells were incubated with 200 μL 50μm EdU for 2 h, followed by xing with 2% paraformaldehyde (PFA) for 20 min and 2 mg/mL glycine was employed to neutralize PFA. After that, cell membrane was destroyed employing 0.1% Triton X-100 for 10 min. Lastly, cells were stained with 100 μL Apollo staining solution for 30 mins and with 4',6-diamidino-2-phenylindole (DAPI) DNA staining solution for 20 mins. The Fluorescence images were collected and EdU-positive cells were counted usingr a microscope (Nikon, Japan) [40] . Three independent assays was performed in the EdU assay in the same method.
Colony formation assay PC-3 and LNCaP cells treated as indication were resuspended in fresh medium and then seeded into the 6-well-plates (1000 cells/well). Then, cells were grown under conditions of 37°C and 5% CO2 for 14 days. Subsequently, the culture medium was discarded and 4% paraformaldehyde was employed to x the cells for 20 mins. After that, cells were colored using Giemsa Stain Kit (Abcam, ab150670) for 20 mins. Lasly, images of typical colony morphology were photographed and the the number of colonies in each conditions was counted under a Leica AM6000 microscope [41] . The colony formation assay was conducted with three independent assays in the same method.