4 6 diamidino 2 phenylindole (dapi)

The proliferation capacity of CPM was detected by ethynyl-deoxyuridine staining, the CCK-8 cell viability assay and flow cytometry.
For ethynyl-deoxyuridine staining, CPM were treated with 50 μM ethynyl-deoxyuridine (Ribobio, Guangzhou, China) at 36 h post-transfection according to the manufacturer’s protocol and incubated for 2 h at 37 °C in a 5% CO₂ incubator. The CPM nuclei were stained with 5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI, Ribobio) for 5 min and washed three times with PBS. Cells were observed under a fluorescence microscope (Nikon, Tokyo, Japan).
For the CCK-8 assay, at 12, 24, 36, and 48 h post-transfection, 10 μL of CCK-8 reagent were added to each well (eight wells per group) to detect the absorbance at 450 nm after incubation for 2 h at 37 °C according to the manufacturer’s instructions.
For flow cytometry, after 48 h, the transfected cells were washed twice in a cold phosphate-buffered saline solution and collected in a 1.5 mL centrifuge tube. The cells were then suspended in 500 μL of a propidium iodide (Sigma, MO, USA) solution with 10 μL RNase A (Sigma) at 37 °C for 30 min. The cell cycle phase was detected using a flow cytometer (Becton Dickinson, FACSCa-libur, Franklin, GA, USA), and the proportion of cells in each cell cycle phase was obtained.

An EdU assay was performed using the Cell Light EdU DNA imaging kit (Guangzhou RiboBio Co., Ltd., Guangzhou China) to measure cell proliferation. 48 hours after transfection, cells were seeded in 96-well plates and exposed to 25 mM EdU for 2 h at 37°C, and were then fixed in 4% paraformaldehyde. Following permeabilization with 0.5% Triton X-100, the 16 Apollo reaction cocktail (Guangzhou RiboBio Co., Ltd., Guangzhou China) was added and the cells were incubated for 30 min. Subsequently, the DNA of the cells was stained with 4',6-diamidino-2-phenylindole (DAPI) (Guangzhou RiboBio Co., Ltd., Guangzhou China) for 15 min and visualized under a fluorescent microscope (IX81; Olympus Corporation, Tokyo, Japan). The cell count was analyzed.

In situ hybridization was used with a Fluorescent In Situ Hybridization Kit (RiboBio, China). Briefly, cells were plated on cover slips in a 24-well plate, and were fixed with 4% paraformaldehyde after three washes using 1 × PBS. Then cells were added 1 ml cold PBS containing 0.5% Triton X-100 at 4 ℃ for 5 min, washed with 1 × PBS three times, and prehybridizated at 37 ℃ for 0.5 h before hybridization. An anti-ZFAS1, anti-18S and anti-U6 oligodeoxynucleotide probe was performed in the hybridization solution at 37 ℃ overnight. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, RiboBio, China) the following day and images were captured by fluorescence microscope (Nikon, Japan).

The second-generation BMSCs were inoculated in the laser confocal culture dish, and when the degree of cell fusion reached 60%–70%, the cells were fixed, made transparent and blocked according to the instructions of the RNA-FISH detection kit (RiboBio, Guangzhou, China). Then, LncRNA Fish Probe Mix (RiboBio) was used for light-avoiding hybridization overnight at 37 °C; 18 S and U6 (RiboBio) were used as positive controls for the cytoplasm and nucleus, respectively. The nucleus was labeled with DAPI (RiboBio). PBS was used to wash the cells. Anti-fluorescence quenching agent (Solarbio) was then added, and the fluorescence was observed with a laser confocal microscope.