Inverted phase contrast fluorescence microscope

An inverted phase contrast fluorescence microscope (Carl Zeiss, Heidenheimer, Germany) was used to directly observe morphological changes in the two cell lines treated with salidroside. Cells were separately seeded into 6-well (12×10⁴ cells/well) and 24-well (3×10⁴ cells/well) plates, cultured to confluence, and treated with (0, 1, 5 and 10 mM) salidroside at 37°C for 48 h. Firstly, we directly observed the morphology of apoptotic cells seeded in the 6-well plate (magnification, ×100). Cells were counted from five random fields for each group, and the average was expressed as the number of apoptotic cells. Then, we observed the nuclear morphology of apoptotic cells seeded in the 24-well plate using Hoechst 33258 staining. In brief, cells were washed three times with PBS, fixed in 4% paraformaldehyde at 4°C for 20 min, stained with Hoechst 33258 staining solution (125 μl/well, Beyotime Institute of Biotechnology, Haimen, China) for 5 min, rewashed three times with PBS, covered with anti-fading solution, and then observed using the aforementioned fluorescence microscope (magnification, ×400). Cells were counted from five random fields for each group and the number of apoptotic cells (Hoechst-positive cells) was expressed as a percentage (%) of the total number of counted cells.

Cell proliferation was evaluated via an MTT assay. MG63 cells (4×10⁴/well) infected with miR-504 and miR-NC were seeded in 96-well plates and cell proliferation was measured at 6, 12, 24, 48, 72 and 96 h. 150 µl of MTT was added to each well and the plate was incubated at 37°C for a further 4 h. Dimethyl sulfoxide was then added to dissolve the sediment. Absorbance was measured at 490 nm using a Spectra Max Plus 384 microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). Cell proliferation was also detected via an EdU assay, using an EdU cell proliferation detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The cells (4×10⁴/well) were seeded in 6-well plates and cultured at 37°C for 48 h, and then incubated with DMEM containing EdU (Nanjing KeyGen Biotech Co., Ltd.). They were subsequently fixed in 4% paraformaldehyde at 4°C for 30 min, washed twice with PBS, reacted with Apollo 643 and dissolved in Apollo reaction buffer (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The cells were subsequently stained with DAPI at 37°C for 3 min and observed under an inverted phase contrast fluorescence microscope (magnification, ×400; Carl Zeiss AG, Oberkochen, Germany). Cells stained red were considered EdU-positive.