Modfit software version 4

The Cell counting kit (CCK)-8 assay (Dojindo Molecular Technologies, Inc.) was used to detect cell proliferation following transfection according to the manufacturer's protocol. After 48 h transfection, cells were seeded in 96 well plate at the density of 4×10³ cells/100 µl per well, and proliferation was detected at 0, 1, 2, 3 and 4 days. CCK-8 reagent (10 µl medium/well) was added prior to detection and after incubation for 1.5 h at 37°C, and the absorbance was measured at 450 nm using a microplate reader. Absorbance at 630 nm was used as the control.
For the cell cycle assays, 3×10⁵ cells/well were harvested from a 6 well plate and fixed in ice-cold 70% (v/v) ethanol for 24 h at 4°C. The cell pellet was collected following centrifugation at 500 × g for 10 min at 4°C and resuspended in PBS. Cells were then stained with a mixture of RNase (10 µg/ml) and propidium iodide (50 µg/ml; Beyotime Institute of Biotechnology) in sodium citrate containing 0.5% Triton X-100 for 20 min, in the dark and at room temperature. The cells were subsequently analyzed using a flow cytometer (Gallios, Beckman Coulter, Inc.). The ModFit software version 4.0 (Verity Software House, Inc.) was used for data analysis.

Passage 2 osteoblasts were cultured at 4×10⁵ cells/dish in 100 mm dishes for 24 h. Osteoblasts were then synchronized and incubated with 10⁻⁸ M ⁹⁹Tc-MDP, 10 ng/ml β-FGF or medium (high-glucose DMEM supplemented with 10% FBS and antibiotics) alone for 48 h. Each condition was replicated three times. Cells were then digested with trypsin, washed with PBS and collected by centrifuging at 500 × g for 5 min at room temperature. Pre-cooled ethanol (70%, 1 ml) was added to the precipitate and the cell suspension was fixed at 4°C overnight. Following centrifugation at 500 × g for 5 min at room temperature, the precipitate was resuspended in 3 ml PBS, passed through a 400-mesh screen and stained with 1 ml propidium iodide (PI) at 4°C for 30 min in the dark. The cell cycle was assessed by flow cytometry, and 3×10⁴ cells were collected for each sample. Modfit software Version 4.0 (Verity Software House, Inc., Topsham, ME, USA) was used for data acquisition, processing, calculation of apoptotic cell proportion, and the determination of cell cycle distribution. The S-phase fraction (SPF) and proliferation index [proliferation index=(S + G₂/M)/(G₀/G₁ + S + G₂/M) ×100%] were used to evaluate the speed of proliferation.

LNCaP and C4-2 cells (1×10⁶) were trypsinized, washed twice with PBS and fixed in 70% ice-cold ethanol (Sangon Biotech Co., Shanghai, China) for 1 h at 4°C. The samples were centrifuged at 300 × g for 5 min at 4°C, the ethanol was removed and they were incubated with 100 mg/ml RNaseA (Sigma-Aldrich; Merck KGaA) for 30 min at 37°C. For cell-cycle distribution analysis, 400 ul PI solution (BestBio Co., Ltd., Shanghai, China) was added to the cells. The cells were vortexed and incubated in the dark for 30–60 min at 2–8°C. For cell apoptosis analysis, cellular DNA was stained with the Annexin V-FITC/PI Apoptosis Detection kit (cat. no. BB-4101-2; BestBio Co., Ltd., Shanghai, China). Briefly, the cells were centrifuged at 300 × g for 5 min at room temperature and resuspended in Annexin V Binding Buffer. A total of 5 µl FITC Annexin V was added to the suspension, they were then gently vortexed and incubated for 15 min at 2–8°C in the dark. The cells were then incubated with 10 µl of PI solution, gently vortexed and incubated for 5 min at 2–8°C in the dark. Cell-cycle distributions and cell apoptosis were determined by flow cytometry using a BD FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA) and data was analyzed using the ModFit software version 4.1 (Verity Software House, Inc., Topsham, ME, USA).