Eu5888

Cells were cultured in a 12-well plate, and FOXA2, RUNX2, COL1A1, t-ERK and p-ERK were detected using a fluorescence microscope (EU5888; Leica, Wetzlar, Germany). Briefly, cells were fixed in 4% paraformaldehyde (Sigma) for 15 min at room temperature, permeabilized and blocked for 30 min in 0.05% Triton X-100 and 5% bovine serum albumin (BSA). Fixed cells were washed three times with PBS and incubated at 4 °C overnight with anti-FOXA2 (1:400; Cell Signalling Technology), RUNX2 (1:1600; Cell Signalling Technology), COL1A1 (1:500; Abcam, Shanghai, China), t-ERK (1:800; Cell Signalling Technology) or p-ERK (1:200; Cell Signalling Technology). Cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) at room temperature for 2 h and nuclei were stained with 4′,6-diamidino-2-phenylindole (KeyGen Biotech, Nanjing, China) for 4 min. Samples were then observed and photographed under a fluorescence microscope (Leica).

To visualize F-actin rings, BMMs were treated with 25 ng⋅mL^-1 M-CSF and 50 ng⋅mL^-1 RANKL for 4 days. An equal number of BMM-derived osteoclasts were seeded onto bovine bone slices and allowed to adhere overnight. Cells were then treated with different concentrations of stevioside (0, 50, 100, and 200 μM) for another 2 days. Next, cells were fixed with 4% PFA for 15 min, permeabilized for 5 min with 0.5% Triton X-100, and stained with rhodamine-conjugated phalloidin (1:200; Invitrogen Life Technologies, Carlsbad, CA, United States) diluted in 0.2% bovine serum albumin (BSA)–PBS for 1 h. Fluorescent images were captured with a fluorescence microscope (EU5888, Leica, Wetzlar, Germany) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, United States). Then, these bone slices were washed twice with PBS for the resorption pit assay. Cells that had adhered to the bone slices were removed by mechanical agitation. Bone slice images were captured using a scanning electron microscope (SEM; S-4800, Hitachi, Japan) and analyzed using ImageJ software.