Vistarimage 3

hBMSCS cells were seeded in a 6-well plate at a density of 1 ×10⁵ cells/well. Cells were washed three times with PBS. After treatment, Cells were fixed in 4% paraformaldehyde solution for 15 min at room temperature,followed by fixation with pre-cooled acetone for 15 min. Later, they were washed twice in PBS followed by a 30-minute incubation in 2% bovine serum albuminsolution (Sigma-Aldrich, Cat# A7906-50G). Cells were then treated with 0.5% PBS-Triton X-100 for 20 min and incubated with 3% H₂O₂ for 15 min. The samples were then blocked with 5% serum for 20 min and incubated with JDJM2B (1:1000; Ab191434; Abcam) and FAM210A(1:200; NBP2-13992; Novusbio) overnight at 4 °C. Then add 200 µL of APC-labeled secondary antibody (1:500) to the sample, and incubate for 1 h at 37 °C, 5% CO₂, and saturated humidity. The nucleus was stained with DAPI (S2110; Solarbio) for 1 min. Finally, the samples were washed to remove excess solution and mounted on microscope slides for imaging Images were collected using VistarImage 3.0 software (Nikon, Japan) software connected to a Olympus CKX53 inverted microscope. Images were acquired using an UPlanFLN objective at magnification 40 ×.

MC3T3-E1 cells were seeded in a 6-well plate at a density of 2  ×10⁵ cells/well. After treatment, cells were washed three times with PBS, followed by fixation with 4% paraformaldehyde for 15 min. Cells were then treated with 0.1% PBS-Triton X-100 for 5 min and incubated with rhodamine-conjugated LCA (1:1,000; RL-1042-5; Vector Laboratories, Burlingame, CA, USA) for 1 h at 26–28°C. The nucleus was stained with DAPI (S2110; Solarbio) for 1 min. The fluorescent staining was observed using an Olympus CKX53 inverted microscope. Images were acquired using an UPlanFLN objective at magnification 40 × and analyzed using VistarImage 3.0 software (Nikon, Japan).