Vectamount aq mounting medium

After exposure to 1, 10, 20 and 40 µM SM or 200 µM H₂O₂, senescence development was checked every third to fourth day using the senescence detection kit I (PromoCell, Heidelberg, Germany) according to manufacturer’s protocol. To exclude false positive staining, cells were plated at the same density 1 day before staining onto cover slips in 4-well plates. Briefly, 10,000 cells were grown overnight on coverslips in 4-well plates, washed once with PBS and fixed for 10–15 min at room temperature with the fixative solution. Meanwhile, the senescence staining solution was prepared by mixing 470 µL of staining solution, 5 µL of staining supplement and 25 µL of 20 mg/mL X-gal in DMSO per well. Fixed cells were washed twice with PBS and senescence staining solution was added and developed in the incubator overnight. Additionally, cells were counterstained with nuclear fast red (Vector Laboratories, Inc., Burlingame, USA) for 10 min at room temperature and mounted with VectaMount™ AQ mounting medium (Vector Laboratories, Inc., Burlingame, USA) onto microscopy slides (Thermo Fisher Scientific, Waltham, USA). Senescence development was monitored over a time period of 4 weeks. The percentage of senescent versus total cells was counted in three randomly selected images (mean of 20 cells per image) and was performed in three independent experiments.

TRAP staining was used to detect osteoclastic activity using the standard naphthol AS-BI phosphate postcoupling method. Paraffin sections (5 μmol·L⁻¹) were rehydrated and incubated with TRAP staining solution containing 0.2 mol·L⁻¹ sodium acetate buffer (pH 5.0, S2889-250G, Millipore Sigma, USA), 50 mmol·L⁻¹ L-(+) tartaric acid (228729-100 G, Millipore Sigma, USA), 0.5 mg·mL⁻¹ naphthol AS-MX phosphate (N4875-100MG, Millipore Sigma, USA), and 1.1 mg·mL⁻¹ Fast Red TR salt (ab146351, Abcam, USA) for 2 h at 37 °C. Nuclei were then counterstained with hematoxylin (ab245880, Abcam, USA) for 5 min before mounting with VectaMount™ AQ Mounting Medium (Vector Laboratories, USA). Slides were visualized using an inverted phase microscope (BZ-X800E, Keyence, USA).

Cells were cultured on coverslips at 60–80% confluence and treated with the indicated drugs for 24 h. After that, cells on the coverslips were fixed with 3.7% formaldehyde in complete DMEM medium for 30 min and subsequently stained with 250 nM mitotracker deep red dye for 30 min. After incubation, the coverslips were washed twice in PBS and then embedded in VectaMountAQ mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA) and finally attached on glass slides. All images were obtained under the fluorescence microscope (BX51-DSU; Olympus, Tokyo, Japan).