Ab125145

For the immunostaining procedure, after senescence induction and incubation with SCA^® for 48 h, the wells were washed with PBS and incubated with 0.5 mL of paraformaldehyde (4%) in PBS for 15 min at room temperature. After washing twice with PBS, a Triton X-100 (0.1%) solution with 2% of BSA in PBS was used to permeabilize the cells for 10 min at 4 °C, and 25 mL of BSA (1%) in PBS for 1 h at room temperature was used to block unspecific binding. Primary antibodies anti-a-tubulin (ab195887, 1:250, Abcam, Cambridge, UK), anti-SIRT1 (19A7AB4, 0,5 μg/mL, Abcam, Cambridge, UK), anti- SIRT6 (MA5-24768, 1:100, Thermo Fisher Scientific Inc., Waltham, MA, USA), anti-mTOR (AHO1232, 1:500, Thermo Fisher Scientific Inc., Waltham, MA, USA), anti-CML, N6-carboxylmethyl-lysine (ab125145, 1:200, Abcam, Cambridge, UK), and anti-procollagen type I (PCIDG10, 1:200, Sigma-Aldrich, St. Louis, MO, USA) (diluted with 1% BSA) were used to probe the tissues at 4 °C overnight. Subsequently, the secondary red fluorescence antibody (Alexa Fluor 568, 1:200, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to reveal the specific binding of the primary antibody. Finally, a nuclear counterstaining was performed with Hoechst solution (Hoechst 33258, 1:2000, H1398, Invitrogen by Thermo Fisher Scientific Inc., Waltham, MA, USA) for 20 min at room temperature.

The human osteosarcoma tissue microarray OS804d was purchased from US, Biomax Inc. containing 40 cases of osteosarcoma. We performed IHC staining using a commercial IHC kit (cat. no. ab64264, Abcam, Cambridge, U.K.), following the manufacturer's procedural guidelines. Tissue sections were removed the wax using Sub-X and then rehydrated with a series of ethanol solutions. Antigen retrieval was performed by incubating slides with protease, and then blocking with protein block reagent for 10 minutes. The CML (cat. no. ab125145, Abcam) and RAGE (cat. no. ab3611, Abcam) antibodies were incubated with the sections overnight at 4°C. HRP-conjugated secondary antibody was then applied, and the sections were treated with DAB substrate. Subsequently, the slides were counterstained with hematoxylin, and sealed with mounting medium. The stained sections were analyzed using the IHC profiler plugin in ImageJ software. A score of 0 indicated negative staining, score of 1 indicated low positive staining, score of 2 indicated positive staining, and score of 3 indicated high positive staining.