His tag

The cells were lysed in a buffer containing 0.1% Triton (Bio-Rad, CA, USA) for 30 min on ice. Each lysate (20 μg) was then electrophoresed in a polyacrylamide gel and transferred onto a nitrocellulose membrane. The primary antibodies used were IGFBP5-D6 (cod. sc-515116, Santa Cruz Biotechnology, TX, USA), IGFBP5-C18 (sc6006, Santa Cruz Biotechnology, TX, USA), His-Tag (Elabscience, TX, USA), Histone H4 (2935S, Cell Signaling, MA, USA), GAPDH (Sigma Aldrich, MO, USA), RARα (E-AB-93398, Elabscience, TX, USA), and RXRα (sc515929, Santa Cruz Biotechnology, MO, USA). Immunoreactive signals were detected using a horseradish peroxidase-conjugated secondary antibody (ImmunoReagents, NC, USA) and reacted with ECL plus reagent (Merck Millipore, MA, USA). All antibodies were used according to the manufacturer's instructions.

MSCs were cultured on cover slides in a 24 multi-wells plate. The Duolink assay was performed following the manufacturer's instructions (Sigma Aldrich, MO, USA) using His-Tag (Elabscience, TX, USA) and CAV1 (Elabscience, TX, USA), or RARα (E-AB-93398, Elabscience, TX, USA) as primary antibodies. Micrographs were captured under a fluorescence microscope (Leica, Germany).

For the beta-galactosidase assay, 20,000 cells per well were seeded in 24-well plates with a glass coverslip. After the treatments, the cells were fixed in a 2% formaldehyde solution for 10 min. Then, cells were washed with PBS 1X (Microgem, Italy) and incubated at 37°C overnight with a staining solution composed of citric acid/phosphate buffer (pH 6), K4Fe(CN)6, K3Fe(CN)6, NaCl, MgCl2, and X-Gal. Next, the cells were permeabilized with 0.3% Triton-X100 (Roche, Switzerland) on ice for 5 min, followed by adding a blocking solution (5% FBS in PBS and 0.1% Triton-X100) for 1 h at room temperature (RT). Subsequently, the cells were incubated with the following primary antibodies: Ki67 (1:200, sc7846, SantaCruz Biotech, TX, USA), or γH2AX (1:600, 9718S, Cell Signaling, MA, USA), and HisTag (1:1000, E-AB-48003, ElabScience, TX, USA) at 4°C overnight in the blocking solution. The TRITC or FITC-conjugated secondary antibody, goat anti-mouse (1:400, Gtx-Mu-003D594), was obtained from ImmunoReagents (Raleigh, NC, USA). Nuclear staining was performed using DAPI mounting medium (ab104139, ABCAM, UK), and micrographs were captured under a fluorescence microscope (Leica DM2000-DMC5400, Leica, Germany).