β galactosidase

Cells (2 × 10⁵/well) were plated in 6-well plates in triplicate in media containing 10% FBS + 2 ng/mL FGF2 and then treated with DMSO, or 1 µM fulvestrant or 1 µM fulvestrant plus 1 µM palbociclib ± 1 µM lucitanib. Media, FGF ligands, and drugs were replenished every 3 days. After 7 days, cells were stained with β-galactosidase (Cell Signaling Technology) at pH 6.0 following the manufacturer’s protocol. Cells were photographed and β-galactosidase-positive cells were counted manually using a light-field microscope.

Three groups of transfected BM-MSCs (2 × 10⁴) were plated into 6-well plates, with three replicates per group. When cells reached 50% confluence, the senescence levels were analyzed by β-galactosidase staining (9860, Cell Signaling Technology). After the medium was discarded, cells were fixed with diluted 1x fixative solution for 15 min. The fixative cells were rinsed twice with 1x PBS and then stained with 1 mL staining solution (pH 7). The staining was completed after the final incubation at 37°C in a dry incubator for 24 h. The positively stained cells were observed under a microscope. Five randomly selected fields were used for subsequent counts and statistical analysis.