Hoechst 33342 staining

Cells were plated into 6-well plates at a density of 1×10⁵ cells per well and pretreated with 1 mM 3-MA (Selleck Chemicals) for 2 h, prior to treatment with muscone. Cells were rinsed three times with PBS and then stained with 10 µg/ml Hoechst 33342 (Sigma-Aldrich; Merck KGaA) for 15 min at 37°C before being washed three times with PBS for a second time. Stained cells were observed under a fluorescence microscope at an ×200 magnification (Olympus IX71). Apoptotic cells were identified by the fragmentation of nuclei and condensation of chromatin, and the apoptotic index was calculated using the following equation: Number of apoptotic cells/total cell count. Experiments were performed in triplicate.