Cell apoptosis assay

For parental cell apoptosis assay, HT29 and HCT116 cells were seeded into 6-well plates (2×10⁵/well) and exposed to the fenretinide treatments with indicated concentration for indicated time. Absolute ethanol was used as the negative control. The treated cells were dissociated with 0.25% trypsin for 3 min at 37°C, collected in BD falcon centrifuge tubes, washed with Annexin V binding buffer (BD Biosciences, San Jose, CA, USA) and centrifuged at 350 × g for 5 min. Washing and centrifugation was repeated twice. Then, the samples were incubated with 5 µl fluorescein isothiocyanate (FITC)-stained Annexin V antibody and 5 µl propidium iodide stain for 15 min at room temperature according to the protocol of the manufacturer of the Apoptosis Detection Kit (BD Biosciences; cat. no. 556547), and detected and analyzed by flow cytometry using the FC500 flow cytometer (Beckman Coulter, Inc., Brea, CA, USA). For the sphere cell apoptosis assay, the sphere cells from HCT116 and HT29 were cultured in SFM, trypsinized, and seeded into 6-well plates with 2×10⁵ cells/well. Following treatment with 3 µm fenretinide, sphere cells were collected and centrifuged with 1,000 × g for 5 min at room temperature. Cell apoptosis was detected using a Fluorescein Isothiocyanate (FITC)-Annexin V Apoptosis Detection kit (BD Biosciences) according to the protocol of the manufacturer, and analyzed by flow cytometry. The total percentage of Annexin V^+/PI^–/+ cells was quantified and CXP Analysis software (version 1.0; Beckman Coulter, Inc., Brea, CA, USA) was used to analyze the apoptosis data.