For apoptosis analysis, cancer cells were treated with vehicle or various concentrations of CYD19 for 48 hours, and the percentage of apoptotic cells was determined by the fluorescein isothiocyanate annexin V apoptosis detection kit I (BD Biosciences, #556547) according to the manufacturer’s instructions. The cell apoptosis was analyzed with FlowJo software. For ALDH activity analysis, tumors were chopped into small fragments (around 1 mm3), digested into single-cell suspension by incubation in digestion buffer [0.1% collagenase type 2 (Sigma-Aldrich, #C6885) and deoxyribonuclease I (3 U/ml; Sigma-Aldrich, #D5025)] for 30 min at 37°C, and then filtered with a 40-μm nylon mesh to remove cell clumps. The single-cell suspensions or cancer cell lines were subjected to serial incubations with an antibody cocktail containing CD31, CD45, and Ter119 (STEMCELL Technologies, #19757C.1); a secondary biotin-labeled antibody cocktail (STEMCELL Technologies, #19153); and magnetic beads (15 min each) on ice (STEMCELL Technologies, #19150). The unbound cells were collected, and the bound cells were discarded. Cells were washed extensively and subjected to ALDH activity assay using a kit from STEMCELL Technologies according to the manufacturer’s instructions. For each sample, half of the cells were treated with diethylaminobenzaldehyde (DEAB), and the other half were incubated with an activated ALDEFLUOR reagent. Gating was established using fixable viability dye exclusion for viability, and DEAB-treated cells were used to define negative gates. Flow cytometry data were collected with a MACSQuant flow cytometer (BD Biosciences), and analysis was conducted using FlowJo software.

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