L calc version 1

The proliferation and cell cycle transition of hepatoma cells treated with ψ-Bufarenogin were determined as previously described [33 (link)]. The apoptosis of hepatoma cells triggered by ψ-Bufarenogin was examined by Vybrant Apoptosis Kit (Molecular Probes, Eugene, OR) and flow cytometry. To perform an anchor-independent growth assay, hepatoma cells were plated at 1 × 10⁴ cells per 60-mm dishes in DMEM containing 10% FBS and 30% (V/V) matrigel at the presence or absence of ψ-Bufarenogin. After 2 weeks, the multicellular colonies were counted under a microscope. For spheroid formation assay, primary hepatoma cells from patients were plated at 3 × 10³/ml in Corning 3261 ultra-low attachment culture dishes followed by ψ-Bufarenogin treatment. One week later, the spheroids formed were counted under the microscope. For limiting dilution assay, hepatoma cells were seeded into 96-well ultra-low attachment culture plates for 7 days. Spheroid formation was assessed by visual inspection. Based on the frequency of wells without colony, the proportion of stem cells was determined by using Poisson distribution statistics and L-Calc Version 1.1 software (Stem Cell Technologies, Inc., Vancouver, Canada) [34 (link)].