The level of significance was set at 0.05. Statistical analyses were performed using SAS 9.1 statistical software (SAS Institute Inc., Cary, NC, USA). Results Inhibition of cell proliferation and colony formation Baicalin inhibited the proliferation of CA46 cells in a concentration- and time-dependent manner, with almost complete

inhibition observed at 48–96 h of treatment with 20–40 μM drug (Figure 1A). An IC50 of 10 μM was obtained (Figure 1B). After 48 h of treatment, rates of proliferation declined in a baicalin concentration-dependent manner, with 15.5 ± 4.7% and 89.4 ± 2.8% inhibitions observed at 5 and 40 μM drug, respectively. Baicalin also suppressed formation of colonies of CA46 cells at 10 days post-seeding (Figures 2A and 2B). Control preparations formed colonies at a rate of 36.2 ± 4.0%. In contrast, rates of colony formation for

RG7112 preparations treated with baicalin at 5 and 10 μM were 14.0 ± 2.3% and 0.5 ± 0.5%, respectively (P <0.01). Figure 1 Proliferation of CA46 cells in the absence and presence of baicalin. Cells were seeded at a density of 1 × 104/well and treated with baicalin at the concentrations and for the times indicated. Cytotoxicity was determined according to the MTT assay. Sampling was performed in triplicate for each experimental condition, and findings are expressed as means ± standard deviation for three independent experiments. (A) Proliferation Y-27632 in vitro as a function of incubation time and baicalin concentration. Absorbance maxima are provided on the ordinate. (B) Rates of proliferation as a function of baicalin concentration. Cells were treated for 48 h with baicalin at the concentrations indicated. Proliferation rates were determined as described in Materials and methods. *P <0.01 compared to the solvent

control; † P <0.01 compared to 5 μM baicalin; ‡ P <0.01 compared to 10 μM baicalin. Figure 2 Formation of CA46 cell colonies after treatment with baicalin at varying concentrations. Cells (4 × 102/well) were cultured with baicalin at the indicated concentrations for 10 days. Colony formation rates were determined as described in Materials and methods. Sampling was performed in triplicate for each experimental condition. (A), phase GSK3235025 contrast inverse microscopy. (B), colony formation rates with findings presented as means ± standard deviation for three independent experiments. *P PtdIns(3,4)P2 <0.01 compared to the solvent control; † P <0.01 compared to 2.5 μM baicalin; ‡ P <0.01 compared to 5 μM baicalin. Induction of apoptosis The percentage of CA46 cells undergoing apoptotic cell death was increased by baicalin in a concentration-dependent manner (Figure 3A-D). The percentages of all cells in apoptosis, as determined by the sum of cells in early and late apoptosis, at various baicalin concentrations are presented in Figure 3E. After 48 h of treatment, 15.2 ± 1.6% of cells were apoptotic at 10 μM baicalin and 35.4 ± 2.6% of cells were apoptotic at 40 μM baicalin.