fig2

Figure 2. BJ affected the growth of multicellular H1975 spheroids. (A) Soft agar colony-forming assay. The multicellular spheroids treated with 5, 10, and 15 mg/mL of BJ extract were grown in soft agar for 28 days and the plates were stained with crystal violet, as described in “Methods”. (B) Statistical analysis. The number of positive soft agar colonies of H1975 spheroids under each concentration was counted and plotted. Colonies containing more than 50 cells were counted as positive. **P < 0.01 indicates a significant difference between BJ treatment groups and water control from three independent experiments. (C) BJ extract reduced H1975 spheroid stemness markers. Cell lysates from H1975 spheroids as treated with BJ extract for 12 h were subjected to Western blot analysis. The blots were incubated with primary antibodies and analyzed for expressions of apoptosis and stemness markers with GAPDH as loading control, as described in “Methods”. The numbers underneath indicate the relative intensities compared with water treatment.