Figure 1. Maintenance of stemness population and property is observed within acquired lapatinib-resistant clones.
A. Total RNA was extracted from SkBr3, SkBr3/Lap#6 and SkBr3/Lap#9 cells and subjected to RT-PCR. mRNA expression of IL6 and internal control ACTIN was examined by 1% electrophoresis. B. Media from SkBr3, SkBr3/Lap#6 and SkBr3/Lap#9 cells were collected to examine IL-6 production by ELISA. Statistical analysis was performed by Student's t test. ***, p<0.001 as compared to control group. C. SkBr3, SkBr3/Lap#6 and SkBr3/Lap#9 cells were cultured at a density of 2 × 104 cells/ml in ultra-low attachment plates with spheroid-inducing medium. Cells were treated with 1 μM lapatinib for 7 days, and spheroid formation observed under microscope. D. SkBr3 and SkBr3/Lap#6 cells were treated with 1 μM lapatinib for 1 day, then re-seeded and 2 × 106 cells per group were subjected to an ALDH activity assay. Statistical analysis was performed by Student's t test. **, p<0.01; ***, p<0.001 as compared to control group. E. The whole cell lysates of both SkBr3 and SkBr3/Lap#6 cells were harvested and subjected to western blot analysis with indicated antibodies. F. SkBr3 and SkBr3/Lap#6 cells were fixed and subjected to flow cytometry with indicated antibodies.