Figure 4.

NANOG overexpression counteracts the suppressive effect of ugonin J on propagation of breast cancer stem cells. (a) Control and NANOG-overexpressing MCF-7 cells (1 × 10⁴ cells in 24-well plates) were treated with ugonin J for 3 days prior to mammosphere formation in mammosphere forming medium for 28 days. Total mammospheres were counted under a microscope at days 15 and 28. Mean of three independent experiments ± SEM. ***P < 0.001 versus control mammospheres. (b) Invasion assay of MCF-7 was performed on matrigel with or without ugonins J or K treatment. Data was shown as mean ± SEM from three independent experiments. ***P < 0.001, **P < 0.01 versus control. (c) Control, NANOG-overexpressing cells (4 × 10⁵ cells in 6-well plates), mammospheres, and NANOG-overexpressing mammospheres (1000 spheres in 6-well plates) were treated with ugonin J for 24 hours before protein extraction. Western blot probed for STAT3 and phospho-STAT3 Tyr705. Equal amounts of protein were used (40 μg per lane). (d) Mammospheres were formed for 15 days from control and NANOG-overexpressing mammospheres (20 spheres in 96 plates) before treatment with or without 10 μM ugonin J for 24 hours. Medium was collected and analyzed by ELISA to determine the production of IL-6 (n = 3). Data was shown as mean ± SEM. *P < 0.05 versus control.