Figure 6.

YMGKI-1 treatment dysregulates the autophagy and diminishes the sphere formation ability in HN-CICs. (a) Crude cell extract proteins of YMGKI-1-treated SAS-HN-CICs were collected, electrophorized, and analyzed by immunoblotting against anti-LC3, anti-phosphor-mTOR, anti-HER2, PI3K, anti-phosphor-p44/42 MAPK, anti-phosphor-p38, or anti-GAPDH serum as indicated. (b) Crude cell extract proteins of SAS-HN-CICs singly treated with YMGKI-1 or cotreated with 3-MA were isolated and analyzed by immunoblotting against anti-phosphor AMPK or anti-GAPDH serum as indicated. (c) SAS-HN-CICs were treated with metformin (10 mM) or rapamycin (100 nM) for 96 hr. Consequently, immunoblotting analysis was performed by against anti-phosphor-AMPK, anti-phosphor-mTOR, and anti-LC3 or anti-GAPDH serum as indicated. The immunoactive signal of GAPDH protein of different crude cell extracts was referred as loading control. (d) Single cell suspension of SAS-HN-CICs was treated with YMGKI-1 or cotreated with 3-MA for 24 hr, and the sphere formation ability of YMGKI-1 or cotreated with 3-MA-treated HNCICs cells was examined. Arrows indicated the sphere cells. Data are means ± SD of triplicate samples from three experiments (***P < 0.005). The same concentration (0.03%) of vehicle (DMSO) was added to all control groups.