Cally2R treatment induces autolysosome fusion with suppression of the mTOR/AKT pathway and activation of AMPK. (A) The typical TEM images of autophagic vacuoles in SKBR3 and MDA-MB-453 cells after treatment of 3 mM metformin or 100 nM cally2R. Scale bar = 2 μm. (B) TEM images of the three stages of autophagic vacuoles in colon (HT29) and breast (SKBR3) cancer cells after cally2R treatment for 24 h. Stage (I): A cup-shaped membrane structure enclosing a portion of cytoplasm. Stage (II): Visible autophagic bodies in the vacuole. Stage (III): Autophagic bodies in the vacuole, showing degradation of the segregated cytoplasm. Scale bar = 2 μm. (C) Immunocytochemistry analysis by staining with anti-LC3 antibody (green) and anti-LAMP2 antibody (red) shows the formation of autolysosomes (merged as yellow) after cally2R treatment in SKBR3 cells. Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm. (D) Immunoblotting showing protein levels of mTOR pathway and mitophagy after cally2R treatment. C, cytosol; M, mitochondria; N, nucleus; yellow arrow, mitochondria in autophagosome; red arrow, autophagosomes; blue arrow, autolysosomes.