Epon 816

Transmission electron microscopy (TEM) was used to characterize CuONPs morphology. Briefly, a few drops of CuONPs solution in deionized water were added to carbon-coated copper electron microscope grids and observed under a TEM (Hitachi-7500, Japan) after drying at room temperature. Cell ultrastructure was also observed under TEM. After treatment and subsequent trypsinization, cells were centrifuged at 1200 rpm/min for 5 min and fixed using 4% glutaraldehyde for 2 h at 4°C. Then, cells were postfixed using 1% osmium tetroxide for 1 h at 4°C, dehydrated in a graded series of alcohol and acetone and embedded in Epon 816 (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections were prepared using a Leica ultramicrotome (Leica Microsystems, Buffalo Grove, IL, USA) and stained with uranyl acetate-lead citrate. TEM images were obtained using a JEM-1400Plus transmission electron microscope (JEOL Ltd. Tokyo, Japan).

Cells were harvested and centrifuged at 1,000 × g for 10 min at room temperature. The cell pellet was fixed with 4% glutaraldehyde for 24 h at 4°C and 1% osmium tetroxide for 2 h at 4°C. Thereafter, the cell pellet was dehydrated in a graded series of alcohol and acetone, followed by embedding in Epon 816 (Electron Microscopy Sciences). Ultrathin sections were cut by a Leica ultramicrotome (Leica Microsystems, Inc.) and stained with uranyl acetate for 4 min and followed by lead citrate for 2 min at room temperature. The exact thickness of the ultrathin section of the cell samples under the transmission electron microscope was 40 nm. TEM was conducted using a JEM-1400Plus transmission electron microscope (JEOL, Ltd.). The mean number of autophagosomes in tumour cells was counted and quantified.