Ix71 ix2

Rat IEC-6 cells (#CRL21592, purchased from Peking Union Medical College, Beijing, China) were cultured in Dulbecco’s modified Eagle medium containing 5 % (v/v) fetal bovine serum (HyClone, Logan, UT, USA), 2 mg/l insulin, 50 IU/ml penicillin and 50 μg/ml streptomycin and incubated at 37 °C under 5 % (v/v) CO2. The medium was replaced 24 h following initial cell plating. Control group cells were kept strictly at 37 °C under 5 % CO2, while cells of the heat treatment groups were exposed to 42 °C under 5 % CO2 in the incubator (Thermo, Marietta, Ohio, USA) for 15 min, 30 min 1 h, 2 h, 4 h, and 8 h, respectively. To inhibit specific intracellular agents, cells were pretreated with 10 μM SP600125 (JNK inhibitor, #1496, Tocris Bioscience, Bristol, UK) for 1 h prior to heat treatment. Changes in cell morphology following all treatments were observed using a phase-contrast inverted biological microscope (IX71/IX2, Olympus).

Exponentially growing HepG2 cells (1.0 × 10⁵ per well) were seeded into 6-well culture plates and incubated with various concentrations of nano-CeO₂ (6.25, 12.5, 25, 50, 100 μg/mL) for 24 h. Changes in gross cellular morphology after each treatment were observed using a phase-contrast inverted biological microscope (IX71/IX2, Olympus, Tokyo, Japan). After that, the cells from each sample were harvested and washed with phosphate-buffered saline (PBS). After resuspension, the HepG2 cells were fixed in 3% malondialdehyde fluid. The samples were prepared for transmission electron microscopy (TEM) and the internal structure changes of cells were observed by TEM (JEOL JEM-1200EX, JEOL Ltd., Tokyo, Japan).

Briefly, cells were treated with JUA or ISO according to the experimental design. Following each specific treatment, cell morphology was observed using a phase-contrast inverted biological microscope (IX71/IX2, Olympus, Japan), scanning electron microscopy (SEM; S-3400N, Hitachi, Tokyo, Japan), and transmission electron microscopy (TEM; 1230, JEOL, Tokyo, Japan). For ultrastructure studies, cardiomyocytes were harvested and fixed with 3.0% glutaraldehyde and 1.5% paraldehyde, washed three times in PBS, and postfixed in cold 1% osmium tetroxide. After dehydration with graded series of alcohol, all samples were freeze-dried, coated in a sputter-coater with a layer of gold embedded in epoxy resin (EPON812, Emicron, Shanghai, China), and examined using a Hitachi S-3400N scanning electron microscope operated at 15 kV. Other ultrathin sections were stained with saturated uranyl acetate in 50% ethanol and lead citrate, and the ultrastructure of the H9C2 cardiomyocytes was examined by TEM.