Hoechst 33258 solution

HaCaT cells were seeded in 6-well plates and pretreated with 12.5 and 25 mg/L fullerenol for 2 h. Then, the cells were exposed to 50 Gy of X-ray radiation. After 24 h of incubation, the cells were washed with PBS twice and then incubated with 10 μg/mL Hoechst 33,258 solution (Solarbio, Beijing, China) for 30 min at 37 °C. The stained cells were rinsed with PBS three times and assessed by fluorescence microscopy (Olympus IX51, Tokyo, Japan) at 200 × magnification. The stained cells with nuclear fragmentation and condensation were considered apoptotic cells. Finally, the apoptotic cell rate was calculated as follows: apoptotic cell rate (%) = apoptotic cell/total cells × 100%.

H520 or A549 cells were inoculated in 6‐well plate; cells were cultured for 48 hours. 4% paraformaldehyde was used to fix cells and 10 ng/mL Hoechst 33 258 solution (Solarbio, #C0021) was used to stain cells, and the increased condensation of chromatin was observed in apoptotic cells. Apoptotic cells were captured with inverted fluorescence microscope (×100).

Infected C3H10T1/2 cells were cultivated as described above, harvested, washed with phosphate-buffered saline (PBS), and fixed with a mixed solvent of 75% (v/v) methanol and 25% (v/v) acetic acid for 30 min. The fixed cells were washed with PBS for 5 min and stained with Hoechst 33258 Solution (Solarbio, Beijing, China) at room temperature for 15 min. After staining, the cells were washed with PBS three times for 5 min each, and observed under a fluorescence microscope in a mounting medium of 10% (v/v) glycerol in PBS. The percentage of dividing cells per 100 cells was measured and shown in histograms.

Cells (2 × 10⁵/well) were seeded in the 6-well plate, grown for 24 h, and co-incubated with different concentrations of the compound for 48 h. Cells were washed three times with PBS, fixed with 4% paraformaldehyde (v/v) for 5 min at room temperature, and then washed with PBS, stained with Hoechst 33258 solution (Solarbio, Beijing, China) at a final concentration of 1 mg/mL for 10 min at room temperature, finally subjected to UV microscopy immediately with filters for blue fluorescence (Leica, Wetzlar, Germany).

Coix seed was obtained from Liaoyang, Liao Ning Province, China. The CSO was obtained by the ultrasonic-assisted extraction method, using acetone as the extraction solvent, and it was sonicated for 20 min under the conditions of a solid–liquid ratio of 1:20 and an ultrasonic power of 130 W. Standard fatty acid methyl ester (FAME) mixture (#463) was acquired from Nu-Chek Prep Inc. (Elysian, MN, USA). Dulbecco’s modified Eagle’s medium (DMEM) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). 5-Fluorouracil (5-FU) was obtained from Xudong Haipu Pharmaceutical Co., Ltd. (Shanghai, China). Trypsin-EDTA was purchased from Beyotime Institute of Biotechnology (Shanghai, China). A cell cycle staining kit was bought from MultiSciences Biotech Co., Ltd. (Hangzhou, China). Phosphate-buffered saline (pH: 7.2–7.4) (PBS) and Hoechst 33258 solution were bought from Solarbio Science and Technology Co., Ltd. (Beijing, China). An annexin V-FITC apoptosis detection kit was obtained from the Beyotime Institute of Biotechnology (Shanghai, China). Fetal bovine serum (FBS) was bought from Wisent Inc. (Montreal, QC, Canada). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. (Kyushu, Japan). Primary antibodies of Bax, Bcl-2, Caspase-3, PI3K, p-AKT⁴⁷³, Cyclin-B1, and GAPDH were obtained from Abcam (Abcam, Cambridge, UK).