Cells and reagents
Human laryngeal squamous carcinoma cell line Hep2 obtained from ATCC, were cultured in RPMI1640 (Hyclone, US) containing 10%FBS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin (Corning, US) at 37°C in a humidified atmosphere of 5% CO2. The cells at logarithmic growth stage were digested by trypsin to produce a single cell suspension, which was planted in a 35 mm culture dish, and then the cells were cultured for 24 h for irradiation with high dose rate single dose radiation (SDR), high dose rate fractionated dose radiation (FDR), and iodine-125 seeds continuous low dose rate radiation (CLDR). The initial dose rate of particle irradiation was 2.77 cGy/h. When the irradiation dose was 4 Gy and 6 Gy, 150.0h and 229.4h were needed respectively. RS2000 X-ray bioradiometer (Rad Source Technologies, USA) was used for high dose rate irradiation with a dose rate of 6312 cGy/h. The single irradiation group was the total dose completed by one irradiation. The fractional irradiation group received irradiation every 24 h, with each dose of 2 Gy.
Colony Forming Assay
Hep2 cells were taken and planted in a 35mm culture dish, which were irradiated for 24 h and then further cultured after the irradiation of 4 Gy and 6 Gy. The cell planting date was set as the 1st day. From the 14th day, the cells of each group were fixed with methanol for 10 min, and the cells were stained with Giemsa-staining solution for 5 min, and then the colony number was counted under conventional microscope. One colony unit was obtained with more than 50 cells and its colony rate (PE) and survival fraction at 2 Grey (SF2) at each clinical irradiation dose. In addition, the calculation formula of PE is :(number of clones/number of cells inoculated) ×100%;The calculation formula of SF2 is :(PE in the irradiation group/PE in the control group) ×100%.
Apoptosis and Cell Cycle Analysis
At 24h, 48h and 72h after irradiation of 4Gy, cells were digested and all of them were prepared into single-cell suspension with a concentration of 5×105 cells per ml. Apoptosis was detected using Annexin-V-FITC Apoptosis Detection Kit (Biolegend, USA) by flow cytometry, according to the manual. For cell cycle analysis, cell suspension was taken, washed twice with pre-cooled PBS, and the pre-cooled 2 ml 75% alcohol was fixed, and then placed at 4℃ overnight. The residual alcohol was washed with PBS twice, and the cells were resuspended with 300μl of PBS. PI and RNaseA were added until the final concentration was 50 ug/mL. The cells were incubated at 37℃ for 30 min in dark. The cell cycle was then measured by flow cytometry (BD celesta, USA).
Immunoblotting analysis
The cells were washed with PBS, lysed with PBS containing protease and phosphatase inhibitors cocktail, and then centrifuged to remove the nuclear fraction. The supernatant was used as the cytoplasmic fraction. To obtain the nuclear fraction for immunoblotting analysis, the cell pellet was processed with the EpiQuik Nuclear Extraction Kit I (Epigentek, USA) according to the manual. The soluble proteins from each sample were separated by SDS-PAGE in a 10% polyacrylamide gel and then transferred onto a PVDF membrane (Millipore, USA). The membrane was blocked with 5% BSA and incubated with the target antibodies. All bound antibodies were incubated with HRP-conjugated anti-rabbit IgG secondary antibody (abcam,USA). The bands were detected by using Immobilon Western (Millipore, USA) and the Las-1000 mini image analyzer (Fujifilm, Tokyo, Japan).
Statistical analysis
Data are described as the mean ± SEM. Statistical analysis and significance were measured by One-way ANOVA. All data were performed using Origin professional software version 2018 (Origin Lab Software, USA). In all comparisons, p values less than 0.05 are considered a statistically significant difference. Statistical analysis was performed using SPSS Statistics (SPSS Inc. Chicago, IL, USA).