Antifading mounting medium with dapi

The TUNEL apoptosis assay kit (40307ES20, Yeasen Biotechnology Co, Ltd. Shanghai, China) was used to examine apoptosis of human CC cells. Human CC cells were seeded in 12-well plates containing climbing sheets for 24 h. The experimental group was exposed to 5mM butyrate for 48 h and then washed three times with PBS. Treated cells were fixed with 4% paraformaldehyde for 30 min, resuspended in PBS with 0.3% Triton X-100 and incubated at room temperature for 5 min. TUNEL solution (50µL) was added to the sample, followed by incubation at 37 °C for 60 min in the dark. Antifading mounting medium (with DAPI, Solarbio Science & Technology Co., LTD. Beijing, China) was used for sealing and the stained CC cells were examined with a fluorescence microscope (Becton Dickinson, FACSCantoII).

Cells were seeded on polylysine-coated coverslips and then exposed to 6 Gy IR after adherence. After 48 h, the cells were fixed in 4% paraformaldehyde at RT for 15 min, permeabilized with 0.25% Triton X-100 at RT for 10 min, blocked with 5% bovine serum albumin (BSA) at RT for 1 h, and then incubated with primary antibody at 4 ℃ overnight. The next day, the cells were incubated with secondary antibodies (away from light, RT, 1 h). Finally, the cells were washed with PBS 3 times and mounted with anti-fading mounting medium (with DAPI) (Solarbio) to stain the nuclei. Images were acquired with Zeiss LSM800 fluorescence microscope (Jena, Germany). Antibody details are shown in Additional file 5: Table S2.