Zen 3.0 blue edition imaging software

For immunofluorescence analysis, the cells were incubated in 24-well plates with or without treatment with radiation at a dose of 6 Gy. Cells were then fixed with 4% paraformaldehyde (Biosharp) for 30min, permeabilized with 1% TritonX-100 (Biosharp) for 30min, blocked with 5% BSA for 1 h, and then incubated overnight at 4°C with primary antibodies (anti-γ-H2AX (Abcam; ab26350, dilution 1:500), anti- Vimentin (Cell Signaling Technology, 5741, dilution 1:500), and primary antibodies were diluted by primary antibody dilution buffer for immunol staining (Beyotime). Subsequently, the cells were incubated for 1 h at 37°C with the secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Solarbio, Beijing, China). Three areas were selected randomly for photography and calculation. Single-staining and merged images were acquired using laser confocal microscopy (LSM 900 with Airyscan 2, Carl Zeiss, Jena, Germany) using Zen 3.0 (blue edition) imaging software (Carl Zeiss).

The EDU assay was performed using an EDU Kit (Beyotime), according to the manufacturer’s instructions. 5 × 10⁴ cells/well Kyse150 and TE1 cells transfected with si-negative-control or si-NRP1 were seeded onto 12-well plates, irradiated with 4 Gy, and cultured for 48 h, and then, the medium was switched to fresh DMEM supplemented with EDU (50 mmol/L). The cells were then incubated for 2 h, followed by fixation, permeabilization, and EDU staining with Azide 488 (Beyotime). After staining cell nuclei with DAPI, five areas were selected randomly for photography and calculation. And the EDU-positive cells were identified using Zen 3.0 (blue edition) imaging software (Carl Zeiss).