Tunel reagent

The terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay is often used to detect the level of DNA damage. The cells were fixed with 4% paraformaldehyde for 25 min at 37 °C, incubated with 0.1% Triton X-100 for 8 min, and washed with PBS twice between each step. The cells were incubated with TUNEL reagent (Beyotime, China, catalog No. C1088) according to the manufacturer’s instructions, and the nuclei were counterstained with 4',6'-diamidino-2-phenylindole (DAPI, Sigma, United States, catalog No. D-9542) after the required time. The samples were observed and imaged under a fluorescence microscope (Olympus Europe GmbH, Germany).

To investigate the effects of DSG on the apoptosis of podocyte cells, a TUNEL assay (cat. no. C1082; Beyotime Institute of Biotechnology) was employed. Briefly, CIHP-1 cells (2x10⁴ cells/well) seeded into a 24-well plate were fixed with 4% paraformaldehyde for 15 min and permeabilized in 0.25% Triton X-100 for 20 min at room temperature. After rinsing in phosphate-buffered saline (PBS; cat. no. C0221A; Beyotime Institute of Biotechnology) three times, the cells were labeled with TUNEL reagent for 60 min at 37˚C, according to the manufacturer's instructions. DAPI staining solution (1 mg/ml; cat. no. C1002; Beyotime Institute of Biotechnology) was utilized to counterstain the cells at 37˚C for 30 min, which were then mounted in an anti-fade reagent (Beijing Solarbio Science & Technology Co., Ltd.). Finally, the images of TUNEL-positive cells were observed in five fields of view selected at random under a fluorescence microscope.

The media of prepared HSFs were discarded, and pre-cooled methanol was added to fix the cells for 30 min at room temperature. Cells were incubated with PBS containing 0.2% Triton X-100 for 15 min at room temperature for permeabilization, and with TBST (0.5% Tween-20) containing 1% BSA for 30 min at room temperature for blocking. For Ki67 staining, the cells were incubated with a primary anti-Ki67 antibody (1:100; cat. no. ab197234; Abcam) at 4˚C overnight followed by incubation with the corresponding CY3-conjugated secondary antibody (1:2,000; cat. no. ab6939; Abcam) at 37˚C for 2 h. For TUNEL staining, the cells were incubated with a TUNEL reagent (Beyotime Institute of Biotechnology) at 37˚C for 1 h. Finally, after the nuclei were counterstained with DAPI (1:1,000; Beyotime Institute of Biotechnology) for 10 min at room temperature, the slides were mounted with Antifade Mounting Medium (Beyotime Institute of Biotechnology). Images of each slide were captured at three random fields of view using an inverted fluorescence microscope (magnification, x100; IX73; Olympus Corporation). Total nuclei (blue) and Ki67 or TUNEL-positive (red) cells were quantified using ImageJ software (version 1.51w; National Institutes of Health).