5 ethynyl 2 deoxyuridine (edu)

For EdU cell proliferation assay, cells were seeded in 96-well plates. After transfection for 48 h, the cells were incubated at 37°C for 2 h in the presence of 50 μM EdU (RiboBio, Guangzhou, China). The cells were then fixed 4% paraformaldehyde for 30 min and neutralized using 2 mg/mL glycine solution for 5 min. Then the cells were permeabilized by 0.5% Triton X-100 for 10 min. The solution with EdU (RiboBio, Guangzhou, China) was added and the cells were incubated at room temperature for 30 min. Subsequently, the cells were permeabilized 3 times with 0.5% Triton X-100 for 10 min each time and then added with methanol for 5 min to rEdUce the dye background. The nuclear stain Hoechest 33342 was then added and incubation for another 30 min. Three randomly selected interfaces were captured using a fluorescence microscope (DMi8; Leica, Germany) to visualize the number of EdU-stained cells.

DNA synthesis during cell proliferation in each group was quantified by incorporating EdU (Invitrogen, Carlsbad, CA, USA) using a Cell-lightTM EdU Kit (Rui Bo Biotechnology Limited Company, Guangzhou, China), as described in our previous studies. Initially, IPEC-J2 cells were cultured in DMEM containing 50 μM EdU for 1 h. A Leica DMI4000B microscope (LEICA, Wetzlar, Germany) was used to capture an image of EdU-positive cells counter-stained with Apollo^® 567 fluorochrome (Invitrogen, Carlsbad, CA, USA). The percentage of EdU-positive cells was expressed as the ratio of cells with red nuclei in at least five separate microscopic fields randomly selected for counting.

Human SSC line was incubated in 96-well plates (5000 cells/well), and each well had DMEM/F12 medium with 50 μmol/L EdU (RiboBio, Guangzhou, China). Cells were incubated for 12 h, washed with DMEM, and fixed in PFA (40 g/L). After neutralization using 2 mg/mL glycine, cells were permeabilized with 5 mL/L Triton X-100 for 10 min at room temperature. Apollo staining EdU staining was then displayed with reaction buffer, while Hoechst 33,342 was employed to stain cellular nuclei. Fluorescent microscopy (Leica, Wetzlar, Germany) was used for imaging, and the percentages of EdU-positive cells were determined by counting ≥500 cells.

The GCs were seeded in 12-well plates (1× 10⁶ viable cells/well in 12-wells). When the cells grew to a density of 50% confluence, they were transfected with overexpression plasmids or siRNA. After transfection for 24 h, GCs were exposed to 50 μM EdU (RiboBio, China) for 2 h at 37 °C. Subsequently, the cells were fixed in 4% paraformaldehyde for 30 min, neutralized using 2 mg/mL glycine solution, and then permeabilized by adding 0.5% Triton X-100. A solution containing EdU (Apollo Reaction Cocktail; RiboBio, Suzhou, China) was added, and the cells were incubated at room temperature for 30 min. The nuclear stain Hoechst 33,342 was then added and incubation was continued for another 30 min. A fluorescence microscope (DMi8; Leica, German) was used to capture three randomly selected fields to visualize the number of EdU-stained cells.

PTr2 cells were seeded in 24-well plates (50,000 cells/well), and after being cultured overnight, they were transfected with pcDNA3.1(+), pcDNA3.1(+)-PAG-2, siRNA-NC, or siRNA-PAG-2. After transfection for 48 h and 72 h, PTr2 cells were exposed to EdU (BeyoClick, China) for 3 h at 37°C. Subsequently, the cells were fixed in 4% paraformaldehyde for 15 min, were wiped with washing solution, and then permeabilized by adding 0.3% Triton X-100. Next, plates were washed with PBS, 0.3 ml of Click was added and the cells were incubated at room temperature in the dark for 30 min. The nuclear stain DAPI was then added, and a confocal laser scanning microscope (Leica, Germany) was used to photograph and visualize the number of EdU-stained cells.