Apollo reaction cocktail

EdU‐labeled HSV‐1 were obtained by propagating HSV‐1 in Vero cells cultured in DMEM containing 10% FBS and 25 µm EdU (C00052, RIBOBIO). The cells and the supernatants were collected for three freeze‐thaw cycles to obtain EdU‐labeled HSV‐1. The BMDCs were incubated with EdU‐labeled HSV‐1 and cell Mask Green at 4 °C for 1 h (attachment) or at 37 °C for 15–30 min (penetration). Subsequently, the cells were then fixed in 4% paraformaldehyde and stained with Apollo reaction cocktail (C00031, RIBOBIO) at room temperature for 30 min. Images were captured on a Leica SP8 fluorescence microscope and processed with the ImageJ software for quantitative analysis.

Use 5-ethyl-2'-deoxyuridine (EdU) detection kit (EdU kit; Ribobio, Guangzhou, China) for determining DNA synthesis. Cells were firstly transfected, incubated with EdU for 2 h, and collected. Then, fix the cells in 4% formaldehyde for 20 min and permeabilize the cells. Wash the cells with PBS, treat the cells with 200 μl Apollo ® reaction cocktail (Ribobio) for 10 min, and permeabilize the cells. Stain the cells with 100 μl Hoechst 33,342 (5 μg/ml; Thermo Fisher Scientific). Observe the cells under a fluorescence microscope (Olympus, Tokyo, Japan).

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.

After transfection for 48 h, intramuscular adipocytes were incubated at 37°C with 50 μM EdU (RiboBio, China) for 2 h, then cells were fixed with 4% PFA for 30 min and neutralized by 2 mg/mL glycine solution, permeabilized with 0.5% Triton X-100. Then cells were incubated with Apollo Reaction Cocktail (RiboBio, China) for 30 min at room temperature. The DNA was stained with DAPI (Beyotime) for 15 min. The EdU-positive cells were observation with a fluorescence microscope (Nikon, Tokyo, Japan).

Cells were transfected with siRNAs (Mock-siRNA, siL21-1 and siL21-2) at a concentration of 40 nM for 72 h. The transfected cells were exposed to 50 μM of 5-ethynyl-2′-deoxyuridine (EdU) (RiboBio, Guangzhou, China) for 2 h at 37°C, and the cells were fixed in 4% paraformaldehyde for 30 min at room temperature, followed by the addition of 2 mg/ml glycine to neutralize the reaction. After permeabilization with 0.5% Triton-X, the cells were reacted with Apollo reaction cocktail (RiboBio, Guangzhou, China) for 30 min in the dark at room temperature. Subsequently, the DNA contents of the cells were stained with Hoechst 33342 for 30 min and visualized under a laser scanning confocal microscopy (Leica Microsystems, Buffalo Grove, IL, United States).