5 ethynyl 2 deoxyuridine (edu)

The pCGs were seeded in a 96-wells plate for 24, 48 or 72 hours, and then incubated with 10 μL CCK8 reagent (Beyotime) for 2 hours. Absorbance at 450 nm was detected by microplate reader (Thermo Fisher Scientific). For EdU staining, cells were seeded in a 48-wells plate, and then incubated with 200 μL EdU (Beyotime) for 2 hours. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Following incubation with Apollo Staining reaction liquid, cells were counterstained with DAPI before measurement under Olympus ZKX53 microscope (Olympus, Tokyo, Japan).

For the EdU incorporation experiment, mice bearing tumors formed by MDA-MB-231 cells with or without Tanshinone IIA pretreatment were received intraperitoneal injection of EdU (Beyotime, Shanghai, China) at a dose of 5 mg/kg body weight every 2 h for three times. 24 h later, mice were euthanized, tumors were removed, and fixed in 4% paraformaldehyde at 4℃ overnight. The tumors were then sectioned at 3 μm from the paraffin-embedded tissue blocks, stained with EdU. The fluorescence intensity of EdU-labeled cells was quantified using fluorescence microscope.

After treatment, cardiac fibroblasts were fixed with 4% paraformaldehyde for 20 min at 4 °C. EdU (Beyotime) was then added and incubated for 2 h. After washing with PBS, cells were incubated with fixative solution, glycine (2 mg /mL) and EdU osmotic solution successively. The cells were then subjected to Apollo staining in the dark at room temperature for 30 min. The nuclei were stained with DAPI for another 30 min. The green fluorescence by EdU and blue by DAPI were observed under a fluorescence microscope (EVOS^TM Thermo-fisher, USA). Image J 1.50 was used for quantitative analysis.

EDU (Beyotime, C0081s) was first diluted into a DMEM/F12 cell medium at a ratio of 250:1 to reach a concentration level of 40 μM. Then, 100 μL of 40 μM EDU medium was added to each well and incubated for 24 h. After washing with PBS, 200 μL of paraformaldehyde (4%) was added to fix them. Then, they were decolorized and shaken for 5 min, followed by a sequential addition of 200 μL permeabilizer (0.3% Triton-100) and 200 μL 1× click reaction solution. After another 30 min incubation, 100 μL of 1× Hoechst 33342 reaction solution was added to each well and incubated for 10 min at room temperature. After washing with PBS, using the 405 nm channel of a fluorescence microscope, it was observed and photographed.

EdU-injected lampreys were cultured under the same conditions, and 50 mg/kg EdU (Beyotime, Shanghai, China) was injected intraperitoneally at the indicated time points and detected according to the manufacturer’s protocols. After another injection 24 h later, at Dam 7 d, samples were collected and stained. EdU incorporation was evaluated using the Click-it EdU imaging technique.

EdU (Beyotime) was diluted to the recommended concentration according to the instructions to generate EdU working solution. The original cell culture medium was removed, the appropriate volume of EdU working solution was added, and the cells were incubated at 37 °C for 2 h. After EdU labeling was complete, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed 3 times. The cells were then incubated with 3% Triton X-100 for 10 min at room temperature and washed 3 times. Click Reaction Solution was prepared according to the manufacturer's recommendation and added to each well, and the cells were incubated for 30 min at room temperature and washed 3 times. The nuclei were stained with Hoechst 33,342 for 10 min at room temperature, and the cells were washed 3 times. After the cells were sealed with anti-fluorescence quenching agent, they were photographed with a 90I inverted fluorescence microscope.

Gastric cancer cells with or without transfection were seeded in 96-well plates at a density of 5 × 10³ cells/well and then treated with Sophoridine (3 μM) for 24 h. Subsequently, the cells were incubated with a final condition of 10 μM EdU (Beyotime) for 2 h at 37 °C. Next, supernatant was discarded, and cells were fixed with 4% paraformaldehyde for 30 min. The cells were then treated with 0.5% Triton X-100 for 10 min and rinsed with PBS three times. Thereafter, the cells were exposed to 100 μL of click reaction cocktail (Azide 647 to label EdU, Beyotime) for 30 min and then incubated with 5 μg/mL of Hoechst 33342 to stain the cell nuclei for 30 min. Images were captured using Olympus IX73 microscope. The percentage of EdU-positive cells in each filed (six random fields were counted in each assay) was defined as the proliferation rate. All the experiments were performed in triplicate.