Apollo dye solution

A172 and LN229 cells were seeded into 96-well plates (20,000 cells) and cultured in the medium (100 μL/well, ThermoFisher) containing 50 μM EdU for 2 h (5% CO₂, 37 °C). Briefly, the cells were treated with 4% paraformaldehyde for 30 min and stained with 100 μL Apollo dye solution (Ribobio) for 30 min. The nuclei were stained with DAPI (ThermoFisher) for 30 min, followed by observation under the fluorescence microscope (Olympus, Tokyo, Japan). The final results are presented as a percentage.

Cells were seeded in 96‐well plates at a density of 3000 cells/well in 100 μl of complete medium and grown overnight. Then, the cells were maintained with 20 μM EdU labeling medium for 3 h. Subsequently, 4% paraformaldehyde was used to fix the cells, and then the cells were incubated with 100 μl Apollo dye solution (RiboBIO, Guangzhou, China) for 30 min. Then the cells were washed with 0.5% TritonX-100 for 10 min. At last, the cells were stained with DAPI and EdU-positive cells were calculated with ImageJ software.

Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2’-deoxyuridine (EdU) incorporation experiments were undertaken to measure cell proliferation. For CCK-8 experiments, 2000 indicated HCC cells re-suspended in 100 µL media were plated onto a 96-well plate. After culturing for 4d, 10 µL CCK-8 reagents (Dojindo, Kumamoto, Japan) were added to each well. After culture for another 2 h, a microplate reader (BioTek, Winooski, VT, USA) was used to measure the absorbance at 450 nm to indicate the number of viable cells. For EdU incorporation experiments, indicated HCC cells were treated with 50 μM EdU (RiboBio, Guangzhou, China) for 2 h. After being fixed in 4% paraformaldehyde for 30 min and permeabilized using 0.5% TritonX-100 for 10 min, the cells were stained with Apollo dye solution (RiboBio). The cell nucleus was further stained using DAPI. The number of EdU-positive and proliferative cells was detected using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Cell apoptosis was detected using the Caspase-3 Activity Assay Kit (Cell Signaling Technology, Danvers, MA, USA) strictly following the provided protocol. Cell migration and invasion were evaluated by transwell migration and invasion assays as we previously described [23 (link)].

In all, 1 × 10⁴ transfected cells/well were seeded into 96-well plates and incubated with 50 mM EdU for 2.5 h. The cells were fixed with 4% paraformaldehyde (PFA) and stained with Apollo Dye Solution and Hoechst using EdU incorporation assay kit (RiboBio, China). The images were obtained using the fluorescence microscope (Nikon, Japan).

Cells were seeded into 24-well plates at a density of 5×10⁴ cells/well and incubated overnight, after which 300 μl of EdU (50 μM) (RiboBio, China) were added to each well, and the cells were incubated for additional 2 h. The cells were then fixed in 4% polyformaldehyde for 20 min, permeabilized with 0.5% TritonX-100 (Beyotime, China) for 20 min, and stained in 300 μl of Apollo dye solution (RiboBio, China) for 25 min. Cell nuclei were stained with Hoechst (RiboBio, China) for 10-30 min. The proportion of EdU-positive cells were determined using a fluorescence microscope.

The cells were seeded onto 96-well plates at a concentration of 5×10³ cells/mL 23 . After incubation for 10 h with 10 uM Erastin, the cells were exposed to two Gy X-rays, and cultured for another 24h. The cells were then incubated with EdU (Beyotime, China) for 2 h, and were fixed with 4% paraformaldehyde and stained sequentially with Apollo dye solution (RiboBio) and DAPI (Invitrogen). The images were captured using a fluorescence microscope (Olympus).