Proliferation and Apoptosis Assays for HCC

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)].