Soft agar colony anchoring assay

Low melting point agarose (Sigma-Aldrich) was first dissolved in ultrapure water (concentration, 1.2% and 0.8%). Then prepare the bottom matrix: 2 × DMEM, which contains 2% PS and 30% FBS, were mixed with 1.2% agarose solution at a 1:1 ratio. 3 ml of the mixture was added to 6‑cm dishes, cooled and solidified in an incubator. Next, cells were suspended in 3ml medium (2 × DMEM: 0.8% agar solution, 1:1) and move to dishes. After the upper layer of agarose fully solidified, incubated at 37˚C for two weeks. Cells were stained with 0.2% crystal violet (Sangon Biotech Co., Ltd.) for 15-30 min at RT. Finally, the colonies were photographed and counted.