Ko 947

The HCC cells in exponential phase were detached with trypsin and washed 1–2 times with phosphate-buffered saline (PBS). Next, cell precipitation was collected, re-suspended with 2% FBS and mixed. The cell concentration was adjusted to 1 × 10⁸ cells/mL, and then 100 μL cell resuspension was added into the centrifuge tube (15 mL), followed by the addition of 10 μL phycoerythrin-labeled cluster of differentiation (CD) 133 antibody (ab216323, Abcam Inc., Cambridge, MA, USA) or corresponding homologous control. The cells were fully mixed and underwent a 30-min incubation away from light at 4°C. After twice washes with PBS containing 2% FBS, the cells were re-suspended with 500 μL PBS containing 2% FBS. Finally, the CD133⁺ and CD133⁻ cell clusters were detected and sorted using a flow cytometer.
The lentiviral interference vector of BMP2 [BMP2-short hairpin RNA (shRNA)] and its negative control (NC) (scramble) were constructed by Cyagen Biosciences Inc. (Guangzhou, China), and the titer determination was performed. The titer of lentiviral interference vector BMP2-shRNA and its NC was 4 × 10⁸ CFU/mL. Next, the constructed vectors were transfected into hepatic CSCs. DIPQUO (HY-128591, MedChemExpress Co., Ltd., Monmouth Junction, NJ, USA) served as the MAPK/ERK pathway activator and KO-947 (HY-112181, MedChemExpress) served as the MAPK/ERK pathway inhibitor.