Ab216323

Protein lysates were prepared using RIPA lysis buffer supplemented with protease inhibitor (Roche, Mannheim, Germany). Protein concentration estimation was performed using Bradford protein assay (Thermo Fisher Scientific). Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The blots were then subjected to blocking and incubation with primary antibodies. Thereafter, the blots were incubated with corresponding secondary antibodies. The signals were visualized using Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific). Antibodies used in Western blot were: anti-E-cadherin (1:2000, ab40772, Abcam), anti-N-cadherin (1:1000, ab18203, Abcam), anti-Vimentin (1:1000, ab92547, Abcam), anti-CD133 (1:1000, ab216323, Abcam), anti-SOX2 (1:1000, ab97959, Abcam), anti-CD44 (1:1000, ab157107, Abcam), anti-WDR66 (1:1000, ab175369, Abcam), anti-E2F7 (1:1000, ab245655, Abcam) and anti-GAPDH (1:1000, ab8245, Abcam).

Gastric cells were tested for a panel of fluorescent-labeled monoclonal antibodies and
respective isotype controls after incubation for 48 hours. After being washed, the labeled
cells were analyzed by flow cytometry with a FACS Vantage cell sorter (Becton &
Dickinson, Mountain View, California). The antibodies used were CD133 (rabbit, ab216323,
1:2000; Abcam, Cambridge, Massachusetts). Sorted cells were resuspended by 1×
phosphate-buffered saline (PBS).

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.

Antibodies against CD133 (ab216323, Abcam), CD44 (ab51037, Abcam), and SOX2 (MAB4423, Millipore Corp, Billerica, MA, USA) were applied to stain the transfected cells. Secondary antibodies were Alexa Fluor 488 goat anti-rabbit IgG antibody and coverslips (Invitrogen Inc., Carlsbad, CA, USA). Microscopic observations were conducted under a confocal laser scanning microscope (Leica Microsystems, Bensheim, Germany). Intensity of fluorescence was assessed in five fields of views with 300 cells per coverslip and analyzed using ImageJ 1.37v software (http://rsb.info.nih.gov/ij/index.html).