Anti aldh1

Cells from both adherent and sphere cultures were dissociated into single cells, washed and suspended in PBS. For the identification of cell surface markers, the cells were labelled with anti-CD24, anti-CD44, anti-CD133, or anti-ALDH1 and secondary fluorescein (FITC)- or APC-conjugated antibodies (BD Pharmingen, CA, USA). Flow cytometry was performed on a FACScan system (BD FACSAria™, BD Biosciences, CA, USA). Annexin V for apoptosis analysis: Apoptosis in treated cells was assessed using an Annexin V/FITC Apoptosis Detection kit (BD Bioscience, MA, USA). The cells were stained with PE, Annexin V and 7-amino-actinomycin (7-AAD) and incubated for 15 min in the dark.

DSF-treated HCC cells were subjected to Western blot analysis using anti-p38 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p38 (Cell Signaling Technology), and anti-tubulin (Oncogene Science, Cambridge, MA) antibodies. ALDH2-knockdown cells and ALDH1-and ALDH2-double knockdown cells were subjected to Western blotting using anti-ALDH1 (BD Biosciences) and anti-ALDH2 (Abcam, Cambridge, MA) antibodies. GPC3-knockdown cells selected by cell sorting for enhanced green fluorescent protein (EGFP) expression were also subjected to Western blot analysis using anti-GPC3 antibody (Santa Cruz Biotechnology).

Cells were collected by sample buffer and analyzed by electrophoresis. Proteins were transferred to polyvinylidene difluoride (PVDF, Merck Millipore) membrane and TBST buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 5% nonfat milk was used for blocking. Anti-Sp1 (Merck Millipore, 1:3000), anti-actin (Cat# 110,564, GeneTex, 1:5000), anti-GFP (Cat#sc-9996, Santa Cruz, 1:5000), anti-CD44 (GeneTex, 1:2000), anti-β-catenin (Cell Signaling, 1:1000), anti-ALDH1 (BD Biosciences, 1:1000), anti-SUMO-1 (ABclonal, Cambridge, MA, USA, 1:1000), anti-RNF4 (R&D systems, Minneapolis, MN, USA, 1:1000), anti-vimentin (Cell Signaling, 1:3000) and anti-E-cadherin (Cat#610,181, BD Biosciences, 1:1000) were used for probing interested proteins. After incubated with primary antibodies, PVDF membranes were then incubated with secondary immunoglobulin antibodies linked with horse radish peroxidase (Merck Millipore, 1:10,000). ECL Western blotting detection system (Merck Millipore) and ChemiDoc-it imager (UVP, Ultra-Violet Products Ltd., Cambridge, UK) were used for detecting signals.

Human and mouse specimens were incubated in 10% formaldehyde for 72 h for fixation, dehydration, and embedded in paraffin. For immunohistochemistry, xylene was used for dewaxing paraffin-embedded sections and serial diluted ethanol was also used for dehydration. Endogenous peroxidases were blocked by incubating in PBS containing 0.3% hydrogen peroxide for 30 min, and then samples were blocked with 1% bovine serum albumin. Proteins of interest were recognized by incubated with anti-Sp1 (Merck Millipore, 1:500), anti-ALDH1 (Cat# 611,194, BD Biosciences, San Jose, CA, USA, 1:500), anti-CD44 (Cat# 102,111, GeneTex, 1:250), anti-vimentin (Cat# 5741, Cell Signaling, 1:50) and anti-β-catenin antibodies (Cat# 8480, Cell Signaling, 1:200) at room temperature for 3 h, and immunoreactivity was visualized by using Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Sections were photographed by Olympus BX-51 microscope (Olympus Corporation, Tokyo, Japan).