Research Paper Volume 12, Issue 22 pp 22700—22718

Drug-selected population in melanoma A2058 cells as melanoma stem-like cells retained angiogenic features – the potential roles of heparan-sulfate binding ANGPTL4 protein

Figure 5. Higher expression of ANGPTL4 in drug-selected cells contributed to angiogenic activity through glycosaminoglycan modification on proteoglycans. (A) GSEA analysis showed that ANGPTL4 was the enriched in the gene sets related to oncogenic features. qPCR and western blot analysis demonstrated the higher expression level of ANGPTL4 in drug-selected cells. (B) Tube formation assay showed significantly connected and aligned cells by drug-selected cells, but not seen by parental cells. (C) Pretreatment with anti-ANGPTL4 antibody reduced tube-forming activity of drug-selected cells. (n=4; data were mean ±SD; **, p < 0.01; *, p < 0.05.) (D) qPCR analysis at expression of several heparan-sulfate proteoglycans (SDC1, SDC2, and SDC4) and sulfate transferases (HS2ST1 and NDST1) in parental and drug-selected cells. (E) Pretreatment with chemical inhibitors to reduce sulfate groups (by NaClO3, sodium chlorate) or carbohydrate chain biosynthesis (by XylPyr, xylopyroside) inhibited tube-forming activity of drug-selected cells. (n=4; data were mean ±SD; **, p < 0.01; *, p < 0.05.).