HACE1 is a potential tumor suppressor in osteosarcoma

a Relative expression of HACE1 is significantly higher (Wilcoxon test <0.05) in mesenchymal stem cells (12 samples, {"type":"entrez-geo","attrs":{"text":"GSE28974","term_id":"28974"}}GSE28974) compared to osteosarcoma tumor biopsies (84 samples, {"type":"entrez-geo","attrs":{"text":"GSE33382","term_id":"33382"}}GSE33382). b Genome-wide analysis of somatic copy number alterations in osteosarcoma. The plot shows fractions of copy number loss/gain of a cohort of 113 primary osteosarcoma specimens across chromosome 6. c Diagrammatic representation of the full-length HACE1 protein structure (i) and mutants lacking the HECT domain (ΔHECT; ii) or the ankyrin repeats (ΔANK; iii) used in this study. d OST cells were transfected with the indicated vectors. Immunoblots showing successful transfection of OST cell line with the indicated vectors as detected by the generated clones of HACE1 antibodies described in the Material and methods, and compared to commercially available HACE1 antibody (Abcam, cat # ab133637). GRB2 and Ponceau staining showing equal protein load. e Immunoblot showing HACE1 protein expression in osteoblasts and osteosarcoma cell lines. HEK-293 was used as a positive control while SK-NEP-1 served as a negative control for HACE1 expression. β-Actin was used as a loading control. f Immunofluorescence analysis of HACE1 expression in MG63 osteosarcoma cell line and OB3 osteoblast cell line displaying strong nucleo-cytoplasmic staining in OB3 cells contrary to remarkably weak cytoplasmic staining in MG63. Blue: DAPI (nuclear stain); green: HACE1. Scale bars, 50？μm. g HACE1 mRNAs expression in osteoblast and osteosarcoma cell lines. The overall relative levels of HACE1 mRNAs were measured using quantitative RT-PCR in the indicated osteoblasts and osteosarcoma cell lines. PCR reactions were performed twice in quadruplicate. The data were normalized to endogenous β-actin, and presented as mean？±？SD. Two-tailed Student's t-test was used to analyze the data with **p-value < 0.00