Human myeloma cell lines utilized were: CAG (obtained from Dr. Joshua Epstein), MM1.R (obtained from Dr. Steven Rosen) and RPMI-8226/DOX40 cells (obtained from Dr. William Dalton). All cell lines were expanded and frozen in multiple vials upon receipt. All experiments were carried out within six weeks of thawing cells and not passaged more than five times. Cells were routinely tested for mycoplasma and retested prior to in vivo experiments. Generation of CAG cells expressing a high level of heparanase (HPSE-high) has been described [13 (link)]. The level of heparanase activity in HPSE-high cells is comparable to levels detected in bone marrow of many myeloma patients [13 (link)] and thus represent a physiologically relevant model for studying heparanase function in myeloma. These cells also express luciferase, which enabled monitoring of their location and growth in vivo. Myeloma cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum.
Antibodies to the various proteins used in this study included the following: anti-human heparanase polyclonal antibody 1453, a kind gift from Dr. Israel Vlodavsky [52 (link)], anti-human CD63 (abcam), and anti-human GAPDH (Cell Signaling). The heparanase inhibitor Roneparstat was provided by Leadiant Biosciences S.A. and heparanase inhibitor OGT2115 and NF-κB inhibitor IT-901 were from Tocris Biosciences.
Antibodies to the various proteins used in this study included the following: anti-human heparanase polyclonal antibody 1453, a kind gift from Dr. Israel Vlodavsky [52 (link)], anti-human CD63 (abcam), and anti-human GAPDH (Cell Signaling). The heparanase inhibitor Roneparstat was provided by Leadiant Biosciences S.A. and heparanase inhibitor OGT2115 and NF-κB inhibitor IT-901 were from Tocris Biosciences.