Kms 12 pe

Human myeloma cell lines LP-1 and RPMI-8226 were kindly provided by Prof. Robert Orlowski (Department of Lymphoma/Myeloma, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA), JJN-3 and OCI-MY5 by Prof. Wee Joo Chng (Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore), and WL-2 by Prof. Andrew Zannettino (Myeloma Research Programme, The University of Adelaide, Australia). NCI-H929 was purchased from American Type Culture Collection (Manassas, VA, USA). Other myeloma cell lines (KMS-12-PE, MOLP-8, OPM-2 and U-266) were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (Braunschweig, Germany). Cells were cultured in RPMI-1640 medium (IMDM for LP-1), supplemented with 10% fetal bovine serum, 50 U/ml of penicillin and 50 ug/ml streptomycin, and incubated in a humidified atmosphere of 5% CO₂ at 37 °C. All cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA).

Human MM cell lines (HMCLs) KMS-12-PE, MOLP-8, and U-266 were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). NCI-H929 was purchased from American Type Culture Collection (Manassas, VA, USA). LP-1 and RPMI-8226 were kindly provided by Prof. Robert Orlowski (Department of Lymphoma/Myeloma, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA). WL-2 was kindly provided by Prof. Andrew Zannettino (Myeloma Research Programme, The University of Adelaide, Australia). Cell lines were cultured in RPMI-1640 medium (IMDM for LP-1), supplemented with 10% fetal bovine serum, 50 U/mL of penicillin and 50 μg/mL streptomycin, in a humidified atmosphere of 5% CO₂ at 37 °C. All cell culture reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

According to the IMWG criteria, patients with MGUS (n = 2), SMM (n = 1), NDMM (n = 26), and RRMM (n = 13) were included in the study population (Table 1) [29 (link)]. The study was designed and conducted in accordance with national and international guidelines and with the ethical standards of the Declaration of Helsinki. Investigations have been approved by the authors’ institutional review board (1163/2017 and 1220/2018 Innsbruck).
KMS12-PE, KMS12-BM, NCI-H929 MM cells and MOLM-13 AML cells were purchased from DSMZ (German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany).

Human MM cell lines RPMI-8226, U266, KMS-12-PE (DSMZ, Braunschneig, Germany), representative of the pattern of expression of ETRs in primary MM cells (8 (link)), were cultured as previously described (8 (link)). Mycoplasma contaminations were excluded (Mycoplasma Species kit, EuroClone, Milan, Italy). The hypoxia-mimetic agent cobalt chloride (CoCl_2, Sigma-Aldrich, Milan, Italy) was used at a concentration of 100 µM. Macitentan was purchased from Selleckchem, Munich, Germany and used at a concentration of 10 µM based on previous titration experiments (9 (link)). MM cells were treated for 24 and 48 h with 20 µmol/L of antisense oligonucleotide EZN-2968 (anti-HIF-1α) or its negative control/scrambled EZN-3088 (19 (link), 20 (link)) (Enzon Pharmaceuticals Inc. Piscataway, New Jersey).

RPMI 8226 cells (referred to here as 8226 cells) were purchased from DSMZ (ACC-402). LP1 cells were generously provided by R Bataille (Centre de recherche en cancérologie Nantes-Angers, Nantes, France). U266 (ACC-9) and KMS-12-PE (ACC-606), from DSMZ, were used as positive control for cyclin D1 expression in immunocytochemistry analysis. Human myeloma cell lines (HMCLs) were maintained in RPMI 1640 medium (Lonza) supplemented with 2 mM L-glutamine (Lonza), 10% fetal calf serum (FCS, PAA Laboratories) and antibiotics (Lonza).
The pEGFP-N1 plasmid was purchased from Clontech Laboratories Inc. and the p-cyclin D1-EGFP plasmid was kindly provided by D. Salomon (USCF School of Medicine, San Francisco, CA, USA). This plasmid was sequenced to check the integrity of the coding sequence, amplified and purified with the QIAGEN maxi kit (Qiagen). We electroporated (250 V, 950 μF, Gene Pulser II, Bio Rad) 10⁷ 8226 or LP1 cells with 10 μg pEGF-N1 or p-cyclin D1-EGFP plasmids in RPMI 1640 medium without FCS. After incubation for 24 h, the cells were transferred to complete medium supplemented with 500 μg/mL G418 (PAA Laboratories). They were cloned by limiting dilution methods. Clones were maintained under selective pressure and selected on the basis of their FL1-fluorescence by flow cytometry (Gallios, Beckman Coulter). Data were analyzed with the Kaluza software (Beckman Coulter).