Mm 1r

Myeloma-cell lines MM.1R and U266 were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA), RPMI-8226 and MM.1S from the National Infrastructure of Cell Line Resource (Beijing, China), NCI-H929 from Chinese Academy of Sciences Cell Bank (Shanghai, China), respectively; ANBL-6, ARP-1, CAG, OPM-2 were kind gifts from Dr. Robert Z. Orlowski at the Department of Lymphoma and Myeloma, UT MD Anderson Cancer Center. MM cells were cultured in RPMI-1640 media supplemented with 15% of fetal bovine serum, 100 U/ml of penicillin, 100 mg/ml of streptomycin, and 2 mM L-glutamine (Gibco, Life Technologies, Carlsbad, CA, USA). The HEK293T cells were cultured in DMEM-high glucose media with 10% fetal bovine serum, 100 U/ml of penicillin, 100 mg/ml of streptomycin, and 2 mM L-glutamine. These cells were all cultured at 37 °C in a humidified incubator with 5% CO₂ (Gibco, Life Technologies, Carlsbad, CA, USA). All cells were STR authenticated (Biowing Biotech, Shanghai, China) and mycoplasma-free confirmed with the Universal Mycoplasma Detection Kit (ATCC, Manassas, VA, USA).

Human MM cell lines RPMI 8226 (RPMI-S), MM.1S, MM.1R, U266 and the human bone marrow stromal cell (BMSC) line HS-5 were purchased from ATCC, Manassas, VA, USA. The RPMI sublines RPMI-MR20, RPMI-LR5 and RPMI-DOX40 and the CAG, OPM1 and OPM2 cell lines were kindly provided by Jana Jakubikova (Dana-Farber Cancer Institute, Boston, MA, USA). MM cell lines were grown in RPMI-1640 medium and HS-5 in Dulbecco’s modified Eagle medium (Gibco/BRL, Gaithersburg, MD, USA), both supplemented with 10% fetal calf serum and antibiotics (Biological Industries, Beit Haemek, Israel).

MM1.S, OPM-2, L-363, U-266 and MM1.R cells were cultured in RPMI1640 GlutaMAX and HEK293T and EA.hy926 cells in DMEM, both supplemented with 10% fetal bovine serum (FBS), 100U/mL penicillin and 0.1 mg/mL streptomycin and grown at 5% CO₂ and 37 °C. MM1.S, MM1.R and EA.hy926 were purchased from ATCC. OPM-2 was kindly provided by Prof. B. Thompson (University of Texas Medical Branch) and L-363 and U-266 cells by Prof. M. Engelhardt (Uniklinik Freiburg, Germany). HEK293T was obtained from the cytokine receptor laboratory (Ghent University). All cell lines were mycoplasma-negative (MycoAlert kit, Lonza). Experiments were performed using charcoal-stripped serum (CTS), unless otherwise specified.
Total solvent concentrations were equal in all conditions. Dex, hydrocortisone (Hcort), prednisolone (Pred), fluocinolone acetonide (FA), aldosterone (Ald), RU486 and cycloheximide (CHX) were purchased from Sigma-Aldrich and dissolved in ethanol (EtOH), unless otherwise specified. Spi and chloroquine (CQ) were obtained from Santa Cruz Biotechnology and dissolved in, respectively, EtOH and water, unless otherwise specified. MG132 was purchased from Selleck Chemicals and dissolved in DMSO.

The MM parental cell lines (RPMI-8226, MM1.S), and MM1.R (DEX resistant), OPM2 vel/R (BTZ resistant), were obtained from ATCC. RPMI-8226R5, a multidrug-resistant MM cell line with cross-resistance to BTZ, was kindly provided by Dr. R Buzzeo [16 (link)]. The resistance of both RPMI-8226R5 and MM1.R to the proteasome inhibitors BTZ was demonstrated in our previous study [17 (link)]. All cell lines were cultured in complete RPMI-1640 medium supplemented with 10% FBS as described previously [18 (link)]. Primary mononuclear cells were freshly isolated and purified from the bone marrow of MM patients, with the institution research ethic board approval.

The cells we used were RPMI 8266 (ATCC CRM-CCL-155), U266 (ATCC TIB-196), H929 (ATCC CRL-9068), MM.1R (ATCC CRL-2975), OPM-2 (DSMZ ACC50), and L-363 (DSMZ ACC 49). We used RPMI with 10% fetal calf serum (FCS) (Gibco, 12657-029, New York, NY, USA), supplemented with Glutamine, Pen-Strep, non-essential amino acids, 10 mM HEPES, and sodium pyruvate (all from Biological Industries, Israel, Kibbutz Beit-Haemek). NK92 cells, transduced with the canonical NKp44 (splice variant 1, NM_004828.3), and named NK92-44-1 were cultured as previously described [29 (link)].