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4 protocols using rpmi 8226

1

Culturing Human Myeloma Cell Lines

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The human myeloma cell lines (HMCLs) U266, NCI-H929, RPMI 8226 and OPM-2 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). U266, RPMI 8226 and OPM-2 cells were cultured in RPMI 1640 (Lonza, Verviers, Belgium) containing 2 mM L-glutamine, supplemented with 15% (for U266 cells) or 10% (for OPM-2 and RPMI 8226 cells) heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies, Grand Island, NY, USA). H929 cells were cultured in RPMI 1640 (Life Technologies) containing 2 mM L-glutamine, supplemented with 15% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich), 0.05 mM 2-mercaptoethanol (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies). The human multiple myeloma INA-6 cell line was a kind gift from Dr. M. Gramatzki (Erlangen, Germany) and was cultured in RPMI 1640 (Life Technologies) containing 2 mM L-glutamine, supplemented with 1 ng/ml IL-6 (Life Technologies), 12 μg/ml gentamycin (Sigma-Aldrich) and 10% heat-inactivated FBS (Lonza).
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2

Cell Lines Maintenance and Verification

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The human cell lines U266, NCI-H929, RPMI-8226, HeLa, Daudi and Raji were obtained from The European Collection of Authenticated Cell Cultures (ECACC, Sigma-Aldrich, Madrid, Spain) and maintained in RPMI 1640 medium (Gibco Life Technologies, Carlsbad, CA) supplemented with 10% heat-inactivated foetal bovine serum (FBS), 2 mM glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco) at 37 °C in a humidified atmosphere containing 5% CO2 and were routinely verified by morphology and periodically checked for Mycoplasma contamination.
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3

Induction of Proteotoxic Stress in Cell Lines

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NCI-H929 and RPMI-8226 myeloma cells were obtained from the American Type Culture Collection (ATCC). HT-1080 cells were obtained from the European Collection of Cell Cultures (ECACC). All cell lines tested negative for mycoplasma by PCR. Cells were passaged in RPMI 1640 medium (NCI-H929 and RPMI-8226 cells) or minimal essential medium (HT-1080 cells) containing 10% fetal bovine serum and 100 mM GlutaMax. To induce proteotoxic stress, cells were seeded at a density of 5 × 105 cells/ml (NCI-H929 and RPMI-8226) or 4 × 104 cells per well in a 24-well format (HT-1080) and incubated for at least 6 h to equilibrate prior to treatment with 1 μg/ml or 10 μg/ml tunicamycin (Sigma) or 0.2 mM or 2 mM dithiothreitol (DTT) (Sigma). For washout experiments, cells were treated with 2 mM DTT for 90 or 60 min and then washed once and resuspended in fresh medium. For inhibition of IRE1 RNase activity, cells were treated with 30 μM 4μ8c (Merck Millipore; IRE1 inhibitor III, product 412512) dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO when added to cells was 0.1%. Actinomycin D (ActD) (Sigma) was used to inhibit transcription where indicated (see Fig. 2).
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4

Cell Lines for Myeloma and Lung Cancer

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For this study, the MM cell lines IM-9 and RPMI-8226, and the lung carcinoma cell line H226 were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The human MM cell line KMS-12-PE was obtained from the Japanese Collection of Research Bioresources (JCRB) (Osaka, Japan). A CHOP−/− murine embryonic fibroblast (MEF) cell line (CHOP-KO-DR) established from a 13.5-day-old CHOP−/− mouse embryo by SV-40 immortalization and a CHOP+/+ MEF cell line (DR-wild-type) established by SV-40 immortalization as a control cell line for CHOP-KO-DR were also obtained from the ATCC. These authorized cell lines were expanded and frozen in aliquots within 1 month after obtaining from the cell banks. Each aliquot was thawed and the cells were used for the experiments within 2 months after thawing. IM-9, H226, RPMI-8226, and KMS-12-PE cells were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Biowest SAS, Nuaillé, France), 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml) (Wako Pure Chemicals Industries, Tokyo, Japan). CHOP-KO-DR and DR-wild-type cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% FBS, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml). All cell lines were cultured in a humidified incubator containing 5% CO2 and 95% air at 37°C.
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