Drug-naïve 5TGM1 (5TGM1.wt)[17] (link) and RPMI-8226 (8226.wt) cells were cultured as previously described [16] (link). Bortezomib-resistant 5TGM1 (5TGM1.BR) cells were generated by exposing 5TGM1.wt cells to increasing doses of Bortezomib (Selleck Chemicals, Houston, TX), until they could be cultured in 20nM Bortezomib. Bortezomib-resistant RPMI-8226 (8226.BR), Kas6 (Kas6.BR) and ANBL6 (ANBL6.BR) cells, and carfilzomib-resistant RMPI-8226 (8226.CR) cells were generated previously in a similar manner [11 (link),18] (link). 5TGM1.BR cells were routinely cultured in media containing 20nM Bortezomib, 8226.BR, Kas6.BR and ANBL6.BR in media containing 10nM Bortezomib, and 8226.CR cells in media containing 10nM carfilzomib (Selleck Chemicals). Cells were removed from proteasome inhibitor for at least 72h before use in experiments. All human cell lines were authenticated by short tandem repeat DNA fingerprinting, tested for mycoplasma by PCR analysis [19] , and used within 3 months of resuscitation.
Bortezomib
Manufactured by Selleck Chemicals
Sourced in United States, Germany, China, United Kingdom, Belgium
Bortezomib is a proteasome inhibitor used in research laboratory settings. It functions by blocking the proteasome, a complex of enzymes responsible for the degradation of unwanted or damaged proteins within cells.
Automatically generated - may contain errors
Lab products found in correlation
Carfilzomib, by Selleck Chemicals (44 mentions)
Cycloheximide (chx), by Merck Group (30 mentions)
Mg132, by Selleck Chemicals (24 mentions)
Mg132, by Merck Group (22 mentions)
Melphalan, by Merck Group (14 mentions)
Pomalidomide, by Selleck Chemicals (14 mentions)
Lenalidomide, by Selleck Chemicals (13 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (13 mentions)
Doxorubicin, by Selleck Chemicals (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Doxorubicin, by Merck Group (9 mentions)
Mln4924, by Selleck Chemicals (9 mentions)
Vorinostat, by Selleck Chemicals (9 mentions)
Dexamethasone (dex), by Merck Group (9 mentions)
Cycloheximide (chx), by Selleck Chemicals (8 mentions)
Ibrutinib, by Selleck Chemicals (8 mentions)
Chloroquine, by Merck Group (8 mentions)
Trametinib, by Selleck Chemicals (8 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (8 mentions)
Panobinostat, by Selleck Chemicals (7 mentions)
346 protocols using bortezomib
1
Generation and Characterization of Proteasome Inhibitor-Resistant Myeloma Cell Lines
2
Evodiamine Enhances Bortezomib Cytotoxicity
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U266 and RPMI8226 cells treated with Evo (50, 100, 200, 400, 800 μg/mL) for 24 h or treated with 400 μg/mL Evo for different time points (12, 24, 36, 48 h). To evaluate the effects of Evo on bortezomib-induced cell apoptosis and proliferative inhibition, the cells were co-treated with 40 nM bortezomib (cat no. S1013, Selleck, Shanghai, China) for 24 h, and then were plated in 96-well plates at a density of 1000 cells in 100 μL of the aforementioned RPMI-1640+FBS media per well at 37°C with 5% CO2.
3
Antibody-based Protein Analysis in Bortezomib-Treated Cells
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The following antibodies were used: β-actin/ACTB (Abcam, Cambridge, UK, #6276), GAPDH (Abcam, #8245), MAP1LC3B/LC3B (Cell Signaling, Beverly, MA, USA, #3868), HMOX1 (Enzo, Farmingdale, NY, USA, #ADI-OSA-110), IgG Fcγ fragment specific (Jackson ImmunoResearch Laboratories, West Grove, PA, USA, #109-006-098), FK2 antibody recognizing mono- and polyubiquitinated proteins (Enzo, #BML-PW-8810), SQSTM1 (Progen, Heidelberg, Germany, #GP62-C), SRXN1 (Proteintech, Chicago, IL, USA, #14273-1-AP), xCT (Cell Signaling, Beverly, MA, USA, #12691). As bortezomib-induced cytotoxicity varied between batches and storage time, bortezomib (Selleck Chemicals, Houston, TX, USA) toxicity was titrated for each batch used, and regularly controlled. Supplements of 1 mm cysteine, GSH and alanine, or 5 mm glutamate (Sigma-Aldrich) were given for 24 h. Where indicated, minimal essential medium (Gibco/Thermo Fisher Scientific, Waltham, MA, USA, #11130-036) was added to a final 1 × concentration to restore levels of essential amino acids, including l -cystine. Sulfasalazine (Fluka/Sigma-Aldrich) was used as indicated. Dimethyl sulfoxide (DMSO) was from Sigma-Aldrich. Hydrogen peroxide was from Merck Millipore (#1.08600, Billerica, MA, USA).
4
Bortezomib-Induced Cell Apoptosis
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Following transfection, cells were incubated with bortezomib (Selleck Chemicals; 4 nM for U266 cells, 1 nM for RPMI-8226 cells) for 48 h. The bortezomib concentration for cell incubation was selected using the IC50, as shown in the ‘Chemosensitivity detection’ subsection. Following incubation, Annexin V-FITC Apoptosis Detection Kit (MilliporeSigma) was used to determine cell apoptosis, according to the manufacturer's instructions. The data were collected by FACSCalibur flow cytometer (Becton, Dickinson and Company) and analyzed by Flowjo 7.6 (Becton, Dickinson and Company).
5
Cell Surface Proteomics of Myeloma Cells
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For cell surface proteomics experiments, 25e6 cells were seeded at 1e6/mL in T75 flasks and treated with compounds for 48 h at the LD25 dose. RPMI-8226 cells were treated with 50 μM Lenalidomide (Sigma CDS022536), 7.5 nM Bortezomib (Selleck Chemicals, S1013), or 300 nM CB-5083 (gift of Cleave Biosciences). AMO1 cells were treated with 50 μM Lenalidomide, 750 nM JG-342 (gift of Jason Gestwicki, UCSF), or 250 nM CB-5083. For flow cytometry experiments, cells were plated and treated in 96-well plates with the doses described in figure legends for 48 h prior to flow cytometry analysis unless otherwise indicated. For Bortezomib drug screens, 1000 cells were seeded in 45 μL of media in 384-well plates using the Multidrop Combi (Thermo Scientific). Bortezomib (Selleck Chemicals, S1013) was added 24 h after seeding and viability was measured using Cell-Titer Glo (Promega) 48 h after the addition of drugs using a GlowMax Explorer (Promega).
6
Drug-Induced Lifespan Modulation in Flies
7
T-cell Activation and Apoptosis Assay
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T cells were isolated from human PBMCs using Human T-cell Isolation kit (StemCell Technologies, Cat#17951). The purity of T cells was greater than 90% as confirmed by flow cytometry. Cells were counted and loaded with 5 μM CellTrace Violet (Thermo Fisher) in phosphate buffered saline (PBS) for 10 min at 37 °C, followed by wash in 10% fetal calf serum (FCS)-supplemented RPMI 1640 media. T cells were cultured in 10% HS-RPMI, with anti-CD3 (1 μg per mL, eBioscience) and anti-CD28 (1 μg per mL, eBioscience), in the presence or absence of different concentration of PKS21221 or Bortezomib (Selleckchem). In some experiments, T cells were cultured in 10% HS-RPMI / anti-CD3 / anti-CD28 for 4 days and then treated with PKS21221 or Bortezomib (Selleckchem) for 12, 24, 48 h. The viability and apoptosis were assessed using Annexin V Apoptosis Detection Kit (BD Biosciences). The experiments were performed in cells from three different healthy volunteer donors.
8
Histone Deacetylase Inhibition and Proteasome Inhibition in Breast Cancer Cells
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BC cell lines (MCF-7, T47D, ZR-75-1, SK-BR-3, Hs-578T, MDA-MB-231, MDA-MB-436, MDA-MB-468, and MDA-MB-453), normal breast cell lines (MCF-10A and HBL-100), and HeLa cells were kindly provided by Professor ZM Shao (Fudan University Shanghai Cancer Center, Shanghai) and were cultured according to standard ATCC protocols. Cells were seeded at an approximate concentration and were cultured under standard incubation conditions (37°C, 95% humidity, 5% CO2) for 24 hours before the following experiments were performed: (1) transfection with sh-HDAC5 (Origene) or a scrambled control shRNA sequence, (2) treatment with LMK-235 (Selleck Chemicals) or DMSO (vehicle control), (3) treatment with bortezomib (Selleck Chemicals) or DMSO, and (4) treatment with both LMK-235 and bortezomib or DMSO.
9
Selinexor and Bortezomib Effects on Multiple Myeloma Cell Survival
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Cell survival was assessed using MTT solution (Sigma-Aldrich, St. Louis, MO), followed by absorbance measurement at 570 nm using a spectrophotometer according to the manufacturers' protocol, where the absorbance is proportional to the number of viable cells. The effects of increasing concentrations of selinexor (KPT-330; 0, 100, 250, and 500 nM), bortezomib (Selleck Chem, Houston, TX; 10 nM), and combination treatment (100 nM of KPT-330 and 10 nM of bortezomib) were tested on MM cell survival/cytotoxicity under normoxic and hypoxic conditions for 24 hours. In addition, the effect of selinexor (0, 50, 100, and 250 nM) and bortezomib (0, 1, 5, and 10 nM) was examined on MM.1S cell survival in normoxic conditions by MTT.
10
Cytotoxicity Assessment of Bortezomib
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The erythroid leukemia cell line HEL and the gastric cancer cell line BGC-823 were used in the present study. Cells were grown in 10% foetal calf serum-containing RPMI-1640 with addition of 2 mM L-glutamine and antibiotics (50 mg/mL penicillin, and 50 mg/mL streptomycin) in a humid atmosphere at 37°C/5% CO2. Bortezomib was bought from Selleck Chemicals (Houston, TX, USA) and exponentially growing cells were incubated with Bortezomib at different concentrations for various time periods. Cells were counted for numbers and viability using trypan blue exclusion test.
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