The parental MM cell lines 8226 and MM1.S were purchased from BioVector NTCC Inc. (Beijing, China). 8226R5 that is a multidrug-resistant MM cell line with cross-resistance to BTZ has been kindly donated by Dr. R Buzzeo [15] (link). MM1.R (BioVector NTCC Inc.) with resistance to dexamethasone has also been shown to be resistant to BTZ [16] (link). These cells were cultivated in Roswell Park Memorial Institute-1640 (RPMI-1640; Gibco, Carlsbad, CA, USA) complemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotics (Gibco) in a 37 °C, 5% CO2 incubator. 8226R5 and MM1.R cells were subjected to the treatment of 10 nM BTZ (PS-341; Selleck, Houston, TX, USA) for 24 h.
Ps 341
Manufactured by Selleck Chemicals
Sourced in United States
PS-341 is a lab equipment product designed for scientific research and analysis. It serves as a tool for sample preparation, separation, and purification processes. The core function of PS-341 is to facilitate the handling and manipulation of various materials and solutions in a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Dmso control, by Merck Group (2 mentions)
Mg132, by Merck Group (2 mentions)
Mg132, by Selleck Chemicals (2 mentions)
Lactacystin, by Cayman Chemical (2 mentions)
Bortezomib, by Selleck Chemicals (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Selleck Chemicals (1 mentions)
Sirna target finder, by InvivoGen (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Bca protein quantitation kit, by Beyotime (1 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Total protein extraction kit, by Applygen (1 mentions)
Carfilzomib, by Selleck Chemicals (1 mentions)
B ap15, by Selleck Chemicals (1 mentions)
Usp14, by OriGene (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Polymyxin b pmb, by Merck Group (1 mentions)
Cpg oligodeoxynucleotide cpg odn, by InvivoGen (1 mentions)
11 protocols using ps 341
1
Evaluating Multidrug Resistance in Myeloma Cell Lines
2
Proteasomal Inhibition in Viral Infection
3
Knockdown and Overexpression of USP1 and ID2 in Gastric Cancer
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Based on the USP1 (NM_001017415.2) and ID2 (NM_002166.5) sequences, two shRNAs were designed using the siRNA Target Finder (InvivoGen). The target sites of shRNAs are detailed in Supplementary Table S1 . The interference effects were confirmed by Western blotting (Figure 1(c) ). The shUSP1 and shID2 construct that produced the most significant knockdown effect was used to transduce GC cells. Stably transfected GC cells were selected based on resistance to hygromycin (600 μg/ml) (Invitrogen, Carlsbad, CA, USA), and GC cells transfected with a negative control vector (shNC) were included as a control. pCMV-flag-USP1, pCMV-HA-Ub, and pCMV-his-ID2-expressing GC cells were selected using G418 (700 μg/ml) (Invitrogen), and an empty vector was used as the negative control. After four weeks of selection, individual colonies were isolated and expanded. All primers are listed in Supplementary Table S1 .
The following antibodies and reagents/kits were used: USP1, ID2, flag, his, HA, and Tubulin (Proteintech, Chicago, IL, USA); ubiquitin (Ub) (Nova Biomedical, MA, USA); protein A/G PLUS-agarose (Santa Cruz, CA, USA); DMEM, fetal bovine serum, and Lipofectamine® 3000 (Invitrogen); total protein extraction kit (Applygen, Beijing, China); BCA protein quantitation kit (Beyotime, Jiangsu, China); CHX [25 (link)] and PS-341 [26 (link)]; and MG132 [27 (link)] (Selleck, Houston, USA).
The following antibodies and reagents/kits were used: USP1, ID2, flag, his, HA, and Tubulin (Proteintech, Chicago, IL, USA); ubiquitin (Ub) (Nova Biomedical, MA, USA); protein A/G PLUS-agarose (Santa Cruz, CA, USA); DMEM, fetal bovine serum, and Lipofectamine® 3000 (Invitrogen); total protein extraction kit (Applygen, Beijing, China); BCA protein quantitation kit (Beyotime, Jiangsu, China); CHX [25 (link)] and PS-341 [26 (link)]; and MG132 [27 (link)] (Selleck, Houston, USA).
4
Proteasome Inhibitors: Modes of Action
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The FDA-approved proteasome inhibitors bortezomib and carfilzomib were purchased from Selleckchem (PS-341 and PR-171, respectively). While bortezomib is a reversible inhibitor of the 20S proteasome's β1- and β5-subunits, carfilzomib irreversibly binds to the β5-subunit and inhibits its chymotryptic-like activity (18 (link)). Lactacystin, an irreversible inhibitor of the β2- and β5-subunits of the 20S proteasome core, was obtained from Cayman Chemical (70980). Because of its capacity to bind to all three catalytic proteasome subunits, Lactacystin is a more potent inhibitor than clinically approved proteasome inhibitors (19 (link)). bortezomib and Lactacystin share the same transcriptional target genes in MM.1S cells, as demonstrated by the almost perfect correlation (R2 = 0.9983) in distribution of FPKM counts in RNA sequencing (RNA-seq) following treatment with the two inhibitors. Both drugs showed similar changes based on individual genes and gene ontologies. No unique gene ontologies were altered specifically by either drug.
5
Proteasomal Inhibition in Viral Infection
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After the 1 h of viral adsorption, cells were treated with either 5 μM MG132 (Sigma-Aldrich), 100 nM PS-341 (Selleck Chemicals) or DMSO control (Sigma) to inhibit proteasomal activity.
6
Generation and Characterization of Plasmid Constructs
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Plasmids containing myc-TDP-43 and myc-TDP-43 M337V were generated as described previously [30 (link)]. Plasmids for tau (4R/2N), α-synuclein, USP14 and UCHL5 were purchased from Origene. Mutant plasmids USP14 C114A, USP14 WT ΔUBL, USP14 C114A ΔUBL and UCHL5 C88A were generated from the wildtype plasmid by Genscript. Cells were treated with IU1 (25–100 μM; Selleckchem or Sigma), MG132 (10 μM; Selleckchem), PS341 (10 μM; Selleckchem) and b-AP15 (0.5–2 μM; Selleckchem or Millipore). All compounds were dissolved in DMSO (Sigma), and control samples were treated with the same volume of DMSO.
7
Adiponectin Regulation in Inflammation
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Recombinant human globular adiponectin (gAd) was purchased from BioVision (USA). A stock solution of gAd was prepared in distilled water containing 50 mg/ml BSA to a concentration of 1 mg/ml and aliquots were stored at −20°C until needed. MG132 was obtained from Calbiochem (Germany). PS-341 and SC-514 were supplied by Selleck Chemicals (USA). Lipopolysaccharide (LPS) and polymyxin B (PMB) were obtained from Sigma-Aldrich (USA). CpG-oligodeoxynucleotide (CpG-ODN) was supplied by InvivoGen (USA). Control and IRAK-1 siRNAs were purchased from Santa Cruz Biotechnology Inc. (USA).
8
Dissolution and Dilution of Anticancer Drugs
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Lapatinib (Selleck Chemicals, USA) was dissolved in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA) and then diluted with culture medium to the desired concentration. DMSO added in treatment groups was equal to the control ones with a final DMSO concentration < 0.2% (v/v). Cisplatin and cycloheximide (CHX) was obtained from Sigma-Aldrich (St. Louis, MO, USA). PS-341 was purchased from Selleck Chemicals (USA). Leupeptin was bought from Amresco (USA).
9
Anticancer Drug Sensitivity Screening
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Parent OVA-BS4 and OVA-BS4 spheroids were dissociated by trypsin and seeded at the concentration of 1.7 × 105 cells/ml onto 96-well plates, according to the specific culture conditions.
After 72 h, exponentially growing cells were treated with different doses of six anticancer agents: cisplatin (DDP; Sigma), paclitaxel (PTX; ChemieTek, Indianapolis, USA), etoposide (VP16; Sigma), PS341 (Selleckchem, Houston, USA), doxorubicin (DOXO; Sigma) and trabectedin (ET; PharmaMar, Madrid, Spain).
Each condition was set up in five replicates and three independent experiments were performed.
After 96 h from treatment, cell viability was monitored by MTS assay (CellTiter® 96 AQueous One Solution Cell Proliferation Assay; Promega) and optical density reading at 490 nm. The control group was represented by untreated cells. Cell viability percentage was calculated using the formula = (mean absorbance of the test well/mean absorbance of the control) × 100. Half-maximal inhibitory concentration (IC50) was calculated for each drug.
After 72 h, exponentially growing cells were treated with different doses of six anticancer agents: cisplatin (DDP; Sigma), paclitaxel (PTX; ChemieTek, Indianapolis, USA), etoposide (VP16; Sigma), PS341 (Selleckchem, Houston, USA), doxorubicin (DOXO; Sigma) and trabectedin (ET; PharmaMar, Madrid, Spain).
Each condition was set up in five replicates and three independent experiments were performed.
After 96 h from treatment, cell viability was monitored by MTS assay (CellTiter® 96 AQueous One Solution Cell Proliferation Assay; Promega) and optical density reading at 490 nm. The control group was represented by untreated cells. Cell viability percentage was calculated using the formula = (mean absorbance of the test well/mean absorbance of the control) × 100. Half-maximal inhibitory concentration (IC50) was calculated for each drug.
10
Proteasome Inhibition and Protein Dynamics
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Cells were treated with 12.5 μM Lactacystin (Cayman Chemical, Cat. 70980) for 6 h to inhibit protein degradation by the proteasome prior to FACS analysis. The fast-acting drug Bortezomib (5 μM) was used for chase experiments (Cat. PS-341, Selleck Chemicals, Houston, TX, USA).
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