Oxaliplatin (L-OHP) and the GRP78 inhibitor vomitoxin (VT) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). L-OHP was stored as a 20 mM solution in dimethyl sulfoxide (DMSO) at −20°C, and was diluted with RPMI 1640 medium prior to use with the highest concentration of DMSO in culture being 0.05% (v/v). The rabbit polyclonal anti-GRP78/Bip antibody (cat no. ab21685) was purchased from Abcam Ltd (Cambridge, MA, USA). Anti-CD24-SWA11 monoclonal antibody used for immunoprecipitation (IP) was obtained from Professor Hans-Peter Altevogt (Department of Translational Immunology, German Cancer Research Center, Heidelberg, Germany), in whose lab the antibody was generated (32 (link)). Anti-CD24 monoclonal antibody (cat. no. SC-7034) used for western blotting and IP, β-actin antibody (cat. no. SC130301) and horseradish peroxidase-conjugated goat anti-rabbit IgG (cat. no. SC2004) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
Oxaliplatin l ohp
Manufactured by Merck Group
Sourced in Germany
Oxaliplatin (L-OHP) is a platinum-based chemotherapeutic agent. It is a laboratory-grade chemical compound used in various research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti grp78 bip antibody, by Abcam (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Aspc 1, by American Type Culture Collection (1 mentions)
Capan 1, by American Type Culture Collection (1 mentions)
Mia paca 2, by American Type Culture Collection (1 mentions)
Capan 2, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle medium, by Nissui Pharmaceutical (1 mentions)
Trastuzumab emtansine t dm1, by Genentech (1 mentions)
Gemcitabine gem, by Eli Lilly (1 mentions)
Mirvana mirna mimic and inhibitor, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
5 fluorouracil 5 fu, by Merck Group (1 mentions)
Sw480, by American Type Culture Collection (1 mentions)
Sw620, by American Type Culture Collection (1 mentions)
Dld 1, by American Type Culture Collection (1 mentions)
Ms 275, by Selleck Chemicals (1 mentions)
Acetylated histone h3, by Santa Cruz Biotechnology (1 mentions)
Hdac1, by Merck Group (1 mentions)
4 protocols using oxaliplatin l ohp
1
Oxaliplatin and GRP78 Inhibitor Protocol
2
Pancreatic Cancer Cell Lines and Treatments
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The human pancreatic adenocarcinoma (PDA) cell lines MIA PaCa-2, PANC-1, AsPC-1, Capan-1 and Capan-2 were obtained from the American Type Culture Collection (Manassas, VA, USA) [21 (link)]. All cell lines were cultured in Dulbecco’s modified Eagle medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA) and 10 % heat-deactivated fetal bovine serum. Gemcitabine (GEM) was purchased from Eli Lilly Japan (Kobe, Japan), five-fluorouracil (5FU) was purchased from Kyowa Hakko Kirin Co. (Tokyo, Japan), and oxaliplatin (L-OHP) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Trastuzumab was a gift from Chugai, Inc. (Tokyo, Japan), and trastuzumab emtansine (T-DM1) was provided by Genentech Inc. (South San Francisco, CA, USA).
3
CRC Cell Line Treatments and Transfection
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The human CRC cell lines SW480 (ATCC CCL-228), WiDr (ATCC CCL-218), DLD-1 (ATCC CCL-221), HT-29 (ATCC HTB-38) and SW620 (ATCC CCL-227) were purchased from American Type Culture Collection (ATCC). Authentication was done by the authors in all cases (LGC Standards). Cell lines were maintained in RPMI-1640 (Invitrogen) with 10% fetal bovine serum and were grown at 37°C in a 5% CO2 atmosphere. Media were supplemented with penicillin G (100 U/ml), and streptomycin (0.1 mg/ml). Cells were treated with oxaliplatin (LOHP) (1 μM) (Sigma), 5-fluorouracil (5-FU) (1 μM) (Sigma) and FTY720 (10 μM) (Calbiochem) as previously reported [6 , 9 (link)]. For transfection experiments, CRC cells were seeded in 6-well plates and transfected with 10 μl of Lipofectamine 2000 (Life Technologies) and 2 μg of SET plasmidic vector or 20 nM of a miR-199b specific mirVana™ miRNA Mimic and Inhibitor (Ambion).
4
Immunoblotting and Immunofluorescence Assay Protocol
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5-FU, oxaliplatin (L-OHP), and propidium iodine (PI) were purchased from Sigma-Aldrich, Munich, Germany; MS-275 and MERCK60 were from Selleck Chemicals, Munich, Germany; and TO-PRO-3 was from Life Technologies, Ober-Olm, Germany. Antibodies for immunoblot were from: Millipore, Darmstadt, Germany (HDAC1 (05–100); acetylated histone H3 (06–599)), Santa Cruz Biotechnology, Heidelberg, Germany (HDAC2 (sc-7899); ɣH2AX (sc-101696)), Enzo Life Sciences, Lörrach, Germany (HSP90 (ADI-SPA-830)), Novus Biologicals, Heidelberg, Germany (p-KAP1 (Ser824), NB100-2350), Abcam, Cambridge, UK (p-ATM (Ser1981), ab81292; HDAC3, ab16047), Cell Signaling Technology, Frankfurt/Main, Germany (ATM (cs-2873); p-AKT1/p-AKT2/p-AKT3 (cs-9271; Ser473); CHK1 (cs-2360); HDAC6 (cs-7558); anti-p-Ser15-p53 (cs-9284); TS (cs-9045)), Sigma-Aldrich, Munich, Germany (acetylated tubulin (6-11B-1), T7451), BD Biosciences, Heidelberg, Germany (β-catenin (BD610154)), Thermo Fisher Scientific, Frankfurt/Main, Germany (RRM2 (PA5-13,570); vimentin (V9), MS-129-P). Antibodies against HDAC2 (#5113) from Cell Signaling Technology, Frankfurt/Main, Germany, and HDAC1 (05–100) from Millipore, Darmstadt, Germany, were used for immunofluorescence analysis.
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