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6 protocols using tin protoporphyrin 9

1

MTT Cytotoxicity Assay in Cell Cultures

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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was purchased from Sigma–Aldrich (Saint Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium alpha (α-MEM), and fetal bovine serum (FBS) were purchased from Welgene Bioscience (Daegu, Korea). Primary antibodies for iNOS, COX-2, HO-1, and EGFR rabbit polyclonal antibodies were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). The enhanced chemiluminescence (ECL) Western blotting detection system was obtained from Advansta Inc. (San Jose, CA, USA). The HPLC-grade acetonitrile and water were obtained from Honeywell-Burdick and Jackson (Muskegon, MI, USA). The TNF-α and IL-6 ELISA (enzyme-linked immunosorbent assay) kits were purchased from R&D Systems (Minneapolis, MN, USA). Lipopolysaccharide isolated from P. gingivalis (PG-LPS) was purchased from InvivoGen (San Diego, CA, USA). Alizarin Red S and tin protoporphyrin IX (SnPP) were obtained Sigma–Aldrich (St. Louis, MO, USA). n-Hexane, ethyl acetate, n-butanol, chloroform, and MeOH were purchased from Daejung Chemicals and Metals CO. LTD. (Siheung, Korea).
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2

Detailed Protocol for Studying Inflammation and Mineralization

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Minimum Essential Medium-Alpha (α-MEM), fetal bovine serum (FBS), and trypsin-ethylene diamine tetra acetic acid (EDTA) culture reagents were purchased from Gibco (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was obtained from Amresco Inc (Cleveland, OH, USA). TNF-α, IL-6, and ELISA (enzyme-linked immunosorbent assay) kits were purchased from R & D system (Minneapolis, MN, USA). Lipopolysaccharide isolated from P. gingivalis (PG-LPS) was purchased from Invivo Gen (San Diego, CA, USA). Mouse monoclonal antibodies iNOS and COX-2 secondary antibodies, anti-rabbit, anti mouse (Santa Cruz Biotechnology Inc., Dallas, TX, USA) were purchased from Cayman Chemical (Ann Arbor, MI, USA) for Western blot analysis. A Hybond ECL Nitrocellulose membrane (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) Western blotting detection system was purchased from Advansta Inc. (Santa Clara, CA, USA). Alizarin Red S and tin protoporphyrin IX (SnPP) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
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3

Comprehensive Cellular Signaling Pathway Analysis

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All cell culture medium and serum were purchased from Invitrogen, whereas cell culture supplements were purchased from Sigma. Antibodies against XBP1 (sc-7160), HDAC3 (sc-136290) phospho-Akt (sc-7985R), Akt1 (sc-1619), Nrf2 (sc-722), mTOR (sc-1549), histone H3 (sc-10809), IRE1α (sc-20790), and GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology; antibodies against FLAG (F2426, F1804, and F7425), HA (H6908) and tubulin (T8203) were from Sigma; antibody against HO-1 (ab13248) was purchased from Abcam. Antibodies against XBP1u and XBP1s were raised in rabbits with peptides CRSSQRSTQKDPVPY and DSGGIDSSDSESDIC, respectively, by GenScript Corp. All secondary antibodies were from DakoCytomation. Inhibitors LY294002, PD98059, SU5416, actinomycin D, cycloheximide, and Tin protoporphyrin IX were purchased from Sigma. AZD2014 was purchased from Selleckchem. All inhibitors were dissolved in DMSO. All other chemicals were also from Sigma.
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4

Investigating TLR4 Activation in Human Myeloma Cells

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Human myeloma cell lines (HMCLs) were grown in RPMI 1640 medium supplemented with 10% (for NCI-H929 and OPM2) and 20% (for U266) fetal bovine serum and 1% penicillin–streptomycin. NCI-H929 and U266 cell lines were obtained from ATCC (Mannas, VA, USA). OPM2 cell line was purchased from Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany).
To activate TLR4, cells were treated with LPS (Sigma-Aldrich, Mylan, Italy). The TLR4 inhibitor TAK-242 and the HO-1 inhibitor tin protoporphyrin IX (SnPP) were purchased from Sigma-Aldrich and Cayman (Ann Arbor, MI, USA), respectively. Hemin was obtained from Sigma-Aldrich and dissolved in 0.1 M NaOH. Commercially available bortezomib (BTZ) was used. CORM3 and CORM-A1 were purchased from Sigma-Aldrich (St. Louis, MI, USA).
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5

Intraperitoneal Administration of Cobalt and Tin Protoporphyrins

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Cobalt protoporphyrin-IX and tin protoporphyrin-IX (Sigma-Aldrich, St. Louis, MO, USA) were diluted in NaOH 1 N at 10 mM. This stock was stored at 4 °C in amber tubes. The volume needed for each animal was calculated based on the weight of the mouse (5 mg/kg for both drugs) and diluted in 150 µL of sterile PBS per dose. The control vehicle was prepared by replacing the volume of the drug for NaOH 1 N diluted in 150 µL of sterile PBS per dose. The administration of the drugs was performed by intraperitoneal (ip) injection using an insulin syringe (BD Ultra Fine, Franklin Lakes, NJ, USA).
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6

Antioxidant Effects of GAS in Cell Apoptosis

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GAS (purity >98%) was purchased from Nantong Feiyu Biological Technology Co., Ltd. (Nantong, China). GAS was dissolved in DMSO to create a 100 mM stock solution and then stored at −20°C. Tin protoporphyrin IX (SnPP), dimethyl sulfoxide (DMSO), TBHP, and carboxymethylcellulose (CMC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against C-caspase3 (#9661), bax (#14796), Bcl-2 (#3498), caspase9 (#9508), cytochrome C (#11940), and β-actin (#3700) were obtained from Cell Signaling Technologies (Beverly, MA, USA). Primary antibodies against Nrf2 (ab137550), HO-1 (ab189491), and lamin B (ab16048) were acquired from Abcam (Cambridge, UK), and 4’,6-diamidino-2- phenylindole (DAPI) was obtained from Beyotime (Shanghai, China). All cell culture reagents were purchased from Gibco (Grand Island, NY, USA).
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