We tested the inhibitory effect of the following compounds at the doses indicated except as otherwise stated, Tetrandrine (TETR 10 μM), Verapamil (VER 100 μM), Curcumin (CUR 75 μM), Bafilomycin (BAF 200 nM), Apilimod (APL 25 μM), Tamoxifen citrate (TMX 10 μM), Raloxifene hydrochloride (RLX 10 μM), Fulvestrant (ICI 182,780; FUL 100 μM) and β-Estradiol (EST 100 μM) were purchased from Sigma-Aldrich. Bapta-AM (BAP 10 μM) and YM201636 (YM 1 μM) and also controls Hydroxycloroquine (HCL 10 μM) endosomal acidification inhibitor and Teicoplanin (TEI 10 μM), an inhibitor of cathepsin L (Zhou et al., 2016 (link)) were purchased from Abcam. Stock solutions were dissolved in DMSO and working solutions were freshly prepared in DMEM 2% fetal bovine serum (FBS) at indicated concentrations. First, we pursued cell viability and cytotoxicity tests of all reagents using the CellTiter 96 Non-radioactive Cell Proliferation Assay (Promega) following the Manufacturer's instructions (Supplementary Fig. S2 , Supplementary Fig. S3 , Supplementary Fig. S4 ). We also studied the cytotoxic activity of the organic solvent DMSO. Based on these experiments we selected optimal non-toxic working concentrations for infection assays.
Raloxifene hydrochloride
Manufactured by Merck Group
Sourced in United States
Raloxifene hydrochloride is a selective estrogen receptor modulator (SERM) that acts as an agonist on bone and as an antagonist on breast and uterine tissue. It is a white to off-white, crystalline solid that is sparingly soluble in water.
Automatically generated - may contain errors
Lab products found in correlation
β estradiol, by Merck Group (2 mentions)
Verapamil, by Merck Group (2 mentions)
Tetrandrine, by Merck Group (1 mentions)
Tamoxifen citrate, by Merck Group (1 mentions)
Apilimod, by Merck Group (1 mentions)
Ym201636, by Abcam (1 mentions)
Bafilomycin, by Merck Group (1 mentions)
Celltiter 96 non radioactive cell proliferation assay, by Promega (1 mentions)
Curcumin, by Merck Group (1 mentions)
Fulvestrant, by Merck Group (1 mentions)
Bapta am, by Abcam (1 mentions)
Ici 182 780, by Bio-Techne (1 mentions)
Atropine methylbromide, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Pilocarpine hydrochloride, by Merck Group (1 mentions)
Nembutal, by Henry Schein (1 mentions)
Montelukast, by Cayman Chemical (1 mentions)
Tenofovir, by Selleck Chemicals (1 mentions)
Allopurinol, by Merck Group (1 mentions)
Disulfiram, by Cayman Chemical (1 mentions)
11 protocols using raloxifene hydrochloride
1
Inhibitory Effect of Compounds on Infection
2
Pharmacological Compound Preparation Protocol
3
Quantification of Tenofovir and Emtricitabine
4
Xenograft Mouse Model for Uveal Melanoma Treatment
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Forty-eight mice xenografted with mixed UM fragments from patient case 6 and 7 (Additional file 1 : Table S1) were equally grouped in to four groups according to the treatment protocols: control with 100 μl 0.9 % saline S.C. per week (n = 12) [35 (link)], AI (Letrozole; Femara® were prepared in 0.3 % hydroxypropylcellulose; mice were injected S.C. five times per week with Letrozole 10 μg per mouse per day; Novartis, Switzerland) (n = 12) [36 (link)], GnRHa (Leuprorelin acetate S.C. 10 mg/kg per week; LEUPLIN®DEPOT 3.75 mg S.C. Injection, Takeda, Japan) (n = 12) [37 (link)], and SERM (Raloxifene hydrochloride, 0.1 μg S.C. per day; Sigma-Aldrich, US) [32 (link)].
Laparotomic examination was performed and these mice were sacrificed 3 weeks after the xenograft procedures.
Laparotomic examination was performed and these mice were sacrificed 3 weeks after the xenograft procedures.
5
Preparation of Raloxifene and Miltefosine
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raloxifene hydrochloride and miltefosine were purchased from Sigma-Aldrich (St Louis, MO, USA). Stock solutions of raloxifene (10 mM) and miltefosine (20 mM) were prepared in DMSO and in sterile water, respectively, and kept at −20°C. Dilutions from the stock solutions were done in culture media. For in vivo experiments, fresh solutions of raloxifene were prepared in saline or Cremophor A25 (Sigma-Aldrich).
6
Cytokine Quantification in Murine Models
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Raloxifene hydrochloride (99% purity), cumene terminated poly(styrene-co-maleic anhydride) with an average Mn∼1600, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDAC), sulforhodamine B, dextran sulfate sodium salt (DSS were obtained from Sigma-Aldrich Ltd (Germany). Quantikine ELISA kits including Mouse IL-6 Immunoassay, Cat. No. M6000B, and Mouse TNF-α Immunoassay, Cat. No. MTA00B were purchased from R&D systems) (USA).
7
Puerarin and Raloxifene Hydrochloride Protocol
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Puerarin (purity ≥ 98%) was purchased from Sigma (batch number: P5555); raloxifene hydrochloride (RLX, purity 98%) was purchased from Sigma (batch number: 1598201); sodium carboxymethyl cellulose (CMC, 800 cps) was purchased from J&K (batch number: 241297).
8
Tamoxifen, Raloxifene, and Estradiol Effects in Mice
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All animal experiments were conducted according to IACUC guidelines. Two-month-old (20 females 20 males) C57B6 (subtype) were purchased from Jackson laboratories and housed in Mt. Sinai’s vivarium under standard conditions. Animals (females n = 4, males n = 4) received twice daily subcutaneous injections on the upper back (2.5 mg/kg) of tamoxifen (Sigma part number T5648-1G), raloxifene hydrochloride (Sigma part number R1402-1G), β-estradiol (Sigma part number E8875-250MG), or vehicle (5% dimethylsulfoxide in vegetable oil) alone twice daily for 3 consecutive days. Animals were sacrificed on the fourth day when tissue was harvested and flash frozen.
Spinal cords were isolated by hydraulic extraction, minced with forceps, and flash frozen for later analysis. Tissue was lysed in NP40 lysis buffer (50 mM Tris, 250 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF, 1 mM dithiothreitol plus protease inhibitors) on ice with roughly a 1:3 volume to volume ratio of tissue powder to lysis buffer. All samples were subjected to probe sonication (Fisher Scientific FB505) with 2 to 3 rounds of 1-second intervals at 20% amplitude on ice. Protein was then cleared by spinning at 20 800g at 4°C for 20 minutes. The soluble fraction was then moved to a new tube and frozen at –20°C for later analysis.
Spinal cords were isolated by hydraulic extraction, minced with forceps, and flash frozen for later analysis. Tissue was lysed in NP40 lysis buffer (50 mM Tris, 250 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF, 1 mM dithiothreitol plus protease inhibitors) on ice with roughly a 1:3 volume to volume ratio of tissue powder to lysis buffer. All samples were subjected to probe sonication (Fisher Scientific FB505) with 2 to 3 rounds of 1-second intervals at 20% amplitude on ice. Protein was then cleared by spinning at 20 800g at 4°C for 20 minutes. The soluble fraction was then moved to a new tube and frozen at –20°C for later analysis.
9
Raloxifene Effects on Ovariectomized Rats
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Fifty-four 10-week-old Sprague-Dawley female rats (Oriental Biotech, Pyeongtaek, Republic of Korea) were raised at room temperature (25 ± 1℃) with a relative humidity of 55% and a circadian light rhythm of 12 h. They were randomly assigned into three groups: two experimental groups and one control group; raloxifene (RAL)-administered group (n = 18) for ovariectomized rats administered with raloxifene, ovariectomy (OVX) group (n = 18) for ovariectomized without raloxifene treatment and the control group (n = 18) for rats that underwent a sham operation. For the RAL group, 1 mg/kg/day of raloxifene hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethylsulfoxide and was directly administered into the rats' stomach through gavage from day one after implant placement. The same amount of 0.9% normal saline was administered to rats in the OVX and the control groups (Fig. 1). All animal handling and surgical procedures were approved by the Institutional Ethical Committee for Animal Care and Use (BSM 16-001).
10
Characterization of Drug Metabolism Assays
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Uridine 59-diphosphoglucuronic acid trisodium salt (UDPGA), NADPH, alamethicin from Trichoderma viride, adenosine 3-phosphate 5-phosphosulfate, diclofenac sodium salt, midazolam hydrochloride, felodipine, S-(+)-mephenytoin, raloxifene hydrochloride, lovastatin, nisoldipine, nifedipine, methadone, quinidine, testosterone, benzydamine hydrochloride, cisapride, cyclosporin, terbutaline, sildenafil, verapamil, trazodone, atorvastatin, simvastatin, buspirone, rifabutin, saquinavir, terfenadine, zolpidem, repaglinide, indinavir, alprazolam, carbazeran, enalapril, ramipril , and dabigatran etexilate were purchased from Sigma-Aldrich (St. Louis, MO). NADPH-regenerating system containing NADP + , glucose-6phosphate, and glucose-6-phosphate dehydrogenase was purchased from Corning (Wiesbaden, Germany). Organic solvents were of LC-mass spectrometry (MS) or higher quality grade and were acquired from VWR International (Radnor, PA) or Fisher Scientific UK Ltd (Loughborough, UK). Purified water was obtained from Milli-Q Integral 5 Water Purification System (Merck KGaA, Darmstadt, Germany). Phosphate buffer, HQM Hepatocyte/Enterocyte Incubation Medium, and Co-Factor N for MetMax were purchased from In Vitro ADMET Laboratories (Columbia, MD).
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