Trifluoperazine tfp

The SCD inhibitors used were MK-8245 (Selleck Chemicals, Houston, TX, USA, 51158, 2019), A939572 (ApexBio, Houston, TX, USA, B3607, 2019), CAY10566 (abcam, Cambridge, MA, USA, ab144421, 2019), MF-438 (Millipore Sigma, Burlington, MA, USA, 569406, 2019), and GSK1940029 (MedChem Express, Monmouth Junction, NJ, HY-19762, 2019). Other modulators of αS homeostasis were trifluoperazine (TFP; Sigma-Aldrich, St. Louis, MO, T8516, 2019) and nortriptyline (NOR; MedChem Express, HY-B1417, 2019). A drug unrelated to αS homeostasis was tafamidis (MedChem Express, HY-14852, 2019).
Primary antibodies used were monoclonal 15G7 [30 (link)] to αS (hybridoma supernatant; 1:500), polyclonal GAPDH (Sigma-Aldrich, G9545; 1:5000), polyclonal β-actin (abcam Cat# ab8227, RRID:AB_2305186; 1:5,000), and polyclonal D64E10 to cleaved PARP (Cell Signaling Technology, Danvers, MA, Cat# 5625, RRID:AB_10699459; 1:1000). Secondary antibodies used were horseradish peroxidase conjugated secondary antibodies (GE healthcare, Chicago, IL; mouse: NA931V; rat: NA935V; rabbit: NA934V; 1:10,000).

All chemical reagents including hexadecyl trimethyl ammonium bromide (CTAB), tetraethyl orthosilicate, 3-mercaptopropyl-triethoxysilane (MPTES), lipopolysaccharides (LPS) from Escherichia coli O111:B4, MK-801, glutamate, NMDA, rapamycin, BAPTA-AM, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and trifluoperazine (TFP) were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Rabbit IgG isotype control was obtained from Abcam (Cambridge, United Kingdom). NMDAR1 polyclonal antibody was obtained from Invitrogen of Thermo Fisher Scientific Co. (Waltham, MA, USA). CellMask™ green plasma membrane stain and Hoechst 33342 (Trihydrochloride) were purchased from Thermo Fisher Scientific. Phosphate buffered saline (PBS, pH7.4) and 4% paraformaldehyde (PFA) were supplied by Biosesang (Seoungnam, South Korea). FSD Fluor™ 647 dye was purchased from BioActs Co. (Incheon, South Korea).

Primary culture of mouse PASMCs was performed. Briefly, pulmonary arteries were first isolated from male C57BL/6 J mice, and their connective tissues were carefully removed under a light microscope. Following adventitia removal, the isolated pulmonary arteries were clipped and then digested in Dulbecco’s Modified Eagle’s medium (DMEM) containing 1 mg/ml type II collagenase (C8150, Solarbio) for 2 h at 37 °C. The digested PASMCs were centrifuged at 180 g for 5 min, then collected in culture bottles and subcultured with DMEM containing 10% fetal bovine serum (FBS) and antibiotics. All experiments only used PASMCs of early generation (three to eight generations). PASMCs were treated with METH (0–2.5 mM) for 36 h and then assayed by Western blot to observe the concentration-dependent effect of METH on the expression of proliferative- and contractile-related proteins. To explore the mechanism, PASMCs were treated with a Nupr1 inhibitor trifluoperazine (TFP) (T8516, Sigma). We pretreated PASMCs with 10 μM TFP for 30 min before treating them with 1.5 mM METH for 36 h.

The human pancreatic cancer cell lines PANC-1 and Suit-2 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). PANC-1 cells were grown in Dulbeccos' Modified Eagle's Media (DMEM; Invitrogen, Carlsbad, CA)) and Suit 2 cells were grown in RPMI 1640 supplemented with penicillin (5 units/mL), streptomycin (5 μg/mL), and 10% heat-inactivated FBS.
DR5 agonist antibody, TRA-8, was generated as previously described [48 ]. All antibodies used were commercially available, including anti-caspase-8 (BD Bioscience, San Jose, CA), anti-caspase-3 (Enzo Life, Plymouth Meeting, PA), anti-FADD and anti-CaM (Millipore, Billerica, MA), Src (Cell Signaling Technology, Danvers, MA), anti-DR5 (Prosci, Poway, CA), anti-c-FLIP (Enzo Life, Farmingdale, NY) and anti-GAPDH (Santa Cruz Biotech, Santa Cruz, CA).
Tamoxifen (TMX) and Trifluoperazine (TFP) were obtained from Sigma-Aldrich (St. Louis, MO). Protein G-agarose and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA). Caspase-8 Inhibitor, Z-IETD-FMK, was from R&D system. The dual-luciferase activity reporter system was purchased from Promega.

Trans-resveratrol (tRVT, purity ≥ 99%), cis-resveratrol (cRVT, purity ≥ 98%), dehydronifedipine, diclofenac, 6-hydroxychlorzoxazone, 4-hydroxydiclofenac, 4-hydroxymephenytoin, S-mephenytoin, midazolam, mycophenolic acid (MPA), nifedipine, CDCA-24-acyl-β-glucuronide, 7-ethyl-10-hydroxy camptothecin (SN-38) glucuronide, MPA-β-d-glucuronide, and N-acetylserotonine (N-ASER)-β-d-glucuronide were obtained from Toronto Research Chemicals (Toronto, ON, Canada). Acetaminophen, amodiaquine, bupropion, chenodeoxycholic acid (CDCA), chlorzoxazone, dextromethorphan, dextrorphan, 6-hydroxybupropion, 7-hydroxycoumarin, N-ASER, N-desethylamodiaquine, naloxone, nifedipine, phenacetin, trifluoperazine (TFP), estrone-β-d-glucuronide, naloxone-β-d-glucuronide, and TFP-β-d-glucuronide were obtained from Sigma-Aldrich (St. Louis, MO, USA). SN-38 and 1′-hydroxymidazolam were provided by Santa Cruz Biotechnology (Dallas, TX, USA) and Cayman Chemicals (Ann Arbor, MI, USA), respectively. Pooled HLMs (XTreme 200) were purchased by XenoTech (Lenexa, KS, USA). rP450 (rCYP3A4, rCYP2E1, rCYP2C19, and rCYP1A2) were obtained from SPMED (Busan, Korea).