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10 protocols using pnu282987

1

Sourcing of Nicotine and Cotinine Analogs

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(-)-Nicotine free base (CAS no. 54-11-5) and (-)-cotinine free base (CAS no. 486-56-6) were purchased from Sigma-Aldrich® (St. Louis, MO, United States). (±)-Anatabine citrate (purity 98.92% by HPLC) was purchased from Concept Life Sciences (Manchester, UK). (±)-Anatabine free base (purity >95% by HPLC) used for the SmartCube® study was a generous gift from Indena® S. p.A. (Milan, Italy) (Rossia et al., 2018 (link)). (±)-Anatabine free base used for the zebrafish NTT was custom synthesized by WuXi AppTec (purity ≥95%; Shanghai, China). PNU282987 (CAS no. 711085-63-1) and buspirone hydrochloride (CAS No. 33386-08-2) were purchased from Tocris Bioscience (Bio-Techne®, Minneapolis, MN, United States). AZD1446 (CAS no. 1025007-04-8) was purchased from Key Organics Limited (Cornwall, UK).
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2

Cell Line Characterization and Culture

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Human cancer or immortal cell lines (A549, BEAS-2B, U87MG, MCF-7, MDA-MB-231, 293T) were purchased from the ATCC, which characterizes them using cytogenetic analysis. We have not authenticated these cell lines. A549, U87MG, MCF-7, MDA-MB-231 and 293T cells were cultured in DMEM medium with 10% fetal bovine serum (FBS). BEAS-2B cells were cultured in RPMI1640 medium with 10% FBS. All media were supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine. All cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37°C. BCX treatments were carried out at a series of concentrations (0 to 4 μM) and incubated for different times, as specified. MG624 and PNU282987 were purchased from Tocris Bioscience. Rac1 vectors were purchased from GE Healthcare and ARF6 vectors were purchased from OriGene Technologies. PDGF, PMA, LY294002, tetramethyl rhodamine isothiocyanate (TRITC)-labeled phalloidin and all other reagents and chemicals were purchased from Sigma.
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3

Neurotransmitter Regulation of NGF Signaling

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Drugs were dissolved in HBSS, which was used as the experimental vehicle, and applied directly to the media containing NGF. Drugs concentrations are based on published literature: choline (Acros Organics, Geel, Belgium) (10 mM, 3 mM, and 1 mM) [22 (link)]; α-bungarotoxin (Thermofisher) (BGTX) (50 nM) [32 (link)]; calpeptin (Sigma Aldrich, St. Louis MO, USA)(26 μM) [33 ], ryanodine (Santa Cruz) (30 μM) [21 (link)]; Xestospongin C. (Tocris Biosciences, Bristol, UK.) (Xest C.) (1 μM) [20 (link)]; FK506 (Tocris Biosciences) (40 μM) [34 (link)]; Substance P (Tocris Biosciences) (Sub P) (1 μM) [20 (link)]; PNU 282987 (Tocris Biosciences) (10 μM) [35 (link)].
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4

Acetylcholine Signaling Pathway Assay

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Acetylcholine (ACh), 5-Hydroxyindole (5-HI), 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), RIPA buffer drugs, phenylmethylsulfonyl fluoride and protease inhibitor mixture, luminol, p-cumaric acid, dimethylsulfoxide (DMSO), pervanadate, SU6656, were purchased from Merck (USA). PNU-282987 (N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride) was obtained from Tocris Biosciences (Bristol, UK).
Stock solutions were prepared in water (ACh, pervanadate) or in DMSO (PNU-282987, PP2, SU6656).
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5

Pharmacological Evaluation of PNU-282987

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The following drugs were used: PNU-282987, Tocris Bioscience (Bristol, UK); DMSO, LLC Tula Pharmaceutical Factory (Tula, Russia); saline, REACHIM (Old Kupavna, Moscow Region, Russia).
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6

Electrophysiological Analysis of α7 nAChR

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Cells were grown on laminin-covered glass placed on 24-well plates under the same conditions described in Section 2.4. To conduct the electrophysiological recording of the α7 nAChR-mediated macroscopic currents, a whole-cell patch clamp was set up as follows. Cells attached to the glass were transferred to a bath filled with extracellular electrophysiology solution (140 mM NaCl, 2 mM CaCl2, 2.8 mM KCl, 4 mM MgCl2, 20 mM HEPES, 10 mM glucose, pH 7.4). A microelectrode pipette was filled with intracellular buffer solution (140 mm CsCl, 6 mm CaCl2, 2 mm MgCl2, 2 mm MgATP, 0.4 mm NaGTP, 10 mm HEPES/CsOH, 20 mm BAPTA/KOH). A microelectrode was attached to the cell membrane until a resistance of at least 1 GOhm was reached. After establishing the gigaseal, the cell membrane was ruptured using suction and the recordings were created. A typical experiment consisted of 1 s cell wash with control extracellular buffer, then the bath solution was changed to an extracellular solution with 1, 10, or 100 μM of α7 nAChR-selective agonist PNU-282987 (Tocris, Bristol, UK) supplemented with 10 μM of α7 nAChR-selective positive allosteric modulator PNU-120596 (Tocris) for 5 s followed by 5 s wash-out with control extracellular buffer.
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7

Neuroinflammation Inhibition Assay

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The agonist PNU282987 and antagonist MLA were purchased from Tocris Bioscience (Bristol, UK), and LPS was purchased from Sigma–Aldrich (St. Louis, MO, USA). The [A10L]PnIA linear crude peptide was synthesized by Bankpeptide Biological Technology Co., Ltd. (Hefei, China) and purified in our laboratory.
A CCK-8 kit, DMEM-H medium, Penicillin/Streptomycin Solution, and PBS buffer were purchased from Sangon Biotech (Shanghai, China). FBS was purchased from Procell (Wuhan, China). A FastPure Cell/Tissue Total RNA Isolation Kit, HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper), 2 × Taq Plus Master Mix Ⅱ, and ChamQ Universal SYBR qPCR Master Mix were obtained from Vazyme (Nanjing, China). A TNF-α ELISA kit, IL-6 ELISA kit, and IL-1β ELISA kit were purchased from Thermo Scientific (Waltham, MA, USA).
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8

Investigating α7 nAChR Agonist PNU 282987 Effects

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The groups VAChT-KDHOM + LPS + PNU and WT + LPS + PNU received PNU 282987 treatment (Tocris, Bristol, UK), intraperitoneal, an agonist the α7 nAChR, in the dose of 10 mg/kg per animal, six hours after LPS instillation [16 (link)]
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9

Nicotine and α7-nAChR Agonist Effects

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Mz-ChA-1 cells were treated with 10 mmol/L nicotine and 10 mmol/L PNU282987 (a7-nAChR specific agonist; Tocris Bioscience, Bristol, UK) 28 in the presence or absence of 1 nmol/L a7-nAChR antagonist a-bungarotoxin (Tocris Bioscience) 28 and b-actin (loading control; Santa Cruz Biotechnology, Santa Cruz, CA) expression was evaluated by immunoblotting as previously described. 29
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10

Pharmacological administration protocols for cognitive studies

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Donepezil hydrochloride monohydrate (Sigma, UK) was dissolved in 0.5% carboxymethylcellulose and given in a volume of 1 ml/kg via the intraperitoneal (i.p.) route.
Risperidone (Sigma, UK) was dissolved in a minimum volume of acetic acid, made up to volume with distilled water and pH adjusted to 6 with 0.1M NaOH. Risperidone was administered in a volume of 1 ml/kg via the i.p. route. PNU-282987 (Tocris, UK) was dissolved in isotonic water and given in a volume of 1 ml/kg via the sub-cutaneous (s.c.) route. RJR-2403 oxalate (Tocris, UK) was dissolved in 0.9% saline and given in a volume of 1 ml/kg via the s.c. route. All drug doses were calculated as base equivalent weight and were administered 30 min before the acquisition trial, with the exception of PNU-282987 which was administered 60 min before the acquisition trial. In experiment 3 all drugs were given 30 min before the retention trial, except PNU-282987 which was administered 60 min before the retention trial.
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