M 3m3fbs

The group 1 mGluR agonist DHPG (50 μM, Tocris, UK) was used to activate group 1 mGluR. To block mGluR5 and mGluR1a, MPEP (10 μM, Tocris, UK) and LY367385 (100 μM, Tocris, UK) was applied to rat hippocampal slices, respectively. AM251 (3 μM, Tocris, UK) was used to block presynaptic CB1R. U73122 (5 μM, Tocris, UK) was used to block PLCβ activity. m-3M3FBS (30 μM, Tocris, UK) was used as the PLC activator. The NMDAR antagonist D-AP5 (50 μM, Tocris, UK) and the AMPA receptor antagonist CNQX (20 μM, Tocris, UK) were used for the eIPSC recordings. AβO and scrambled AβO were synthesized from a lyophilized powder of Aβ and scrambled Aβ peptide, respectively (Bachem, Japan). A 4× Laemmli sample buffer (Bio-Rad, USA) and running buffer (Bio-Rad, USA) were used for western blot SDS-PAGE. For the antibody incubation step in western blotting, rabbit monoclonal primary antibodies (NBP2-38220, Novus, USA) and HRP-conjugated secondary anti-rabbit antibodies (Cat# 170-6515, Control# 64170140, RRID: AB_2617112, Bio-Rad, USA) were used, respectively.

The following drugs were used: m-3M3FBS, U73122, U0126, GF109203X, FPL64176, 2-methyl-serotonin maleate salt (2-Methyl-5-HT), and GR73632 were purchased from Tocris (Minneapolis, MN); McN-A-343, quinpirole HCl and nifedipine from Sigma Sigma/RBI (St. Louis, MO); thapsigargin, dantrolene and 2-APB from Santa Cruz Biotechnology (Dallas, TX). Palonosetron and netupitant were kindly provided by Helsinn Health Care (Lugano, Switzerland). m-3M3FBS, U73122, nifedipine, U0126, netupitant were dissolved in a mixture of ethanol/Tween 80/saline at a volume ratio of 1:1:18. dantrolene, 2-APB, GF109203X and FPL64176 were dissolved in 25% DMSO in water. thapsigargin was dissolved in 10% DMSO in distilled water. Other drugs were dissolved in distilled water. All drugs were administered at a volume of 0.1 ml/10 g of body weight.

Six-well plate cultures of either the C-20/A4 cell line or AC were treated for 8 h with individual or combination treatments of: the CRF-R1 specific antagonist CP-154526 (1–50 μM; Tocris), the CRF-R2 specific antagonist astressin 2B (1–50 μM; Tocris), the non-selective cation channel blocker Gd³⁺ (100 μM; Tocris), the adenylate cyclase activator forskolin (0.1 μM; Tocris), the PLC activator m3M3FBS (0.1 μM; Tocris), or the PLA₂ inhibitor OBAA (0.1 μM; Tocris). Cells were then assayed for cell death or photographed using an Olympus IX73 phase contrast microscope and camera.

Acetylcholine (1mM, Sigma) was used to exogenously stimulate cholinergic receptors. Atropine (200 nM, Sigma) and Dihydro-β-erythroidine (DHBE, 10 μM, Tocris) were used to competitively block muscarinic receptors and β2 subunit-containing nicotinic receptors respectively. Mecamylamine (5 μM, Tocris) was used to further non-competitively block nicotinic receptors. Phospholipase C activator m-3M3FBS (25 μM, Tocris) was used to cleave GPI-anchored Lynx prototoxins and the inactive ortholog o-3M3FBS (25 μM, Tocris) was used as a control.⁵⁵ (link) Cyclodextrin (1 mM, Tocris) was included in a small subset of experiments to improve solubility of 3M3FBS compounds, but no further improvement in efficacy was observed. Water soluble recombinant Ly6g6e (0.5 mg/ml) was obtained by custom purification (Creative Biomart) and used for exogenous application at 1: 1000 and 3:1000 dilution. Effects on nicotinic receptors were not distinguishable between the two different protein concentrations. Only freshly thawed protein aliquots were used for experiments.

Prior to Ca²⁺ imaging measurement (r)TRPM8-transfected or empty-vector-transfected HEK cells were treated with 5 µM or 10 µM oxaliplatin or saline for 24 h. HEK cells transfected with (h)TRPV1 or empty-vector plasmid DNA were treated with 10 µM oxaliplatin prior to Ca²⁺ imaging experiments.
For identifying the related pathway that contribute to TRPM8 channel desensitization in (r)TRPM8-transfected HEK cells 24 h after oxaliplatin treatment, cells were treated with 10 µM oxaliplatin ±1 µM of the PLC inhibitor U 73122 (Tocris Bioscience, Bristol, UK), 10 µM oxaliplatin ±1 µM of the PKC inhibitor GF 109203X (Tocris) or 10 µM oxaliplatin ±10 µM or 25 µM of the PLC activator m-3M3FBS (Tocris) prior to Ca²⁺ imaging measurement.
For PI (4,5) P2 ELISA measurement analysis, (r)TRPM8-transfected or empty-vector-transfected and untransfected HEK cells were treated with 10 µM oxaliplatin for 24 h. For investigating the contribution of the PLC pathway to the PIP₂ amount, (r)TRPM8-transfected or empty-vector-transfected and untransfected HEK cells were treated with 10 µM oxaliplatin ±10 µM of the PLC inhibitor D609 (Tocris) for 5 h.

HEK293T/17 cells were infected with a lentivirus to express of Rαq, a Gαq-coupled designer receptor, as described [24 (link)]. HEK293 cells expressing TRP channels TRPM3 (T-REx-TRPM3 cells) and TRPM8 (HEK293-M8 cells) have been described elsewhere [4 (link),51 (link)]. HEK293-∆B-Raf:ER cells [52 (link)] express a conditionally active B-Raf protein kinase mutant that could be activated with 4-hydroxytamoxifen (4OHT, Sigma # H7904, dissolved in ethanol) for 24 h in medium containing 0.05% fetal calf serum. HEK293 cells, T-REx-TRPM3 cells, HEK293-M8 cells, and HEK293-∆B-Raf:ER were incubated in DMEM containing 0.05% fetal bovine serum for 24 h prior to stimulation. Stimulation was performed with clozapine-N-oxide (1 μM CNO, dissolved in ethanol, Enzo Life Sciences, Lörrach, Germany, # NS-105-0005), pregnenolone sulfate (PregS, 20 μM, dissolved in DMSO, Sigma-Aldrich GmbH, Taufkirchen, Germany, # P162), icilin (1 μM, Santa Cruz Biotechnology, Heidelberg, Germany, # sc-201557), or 4-hydroxytamoxifen (4OHT) (100 nM, Sigma # H7904, with ethanol as solvent) for 24 h in medium containing 0.05% fetal bovine serum. Cells were preincubated for 3 h with the compound 2,4,6-Trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide (m-3M3FBS) (Tocris, Bristol, UK, Cat.No. 1941, dissolved in DMSO) at a concentration of 5 μM. Cells were stimulated for 24 h in the presence of m-3M3FBS.