Kolliphor el

Oxycodone (USP, Rockville, MD) was dissolved in saline (0.9% NaCl; Hospira, Lake Forest, IL), and morphine (10 mg/mL; Hikma, Eatontown, NJ) was diluted in saline; each was delivered s.c. in a volume of 5 mL/kg body weight. Δ⁹-tetrahydrocannabinol (THC; 200 mg/mL; National Institute on Drug Abuse Drug Supply Program, Bethesda, MD) was diluted with 95% ethanol followed by a mix of Kolliphor EL (Sigma-Aldrich, St. Louis, MO) and saline to achieve a final vehicle of 5% 95% ethanol, 5% Kolliphor EL, and 90% 0.9% saline, delivered s.c. in a volume of 5 mL/kg. Fenobam (SCYNEXIS, Durham, NC) was dissolved in a vehicle of 100% DMSO (Sigma-Aldrich), delivered i.p. in a volume of 20 μL. For all drugs, testing was performed within 30 minutes (30–60 minutes postdelivery of oxycodone, morphine, and Δ9-THC; 5–35 minutes postdelivery of fenobam). Doses were chosen based on previous reports that analgesia and/or antinociception can be achieved at the indicated doses for oxycodone,^{2 (link),14 (link),26 (link),30 (link),39 (link),40 (link)} morphine,^{26 (link),30 (link)} fenobam,^{25 (link),27 (link),28 (link)} and Δ⁹-THC.^{3 (link),9 (link),23 (link)} Mice were returned to home cage after injection and were kept there until testing.

Mice were given an intraperitoneal (IP) injection of DO34 (50 mg/kg) synthesized as previously described (Ogasawara et al., 2016 (link)) dissolved in an 18:1:1 solution of saline, ethanol, and kolliphor EL (Sigma–Aldrich, St. Louis, MO, United States), or vehicle alone (18:1:1 solution of saline, ethanol and kolliphor EL) 2 h prior to behavioral testing at a volume of 10 ml/kg. In one experiment, mice were treated with a combination of DO34 (50 mg/kg) and PF-3845 (FAAH Inhibitor) at 1 mg/kg (A gift from Pfizer Central Research), via IP injection 2 h prior to behavioral testing, or just DO34 (50 mg/kg) alone. In two experiments, mice were treated with a combination of IP injection of DO34 (50 mg/kg) 2 h prior to extinction training and Tetrahydrocannabinol (THC; CB1R partial agonist) at 0.3 mg/kg (in one experiment) or 0.6 mg/kg (Cayman Chemical Company-18:1:1 solution of saline, ethanol and kolliphor EL), via IP injection 30 min prior to behavioral testing, or just DO34 (50 mg/kg) alone. Doses utilized in this study were similar to those previously described in (Bedse et al., 2017 (link)).

All experiments were performed using Chinese Hamster ovary cells, CHO-K1 (Cat. No 85051005; European Collection of Cell Cultures, Salisbury, UK), which do not express endogenous EGFR. Cells were cultured in Ham's F-12 Nutrient Mixture (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA) and 50 μg/ml gentamycin (Life Technologies) at 37°C under 5% CO₂ atmosphere. Two days prior to imaging, cells were seeded on a glass bottom 8-well Lab-Tek chamber slide (Thermo Scientific, Rockford, IL) and transfected with one or combination of following plasmids: eGFP/pcDNA3, Lyn-eGFP/pcDNA3, Lyn-mCherry/pcDNA3, wild type or mutant eGFP-EGFR/pcDNA3, mCherry-HA-PDK1/pcDNA3, PLCγ1-mCherry/pcDNA3, pEKAREV [46 (link)] (~0.2 μg of each plasmid per well) using FuGENE6 transfection reagent (Promega, Madison, WI) according to the manufacturer’s protocol. The EGFR expression level was evaluated by the confocal microscopy. Before imaging, cells were incubated in serum-free F-12 for ≥ 20 min for serum starvation. For PKC activation experiments, cells were incubated for ≥ 20 min in serum-free F-12 containing of 200 nM phorbol 12-myristate 13-acetate (PMA; InvivoGen, Toulouse, France) and 0.02% Kolliphor EL (Sigma-Aldrich). For PKD inhibition experiments, cells were incubated with 10 μM CID755673 (Sigma-Aldrich) in the presence of 0.02% Kolliphor EL.