O 1602

Monoclonal IgE (clone SPE7), RPMI 1640, 2-mercaptoethanol (2-ME), Laemmli buffer, L-α-lysophosphatidylinositol (LPI), and sphingosine-1-phosphate (S1P) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HEPES, non-essential amino acids (NEAA), penicillin, streptomycin, and fetal bovine serum (FBS) were obtained from Gibco-BRL-Life Technologies (Gaithersburg, MD, USA). Interleukin (IL)-3 was purchased from PeproTech (Rocky Hill, NJ, USA). The GPR55 synthetic agonist, O-1602, was purchased from Cayman Chemical (Ann Arbor, MI, USA), while the GPR55 antagonist, ML-193, was purchased from TOCRIS (Bristol, UK). The CB2 antagonist, AM630, was from Sigma-Aldrich (St. Louis, MO, USA). Anti-p-cofilin (pSer 3) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-p-LIMK (pThr 508/505) and anti-β-actin (C-terminal) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The secondary antibodies, HRP-coupled anti-mouse and anti-rabbit, were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Rhodamine-labeled phalloidin and calcein-AM were obtained from Life Technologies (Carlsbad, CA, USA). DAPI was purchased from Invitrogen (Carlsbad, CA, USA). The polycarbonate filters were from Neuro Probe, and bovine skin gelatin from Sigma-Aldrich (St. Louis, MO, USA)

Paclitaxel and thiazolyl blue methyltetrazolium bromide (MTT) were obtained from Millipore-Sigma and dimethyl sulfoxide (DMSO) from Fisher BioReagents. O-1602 and abnormal CBD were obtained from Cayman Chemical Company. For all experiments, the drugs were dissolved in DMSO. The structures of cannabinoids used in this study were drawn using ChemDraw Prime 16.0.

Cocaine, nicotine and Δ⁹-THC was provided by the NIDA pharmacy (Baltimore, MD). O-1602 was supplied by Cayman Chemical (Cat#: 10006803). The selective GPR55 antagonist CID 16020046 (CID, Cat#: 4959) and Tocrifluor T1117 were purchased from Tocris Bioscience. For the more in detail, please see Supplmentary Information.

C2C12 cells were a kind gift from Professor David Cameron-Smith (Deakin University, Melbourne, Australia). Mouse-derived C₂C₁₂ myoblasts were cultured in Dulbecco’s modified eagle high glucose growth medium (D-MEM) supplemented with 10% foetal bovine serum (v/v), 1% penicillin streptomycin (v/v), 0.5% amphotericin B (v/v) and incubated at 37°C, 5% CO₂ in a humidity controlled environment as previously described [39 (link)]. C₂C₁₂ myoblasts were seeded into 6 well plates and differentiated into myotubes (within ~72 h) via supplementation with 2% horse serum (v/v), 1% penicillin streptomycin (v/v), 0.5% amphotericin B (v/v) [39 (link)]. C₂C₁₂ cells were serum starved in 0.1% BSA (w/v) and a D-MEM solution for six hours prior to treatment, then treated for 24 h with vehicle (0.1% ethanol; Sigma Aldrich, St Louise, MO, USA) (n = 8–9) or O-1602 (10 nM–1000 nM; Cayman Chemical, Ann Arbor, Michigan, USA) (n = 9) or O-1918 (100nM; Cayman Chemical, Ann Arbor, Michigan, USA) (n = 8) and all treatments were dissolved in 0.1% ethanol and suspended in a 0.1% BSA and a D-MEM solution. Following treatment, cells were washed with ice cold PBS; then lysed, on ice with TRIzol Reagent^® (Invitrogen, Victoria, Australia) and stored at −80 °C for subsequent RNA extraction.

AEA, O-1602 and AbnCBD were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). NAGly was bought from both Cayman and Enzo Biosciences (in order to exclude the possibility that lack of responses were related to a specific company’s synthesis/production; results were equivalent for both suppliers). Forskolin, CP55, 940, SR141716A, AM251 and AM630 were purchased from Tocris Bioscience (Bristol, UK). Phorbol-12-myristate-13 acetate (PMA) was purchased from Sigma Aldrich (St Louis, MO, USA). U0126 was purchased from Cell Signaling Technology (Danvers, MA, USA). All cannabinoid and lipid ligands were diluted in ethanol prior to storage in single use aliquots at −80 °C. The lipophilic properties of cannabinoids and lipid ligands such as NAGly cause them to adsorb onto the hydrophobic surfaces of plasticware and interact with serum components. To sustain drug availability in assays, the vessels in which cannabinoids were diluted were silanised (Coatasil, ThermoFisher AJA2293; Waltham MA, USA) and autoclaved prior to use, and assay media or HBSS were supplemented with 1 mg/ml bovine serum albumin (BSA, MP Biomedicals ABRE, Santa Ana, CA, USA).