Figure 6
JWH133 and HU308 do not elicit cytoskeletal rearrangements in CHO cells expressing murine GPR18 or GPR55.
CHO cells (1 × 106) were mixed with 2 μg of empty or GPCR-containing vector, transfected via electroporation and left overnight at 37 °C, 5% CO2 to allow tagged GPCR expression. (A-D) GPCR surface levels were determined by flow cytometry and representative histograms are shown. Cells transfected with empty vector alone were used to determine non-specific antibody staining. (A) FLAG-CB2, (B) HA-GPR18 and c-Myc-GPR55 (C) all had similar expression levels. (D) HA-C5aR1 was used as a positive control and displayed high surface levels. (E-L) Transfected CHO cells were plated into a 96 well E-plate (25,000 cells/well) and left overnight at 37 °C, 5% CO2 to reach a stable baseline. Afterwards, cells were stimulated with either vehicle (0.3% DMSO), C5a (10 nM) or cannabinoid (10 μM). (H) Quantification of C5a-induced responses found that only C5aR1 transfected cells responded to C5a (10 nM; E – Representative trace). JWH133 (F,I), HU308 (G,J) and CP55,940 (K) only caused significant responses in CB2 transfected cells. Importantly, cells transfected with GPR18 or GPR55 did not respond to either JWH133 or HU308 (I,J). Abnormal cannabidiol (Abn-CBD; 10 μM) did not elicit a response in any of the GPCR transfected cells (L). Data are mean ± SEM, n = 3-6 biological replicates with 2-3 technical replicates per condition. Statistical analysis was conducted by one-way ANOVA with Dunnett’s multiple comparisons correction. * P < 0.05, *** P < 0.001, comparing all samples to empty vector transfected cells.
