Figure 1
The CB2 selective agonists JWH133, HU308 and DIAS2 differ in their ability to induce the directional migration of murine PECs.
(A) Bio-gel elicited murine PECs were placed into the top well of a modified 96 well Boyden chamber (8 μm pore size) and allowed to migrate towards vehicle or cannabinoid for 4 hours at 37 °C, 5% CO2. JWH133 (10 μM) significantly induced macrophage chemotaxis; however DIAS2 had no effect on macrophage migration. Data are mean± SEM, n = 3 biological replicates. (B-E) Bio-gel elicited murine PECs were placed into the upper chamber of a CIM-16 plate and allowed to migrate for 3-4 hours at 37 °C, 5% CO2 towards indicated compounds. (B) Real-time chemotaxis analysis confirmed that JWH133 and HU308, but not DIAS2 (all 10 μM), elicit PEC chemotaxis. (C) Chemokinetic analysis, comparing all combinations of vehicle or cannabinoid (10 μM) in the top (T) and bottom (B) chambers, demonstrated that JWH133 induced true chemotaxis. Data are mean real-time traces from one experiment with 4 technical replicates per condition and are representative of 2 independent biological replicates. (D,E) Concentration response analysis found that both JWH133 and HU308 elicit PEC chemotaxis in a concentration dependent manner. Data are mean ± SEM, n = 4 biological replicates with 3-4 technical replicates per condition. (F,G) Bio-gel elicited PECs were placed into a 96 well E-plate allowed to adhere for 2-3 hours at 37 °C, 5% CO2. Afterwards, cells were stimulated with either vehicle or cannabinoid at the indicated concentration. (F) Representative raw traces showing cell responses to JWH133 (30, 10, 3 and 1 μM). Data are mean traces from one experiment with 2-3 technical replicates per condition. (G) Concentration response quantification demonstrates that JWH133 and HU308 caused macrophage cytoskeletal rearrangement in a concentration dependent manner, with EC50 values of 5.3 and 1.6 μM. Data are mean ± SEM, n = 3 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 < 0.01 *** P < 0.001, comparing all samples to vehicle control.
