Fig. 4

P2Y₁-R antagonists behave as inverse agonists. a Dose-response curve was performed in HEK293T cells co-expressing Gαq-RLuc8, GFP2-Gγ2, Gβ1, and P2Y₁-R after stimulation or not with increasing concentrations of MRS2179, MRS2279, or MRS2500 for 1 min. Results are expressed as the difference in the BRET signal measured in the presence and in the absence of ligand and are normalized to the mean value of maximal MRS2179 response. Data represent the mean ± s.e.m. of six independent experiments. Statistical significance between unstimulated and stimulated cells was assessed by one-way ANOVA followed by Sidak’s post-tests (**p < 0.01; ***p < 0.001; ****p < 0.0001). b Dose-response curve was performed in HEK293T cells co-expressing β-arrestin 2-RLuc and P2Y₁-R-Venus after stimulation or not with increasing concentrations of MRS2179, MRS2279, or MRS2500 for 15 min. Results are expressed as the difference in the BRET signal measured in the presence and in the absence of ligand and are normalized to the mean value of maximal MRS2179 response. Data represent the mean ± s.e.m. of five independent experiments. Statistical significance between unstimulated and stimulated cells was assessed by one-way ANOVA followed by Sidak’s post-tests (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). c Washed human platelets were incubated in the absence (basal) or in the presence of MRS2179 (10 μM), MRS2279 (10 μM), or MRS2500 (10 μM) for 2 h and IP1 accumulation was quantified. Data represent the mean ± s.d. of 8 healthy donors and are expressed as the percentage of matched basal values. The statistical comparison was assessed using Kruskal-Wallis test followed by Dunn’s post-tests (*p < 0.05; **p < 0.01; ****p < 0.0001)