Fig 2: Gentisic acid reduced the expression of angiotensin-converting enzyme 1 (ACE1) and angiotensin II receptor type 1 (AT1) in TAC-induced heart failure. Three weeks after TAC, mice were administered the vehicle, gentisic acid (10 mg∙kg−1∙day−1 or 100 mg∙kg−1∙day−1), or bisoprolol (0.5 mg∙kg−1∙day−1) daily for 3 weeks. (A) Representative western blots of ACE1, AT1, and endothelin-1 receptor type A (ETAR) expression in hearts from mice in the sham, TAC, TAC + gentisic acid (10 mg∙kg−1∙day−1 or 100 mg∙kg−1∙day−1), or TAC + bisoprolol (0.5 mg∙kg−1∙day−1) groups. (B‒D) The protein levels of ACE, AT1, and ETAR were quantified. Representative images were cropped from different parts of the same western blot. Full-length images are included in a Supplementary Information file. *P < 0.05 and **P < 0.01 versus the sham group; #P < 0.05 and ##P < 0.01 versus the TAC group; NS: not significant.

Fig 3: Functional characterization of AT1R-AT2R heteromer in HEK-293T cells. HEK-293T cells were transfected with cDNAs for AT1R (1 μg) and/or AT2R (1 μg). Cells were pretreated (15 min) with selective receptor antagonists (300 nM candesartan for AT1R or 1 μM PD123319 for AT2R receptors) and subsequently treated with selective agonists (100 nM angiotensin II for AT1R and 300 nM CGP-42112A for AT2R receptors). a–c ERK1/2 phosphorylation was analyzed using an AlphaScreen®SureFire® kit (Perkin Elmer). Values are the mean ± S.E.M. of 5 independent experiments performed in duplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. Signaling output was the dependent variable and the different treatments were the independent variables (*p < 0.05, **p < 0.01, ***p < 0.001; versus vehicle treatment (basal)). d–f DMR tracings represent the picometer-shifts of reflected light wavelength over time. Values are the mean ± S.E.M. 8 independent experiments performed in triplicates

Fig 4: AT1R-AT2R heteromer expression in brain striatal sections of Parkinson’s disease (PD) rat model. a–d PLA assays in striatal sections from the 6-OH-dopamine PD rat model, non-lesioned (a), lesioned (b), and lesioned plus chronically treated with l-DOPA and either lacking (c) or displaying (d) dyskinesias. Confocal microscopy images (stacks of 3 consecutive planes) show heteroreceptor complexes as red clusters and Hoechst-stained nuclei (blue). Scale bar: 20 μm. e Bar graph showing the percentage of red dots/cell. Data are the mean S.E.M . of counts in 9–12 different fields per animal (n = 4 per group). One-way ANOVA followed by Bonferroni’s post-hoc multiple comparison tests were used to compare the red dots/cell values. The number of clusters (r) was the dependent variable and the four animal groups treatments were independent variables (***p < 0.001; versus lesioned condition, ++p < 0.01; versus l-DOPA non-dyskinesia condition)

Fig 5: Functional characterization in HEK-293T cells expressing the AT1R-AT2R heteromer. HEK-293T cells were pretreated with selective receptor antagonists (300 nM candesartan for AT1R or 1 μM PD123319 for AT2R) and subsequently treated with selective agonists (100 nM angiotensin II for AT1R and 300 nM CGP-42112A for AT2R) a–c Cytosolic calcium detection assay were performed in HEK-293T cells transfected with the cDNAs for an engineered calcium sensor, 6GCaMP (1 μg), AT1R (1 μg), and/or AT2R (1 μg). Values are the mean ± S.E.M. of 5 independent experiments performed in duplicates. d–f Intracellular cAMP levels were determined by TR-FRET as described in Methods. HEK-293T cells were transfected with cDNAs for AT1R (1 μg) and/or AT2R (1 μg). When Gi coupling was assessed, decreases in [cAMP] were determined using 0.5 μM forskolin added 15 min after the agonists stimulation. Values are the mean ± S.E.M. of 6 independent experiments performed in triplicates. In cAMP one-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. Signaling output was the dependent variable and the different treatments were the independent variables. (*p < 0.05, **p < 0.01, ***p < 0.001 versus forskolin treatment; +++p < 0.001 versus Ang II treatment; &&&p < 0.001 versus CGP-42112A treatment)