Fig 2: Detection of AT1R-ACE2, AT2R-ACE2, and MasR-ACE2 complexes in mouse lung tissue. Proximity Ligation Assays (PLAs) were performed using lung sections of adult (A–C) or 19-day fetal CD-1 mice (D–F) as described in the Methods. Cell nuclei were stained with Hoechst (blue). Protein complexes appear as red clusters or dots. Representative images corresponding to stacks of 4 sequential planes are shown. Graphs (G,H) display the number of clusters and spots in spot-containing cells. Values presented are the mean ± SEM of 5 independent experiments.

Fig 3: RAS receptors regulate cell surface expression of ACE2. (A) Immunocytochemistry assays were performed in HEK-293T cells expressing ACE2-eGFP together with AT1R-RLuc, AT2R-RLuc, or MasR-RLuc and activated with the respective agonists. Cells that expressed each RAS receptor were treated with their respective selective agonist. ACE2-eGFP expression was evaluated quantitatively via green fluorescence. The RLuc-containing receptors were detected by an anti-RLuc primary antibody and a secondary Cy3-conjugated anti-mouse antibody (red-staining). Colocalization is shown in yellow. Scale bar = 10 µm. (B–G) Biotinylation experiments were performed in HEK-293T cells transfected with cDNA encoding AT1R-RLuc (1 µg; B,E), AT2R-RLuc (1 µg; C,F) or MasR-RLuc (1 µg; D,G) with or without 0.8 µg of ACE2-HA cDNA. Images from a representative experiment are shown (expansion of the image areas in Figure 5 and position of the MW from this representative experiment appear in Supplementary Figure S1). Immunoreactive bands from 6 independent experiments were quantified. Values presented are the mean ± SD. One-way ANOVA followed by Bonferroni’s multiple comparison posthoc tests were used for statistical analysis; * p < 0.05, *** p < 0.001 vs. ACE2-HA singly-transfected cells.

Fig 4: Interactions of RAS receptors and ACE2 as assessed by Bioluminescence Resonance Energy Transfer (BRET) assays. BRET assays were performed in HEK-293T cells transfected with constant amounts of cDNA encoding AT1R-RLuc (0.5 µg) (A), AT2R-RLuc (0.4 µg) (B), MasR-RLuc (0.6 µg) (C), or GHS-R1a-RLuc (0.3 µg; negative control) (D) together with increasing amounts of cDNA encoding ACE2-eGFP (0.1 to 1 µg). Cells were treated (red symbols) or not (black symbols) for 25 min with selective antagonists (candesartan for AT1R, PD123319 for AT2R or A779 for MasR, both at 1 µM; red symbols) or selective agonists (Ang II for AT1R, CGP for AT2R or Ang 1-7 for MasR, all at 100 nM; green symbols). Values correspond to experimental points from 6 independent experiments each performed in quadruplicate. BRET50 and BRETmax values were calculated by non-linear regression using Prism GraphPad software; specific parameters are as described in the text.