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1.
Fig. 4.

Fig. 4. From: Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry.

Concentration-response curves for FAP-receptor internalization or stabilization with selected hit compounds from the PCL agonist screen (a–c) and antagonist screen (d–f). a, levonordefrin, an αAR agonist (EC50 = 4.5 × 10−5 M). b, anisomycin (EC50 = 3.5 × 10−8 M). c, pimozide (EC50 = 2 × 10−5 M). d, propafenone (IC50 = 2.1 × 10−7 M). e, naftopidil (IC50 = 1.2 × 10−7 M). f, pizotifen (IC50 = 8.6 × 10−6 M). Norm, normalized.

Yang Wu, et al. Mol Pharmacol. 2012 October;82(4):645-657.
2.
Fig. 1.

Fig. 1. From: Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry.

Internalization of FAP-tagged receptors detected by confocal microscopy. a, image of living AAM2 cells treated with 150 nM TO1-2p, obtained 1 to 60 min after treatment. b, image of living AAM2 cells stimulated with 20 μM ISO for 60 min before the addition of 150 nM TO1-2p, obtained 1 to 15 min after the final addition. c, image of living AAM2 cells treated with 20 μM ISO and 150 nM TO1-2p, obtained 20 to 60 min after the simultaneous additions. Diagrams describe the experimental scheme to acquire each confocal image and the presumed states of the receptor with the FAP tag, fluorogen, and agonist.

Yang Wu, et al. Mol Pharmacol. 2012 October;82(4):645-657.
3.
Fig. 2.

Fig. 2. From: Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry.

Flow cytometric characterization of AAM2 cells with TO1-2p, agonist, and antagonist. a and b, kinetic (a) and equilibrium (b) binding of TO1-2p to AAM2 cells. a, binding between TO1-2p and AM2.2 had a half-time of less than 5 s. Solid, dashed, and dotted lines represent the binding of TO1-2p to resting AAM2 cells, ISO-stimulated AAM2 cells, and wild-type U937 cells, respectively. b, TO1-2p binds with high affinity, displaying a Kd value of ∼2 nM. c, β2AR agonist ISO-induced receptor internalization, with an EC50 value of ∼800 nM. d, time course of surface β2AR internalization induced by ISO in the absence and presence of the β2AR antagonist ICI 118,551. Open triangles, open circles, and open diamonds represent the signals for AAM2 cells treated with 0.2, 1, and 20 μM ISO, respectively, solid diamonds represent the signals for resting AAM2 cells, and crosses represent the signals for AAM2 cells treated with 10 μM ICI 118,551 plus 20 μM ISO. Norm, normalized.

Yang Wu, et al. Mol Pharmacol. 2012 October;82(4):645-657.
4.
Fig. 5.

Fig. 5. From: Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry.

β2AR-GFP-DHA bead competitive binding assay. a, schematic diagram of the HTFC-compatible β2AR-DHA competitive binding assay. Soluble β2AR-GFP can bind to dihydroalprenolol-derivatized beads at high affinity (left receptor), whereas β2AR ligands (agonists or antagonists) can block the binding between the receptor and the beads (right receptor), leading to decreased fluorescence signals for the beads. b, concentration-response curves for selected agonists measured with the β2AR-DHA competitive binding assay. The y-axis indicates normalized (Norm) bead fluorescence intensity, in arbitrary units (a.u.). c, concentration-response curves for selected antagonists measured with the β2AR-DHA competitive binding assay. ALP served as the positive control for the experiment. All experiments were performed in duplicate at least three times. EC50 values for all β2AR ligands and several active compounds can be found in Table 1. BAM, bambuterol; SALB, salbutamol; CLEN, clenbuterol; SALM, salmeterol; ACE, acebutolol; BET, betaxolol; PRON, pronethalol; (+)LEV, (+)-levobunolol; PENB, penbutolol.

Yang Wu, et al. Mol Pharmacol. 2012 October;82(4):645-657.
5.
Fig. 7.

Fig. 7. From: Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry.

Time course of changes in cell surface and total β2AR levels. Cell total and surface receptor levels were measured 0, 30, 60, 90, 120, 180, 240, 300, and 360 min after drug treatment. a, untreated β2AR-GFP cells (DMSO) and cells treated with 10 μM ISO, 10 μM ANI, 10 μM CYC, 10 μM LEVON, or 10 μM (s) Atenolol (ATEN) together with 1 μM ISO. b, AAM2 cells preincubated with 1 μM TO1-2p before drug treatment and analysis. c, untreated AAM2 cells and cells treated with 10 μM ISO, 10 μM ANI, 10 μM CYC, or 10 μM LEVON. An aliquot of cells was removed from the stock and cooled on ice for 15 min. TO1-2p (1 μM) was added to the cells and the mixture was incubated for 15 min before flow cytometric measurements. Norm, normalized.

Yang Wu, et al. Mol Pharmacol. 2012 October;82(4):645-657.
6.
Fig. 6.

Fig. 6. From: Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry.

Confocal microscopic images of AAM2 cells (a–f) and β2AR-GFP cells (g–l) obtained in parallel, demonstrating the extent of receptor internalization. For AAM2 cells, a saturating concentration of TO1-2p was added to the cells together with the compounds, to track the translocation of FAP-β2ARs. a and g, untreated cells of both cell types showed receptors largely on the plasma membrane, with little receptor internalization. b and h, cells of both cell types that were treated for 1 h with a 10 μM concentration of the β2AR agonist ISO showed more receptors internalized than on the surface (high receptor internalization levels). c and i, test cells that were treated with 10 μM anisomycin showed little internalization, which classified these compounds as nonagonists of receptor internalization. e and k, cells that were treated with the β2AR antagonist ALP plus 1 μM ISO showed little internalization. d and j, cells that were treated with 10 μM LEVON showed a certain degree of receptor internalization, indicating that LEVON acts as a weak agonist of β2AR. f and l, test cells that were treated with 10 μM naftopidil plus 1 μM ISO also showed little internalization, which classified this compound as an antagonist of receptor internalization.

Yang Wu, et al. Mol Pharmacol. 2012 October;82(4):645-657.
7.
Fig. 8.

Fig. 8. From: Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry.

a, time course comparison of cell surface AM2.2-β2AR levels in the presence of 1% DMSO, 1 μM ISO, 1 μM BUC, 1 nM BUC, 1 μM CAV, 1 nM CAV, 1 μM (S)-PRO, or 1 nM (S)-PRO. Cell surface receptor levels were measured 0, 30, 60, 90, 120, 180, 240, and 300 min after drug treatment. ISO (10 μM) progressively induced receptor internalization, and DMSO had no effect on surface receptor expression. All other compounds tested did not induce receptor internalization up to 5 h after treatment. b, concentration-response curves for FAP receptor stabilization with BUC, CAV, or (S)-PRO in the presence of 1 μM ISO. The presence of up to 1 μM BUC, CAV, or (S)-PRO alone did not induce any surface receptor internalization, and all three compounds effectively prevented ISO-induced receptor internalization, with nanomolar efficacy [BUC, IC50 = 1.8 nM; CAV, IC50 = 1.5 nM; (S)-PRO, IC50 = 0.7 nM]. Norm, normalized.

Yang Wu, et al. Mol Pharmacol. 2012 October;82(4):645-657.
8.
Fig. 3.

Fig. 3. From: Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry.

Screens of a Prestwick Chemical Library plate for FAP-β2AR ligands yielding internalization (for agonists) or prevention of ISO-induced internalization (for antagonists). a and c, screen shots of time versus fluorescence signals for a complete 384-well plate targeting β2AR agonists (a) and antagonists (c). Pseudo-color represents the density of cells that emit the same level of fluorescence signal at any given time point; blue represents the highest cell density and red the lowest. Inset, magnified data from the first 26 wells of the plate shown in a. Each cluster represents data from a single well, and the gaps between clusters are air bubbles used to separate the samples. The larger gap toward the end of the trace indicates the end of the row, because the last two wells of each row were intentionally left free of samples. b and d, analyzed data from row N for the screens shown in a and c, respectively. b, point 1, signal for positive control well N1 (12.5 μM ISO); point 2, signal for negative control well N2 (vehicle); point 3, hit for cells treated with a 10 μM concentration of the β2AR full agonist salmeterol. d, points 1 and 1′, signals for positive control wells N1/N23 (9 μM ICI 118,551 and 1 μM ISO/vehicle); point 2, signal for negative control well N2 (1 μM ISO); point 3, hit for cells treated with a 9 μM concentration of the β2AR antagonist timolol; point 4, near-hit for cells treated with a 9 μM concentration of the αAR-selective antagonist prazosin.

Yang Wu, et al. Mol Pharmacol. 2012 October;82(4):645-657.

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