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. 2023 Jul 25;120(31):e2302668120. doi: 10.1073/pnas.2302668120

Fig. 4.

Fig. 4.

DFPQ inhibits internalization of the β2AR and protects from agonist-induced desensitization in cell and tissue models. (A) Effect of DFPQ on agonist-promoted internalization of the β2AR. HEK 293 cells stably expressing FLAG-β2AR were preincubated with 0.1% DMSO or 10 μM DFPQ for 30 min and then stimulated with 10 μM ISO for up to 60 min. Cells were fixed and receptor surface expression was measured by ELISA. These data represent the mean ± SD from three independent experiments. ***P < 0.001; **P < 0.01; *P < 0.05 (B) HEK 293 cells were desensitized by incubating with 1 μM ISO ± 10 μM DFPQ, washed with PBS, then stimulated with ISO at the indicated concentrations for 10 min. Cells were lysed and cAMP production was measured by ELISA. Cells incubated with DFPQ during desensitization were protected from reduced response to agonist after wash. Data are the mean ± SD, n = 3. (C) Comparison of the stiffness in HASM cells following a 30 min pretreatment with (n = 473 individual cell measurements) or without (n = 463 individual cell measurements) 1 μM DFPQ. Histograms are represented as geometric mean ± 95% CIs. (D) Functional desensitization of the β2AR was studied by pretreating HASM cells with or without 1 μM DFPQ for 30 min, followed by relaxation over a 30 min period induced by 10 μM ISO. Data are collected at every 1.3 s (t, 0 to 1,800 s) and presented as estimated mean ± SE (n = 463 to 473 cell measurement per treatment). (E) Murine precision cut lung slices were contracted with 1 μM methacholine for 5 min and then incubated overnight with 1 μM ISO ± 1 μM DFPQ. Airway slices were washed, recontracted with methacholine, and incubated with ISO for 1 h (n = 4 airways). Airway contractility was measured by the luminal area of bronchioles by microscopy at various times. (F) Bar graphs represent normalized ISO-promoted relaxation from methacholine contraction on day 2 of treatment at the 25 min time point from 4 airways. Statistical significance (*) in panels E and F were assessed using an unpaired t test, P < 0.05.