Fig. 5: Pharmacological inhibition of AXL receptor induces a pAKT-mediated upregulation of intracellular cAMP levels in response to β-adrenergic stimulation.

Mature iBAs were serum-depleted for 2-3 hours and acutely treated with AXL inhibitors, anti-AXL antibody, PDE inhibitors and/or GAS6 in the presence or absence of isoproterenol. A Transcription factor enrichment analysis in upregulated genes upon AXL receptor siRNA-mediated knockdown. B Intracellular cAMP levels at indicated time points after isoproterenol stimulation and simultaneous treatment with treatment with AXL inhibitor BMS-777607, or AXL agonist GAS6 (* Control vs. BMS; # Control vs. GAS6) (n = 3/group). C Intracellular cAMP levels in response to acute (15 min) treatment with BMS-777607 and simultaneous stimulation with increasing isoproterenol doses (n = 3/group). D Representative western blots of 3 independent experiments of phosphorylated PKA substrates. E Representative western blots of 3 independent experiments of phosphorylated CREB (pCREB) and ATF (pATF) after (60 min) isoproterenol stimulation and concomitant treatment with BMS-777607 and/or GAS6. HSP90 was used as a loading control. F) OCR (Seahorse assays) measurements in isoproterenol-stimulated iBAs upon concomitant treatment with BMS-777607 or DMSO control (n = 5/group). Arrow signifies the time of injection of isoproterenol and compound. G Intracellular cAMP levels of isoproterenol-stimulated mature iBAs in response to acute treatment with an AKT activator (left panel) and an AKT inhibitor (right panel), in the presence or absence of the BMS-777607 (n = 3/group). H Intracellular cAMP levels of isoproterenol-stimulated mature iBAs in response to acute treatment with a PI3K inhibitor compound (left panel) and a PTEN inhibitor compound (right panel), in the presence or absence BMS-777607 (n = 3/group). I Representative western blots of two independent experiments of total AXL receptor expression in A549 cells treated for indicated times with anti-AXL antibody. siRNA-mediated knockdown of AXL receptor was used as positive control. γ-TUBULIN was used as loading control. J Representative western blots of three independent experiments of pAKT (S473) in response to acute treatment with anti-AXL antibody in mature iBAs. BMS-777607 was used as positive control and HSP90 was used as loading control. K OCR measurements in isoproterenol-stimulated (1uM) mature iBAs in response to treatment with anti-AXL antibody or IgG control (n = 6/group). Arrow signifies the time of injection of isoproterenol (Iso), IgG control or anti-AXL antibody (Ab). L Direct phosphodiesterase activity (relative values) in acutely isoproterenol-stimulated mature iBAs in response to treatment with anti-AXL antibody or IgG control (n = 4/group for PDE4, n = 3/group for all other conditions). M Gene expression analysis of Ucp1 and Pgc1-α in mature iBAs in response to pharmacological inhibition of PKA (PKAi), in the presence or absence of BMS-777607 (n = 4/group). N) Gene expression analysis of Ucp1 and Pgc1-α in mature iBAs in response to siRNA-mediated knockdown of Atf2 (siAtf2), in the presence or absence of BMS-777607 (n = 4/group). For all graphs, results are presented as average ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For two-group comparisons (C, L), an unpaired two-tailed t-test was performed. For three or more group comparisons (E, G, H, M, N) one-way ANOVA was performed. Tukey test was applied to correct for multiple comparisons. For time-course comparisons (B, F, K) two-way ANOVA was performed. Statistically significant differences are annotated in graphs.