Figure 7

Dual endocrine activity of Adrenoids. Schematic representation of catecholamine (A) and steroidogenic (B) synthesis cascade: in blue are indicated the enzymes detected in Adrenoids by Western blot (WB, C and E); in red, the corresponding hormones. (C) Representative Western blot analysis of Tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) protein expression in Adrenoids compared to positive (pheochromocytoma-Pheo-tissue lysates, MTT cells) and negative (H295R cells) controls. Molecular weight of the target proteins are shown in brackets. Stain-free gel total protein load/lane is shown as loading control. (D) Representative immunofluorescence staining for CHGA (green) at 40× magnification. Nuclear counterstaining is shown in blue. (E) Representative Western blot analysis of StAR and CYP11B1 proteins in Adrenoids compared to positive (H295R cells) and negative (MTT cells) controls. Stain free gel total protein load/lane is shown as loading control. For Western blots (panels C and E) to allow multiple detection of proteins on the same membranes, they were cut prior to hybridization with the different antibodies during the blotting procedure; moreover, acquired total images were further processed and cropped to evidence the target antigens. The corresponding fuller-length, original, unprocessed blots for each antibody and stain-free gels are shown in Fig. S8. (F) Representative image showing SF-1 nuclear expression detected by immunofluorescence staining (green) in H295R cell components of the Adrenoids, while MTT cells were traced by viability prestaining in red. Scale bar = 25 μm. (G) Catecholamine production at 5 and 10 days of the Adrenoid growth was measured intracellularly by LC-ECD. Bars are means ± SD of DOPA, dopamine, norepinephrine and epinephrine intracellular production normalized to the Adrenoid total protein content at the 2 time points in three independent experiments: *p < 0.05 and **p < 0.001, Student’s t test. (H) Steroid secretion was evaluated by mass spectrometry in conditioned media of Adrenoids at 5 and 10 days, normalized for the number of Adrenoids. Testosterone, androstenedione, progesterone, 17-OH-P, DHEAS, 11-deoxycortisol and 11-deoxycorticosterone were detectable at both day 5 and 10. Bars are means of steroid concentration ± SD/well in three independent experiments, except for DHEAS measurement performed in single.