Human KGN cells formed stable 3D spheroids that expressed connexin 43 and CYP19 aromatase enzyme. Representative image of formalin-fixed, paraffin-embedded sections of KGN spheroids, stained with hematoxylin and eosin (A, B), immunostained for Cx43 (green) (C) or CYP19 enzyme (green) (D), isotype control for both anti-Cx43 and anti-CYP19 antibody (green) (C, D), cell membrane with wheat germ agglutin (WGA, red) (C), and nuclei with DAPI (blue) (C, D). KGN spheroids were fixed at 24 h postself-assembly (A, C, and D), or at 72 h after incubation in media containing 10−7M testosterone (B). Figure 2A includes an enlarged inset of a spheroid section with black arrows pointing at the pyknotic nuclei. Scale bar = 50 µm. Three independent experiments, n = 2–3 gels per experiment, each with 256 spheroids.
