Figure 1

Characterization of adrenal glands and adrenal gland chromaffin cells of EPHB6 KO mice. For A, B and C, experiments were conducted three times and representative data are presented. AGCCs: adrenal gland chromaffin cells. (A) EPHB6 mRNA deletion in the adrenal glands and spleen of EPHB6 KO mice. Total RNA was extracted from the adrenal glands and spleen of male WT and EPHB6 KO mice and analyzed by RT-qPCR for EPHB6 mRNA levels. Beta-actin levels were used as internal controls. Samples in RT-qPCR were in triplicate, and EPHB6/β-actin signal ratios are shown as means ± S.E. (B) EPHB6 deletion in adrenal gland chromaffin cells from EPHB6 KO mice according to immunofluorescence. Adrenal gland chromaffin cells isolated from adrenal glands of male WT and EPHB6 KO mice were cultured for one day, and then stained with goat anti-mouse EPHB6 Ab followed by Alexa-488-conjugated donkey anti-goat Ab (green). Nuclei were stained with DAPI (blue). (C) Normal histology of EPHB6 KO adrenal glands. Sections of adrenal glands from 8- to 10-week-old male WT and EPHB6 KO mice were stained with hematoxylin/eosin. (D) Adrenal gland chromaffin cells from male WT and EPHB6 KO and WT mice are similar in size. Left panel: phase-contrast micrographs of adrenal gland chromaffin cells from WT and KO mice after 24-hour culture. Right panel: diameters of adrenal gland chromaffin cells from WT and KO mice after 24-hour culture. Means ± S.E. of the diameters of more than 30 adrenal gland chromaffin cells (more than 10 cells/mouse and three mice/group) from male WT and KO mice are shown. No significant difference is observed (2-way Student’s t test). (E) Epinephrine release from the adrenal glands of WT and KO mice. Adrenal glands, isolated from male, female and castrated male KO and WT mice, were cut in half, and were stimulated with acetylcholine (5 mmol/L) in 300 μl Hank’s buffer for one minute at room temperature. The supernatants were analyzed for epinephrine levels by ELISA. Data from three independent experiments (each using one KO mouse and one WT control) were pooled, analyzed by two-way Student’s t test, and reported as means ± S.E. *p < 0.05.