Figure 1

Adrenoid growth and cell composition. (A) Representative optical microscopy images of MTT spheroids and Adrenoids at different time points to monitor growth differences. (B) Diameters of MTT spheroids (squares) and of Adrenoids (circles) were measured at T0, 3, 5, 7 and 10 days and compared at each time point. Adrenoid diameters were significantly greater than those of the MTT spheroids starting from day 3, and by day 5 to day 10 the difference was even more significant. Graphs represent mean ± SD of diameters in three independent experiments performed in six replicates. Two-way ANOVA: ***p < 0.01 and ****p < 0.001. (C) Gene expression of the specific housekeeping markers (Gapdh and Rsp18 for human H295R or mouse MTT cells, normalized on the other housekeeping gene, respectively) to assess the proportion of the two cell populations within the Adrenoid at 5 and 10 days. Bar graphs indicate gene expression mean ± SE fold increase on T0 in three independent experiments. An increased expression of Rsp18 is associated with a decrease in Gapdh at the different time points, indicating an increased ratio between MTT and H295R cells over time; *p < 0.05 and **p < 0.001 vs T0, one-way ANOVA followed by Dunnett’s post hoc test. Gene expression of the cortex specific markers steroidogenic factor 1 (Sf1) and 11beta-hydroxysteroid dehydrogenase 1 (Hsd11B1) significantly decreased over time D), while gene expression of the medulla specific markers chromogranin a (CgA) and tyrosine hydroxylase (Th) significantly increased. Data are the mean ± SE fold increase of target gene expression normalized to the respective housekeeping gene (Gapdh and Rps18 for human and mouse cell components, respectively) in three independent experiments; *p < 0.05 and **p < 0.001 vs T0, one-way ANOVA followed by Dunnett’s post hoc test.