Table 1.
Characteristics of subjects used in microarray and subsequent studies.
Fig 1.
qPCR verification of concentration- and depot-dependent effects of glucocorticoids on selected, known glucocorticoid target genes.
(A) PCK1, (B) LPL, (C) GILZ, and (D) IL-6. Data are mean ± SEM, n = 5–7 independent subjects. The X-axis is a log scale. Significant depot differences at each [Dex] are indicated by an asterisk (*, p < 0.05, paired t-test of log transformed values). Repeated measures ANOVA verified a significant Dex effect in both depots for each gene (Dex effect, p ≤ 0.002). All doses in both Om and Abdsc were significantly different from 0 nM Dex (p ≤ 0.05, Dunnett’s test).
Fig 2.
Parallel plot illustrating the cluster analysis of genes that exhibited a Depot*[Dex] interaction.
460 genes that showed a significant interaction of Depot and [Dex], and expression values above a threshold of 20 for at least one Dex concentration in one depot were included in an unsupervised hierarchical cluster analysis (JMP 10 software), as described in Methods. The analysis with 10 clusters is shown.
Table 2.
Transcripts of interest from the cluster analysis of significant Depot*[Dex] interactions (Fig 2).
Fig 3.
qPCR verification of selected depot-dependent Dex effects.
Transcripts for verification were selected for biological interest, large depot differences in the baseline values and/or the magnitude of the Dex effects: (A) INHBA, (B) GREM1, (C) PKHD1L1, (D) ITLN1, (E) ITGB8, and (F) NRN1. Depot differences are indicated by asterisks (*p < 0.05, paired t-test at each Dex concentration). Within depot, Dex effects were tested by repeated measures ANOVA on log-transformed data (p values indicated in the box on each graph). Post-hoc comparisons of values at each Dex concentration compared to baseline (0 Dex) were carried out by Dunnett’s tests. Within Abdsc, Dex effects were significant for INHBA at Dex concentrations of 10 nM or higher, ITGB8 at 25 and 1000 nM and NRN1 at 1, 10, and 25 nM. Within Om, Dex effects were significant for PKHD1L1 at Dex concentrations of 10 nM and higher and ITLN1 at 25 and 1000 nM. Because of missing values for Om for INHBA, only paired t-tests were used to test the effect of each Dex concentration vs. baseline [p = 0.051 at 1 nM (n = 6), p<0.01 at 10 nM (n = 5), 25 and 1000 nM (n = 6)].
Fig 4.
Depot differences in flash frozen samples of Om and Abdsc reflect patterns observed in tissues cultured with Dex.
(A) INHBA, (B) GREM1, (C) PKHD1L1, (D) ITLN1, (E) ITGB8, and (F) NRN1. *p < 0.05, depot difference (paired t-tests of log transformed values, n = 6). Data presented as mean ± SEM.