Fig. 2

DHEA is metabolized to DHT in THP–1 cells; and testosterone (similarly to DHEA) affects RACK1 expression. a DHEA metabolism. THP–1 cells (10⁷) were treated with (10 nM) or DMSO as vehicle control for 1 h. Testosterone, DHT, and its metabolites 3α-diol and 3β-diol were assessed by LC-MS/MS in cell lysates. Each dot represents the mean ± SD of 4 samples obtained in two independent experiments. b The 5α-reductase inhibitor finasteride blocks DHEA-induced RACK1 expression. THP-1 cells were treated with finasteride (0.1 μM) for 1 h and then DHEA (10 nM) or DMSO as vehicle control was added for 16 h. RACK1 mRNA expression was assessed by Real Time-PCR. Results are expressed as mean ± SD of three independent experiments, with §p < 0.05 vs DHEA alone. c, d Testosterone induces a dose-related increase in RACK1 expression. THP-1 cells were treated with increasing concentrations of testosterone (1–100 nM) or DMSO as vehicle control for 16 h for mRNA expression (c) or 24 h for protein expression (d). RACK1 mRNA expression was assessed by Real Time-PCR. Results are expressed as mean ± SD of four independent experiments, with *p < 0.05 vs Control cells. RACK1 immunoreactivity was assessed by Western blot analysis of cell lysates. β-Actin immunoreactivity was used as protein loading control and to normalize RACK1 expression. A representative Western blot is reported together with a densitometric analysis of data obtained from three independent experiments. Results are expressed as mean ± SD, with *p < 0.05 vs Control cells. e, f Effect of testosterone on LPS-induced cytokine production. THP-1 cells were treated with increasing concentrations of testosterone (1–100 nM) or DMSO as vehicle control. After 24 h, LPS (10 ng/ml) was added for 3 h to assess TNF-α release (e) or 24 h to assess IL–8 release (f). Results are expressed as mean ± SD, n = 3 replicates. Data is representative of three independent experiments. Statistical analysis was performed with Tukey’s multiple comparison test with §p < 0.05 and §§p < 0.01 vs LPS treated cells