Figure 6.

Cholesterol regulates GR transactivation via activation of JNK. HeLa cells were cultured in 10% serum or 10% serum supplemented with 10 μg/ml cholesterol overnight (A and B), or with 50% serum or 50% serum supplemented with 5% MCD (C and D). Cells were harvested after 4 h treatment with either 100 nM Dex or DMSO and analysed by qRT-PCR for expression of MT1X and FKBP5 (normalised to GAPDH). HeLa cells cultured in 10% serum supplemented with 10 μg/ml cholesterol (CHOL) or 50% serum supplemented with 5% MCD overnight, and then protein lysates analysed by immunoblot for JNK and phosphorylated JNK (E). Tubulin and caveolin expression was used as loading controls. Cells were cultured in 10% serum supplemented with 10 μg/ml cholesterol, together with 10 μM JNK inhibitor (SP600125) for 6 h. Following treatment with 100 nM Dex for an additional 4 h, expression of GR transactivation of either MT1X (F) or FKBP5 (G) was measured by qRT-PCR (normalised to GAPDH). Graphs show average±s.e.m. Experiments were carried out in triplicate and repeated three times. *P<0.05 and **P<0.001.