Figure 2.

Inhibition of NOTCH1-HES1 signaling restores glucocorticoid receptor autoregulation. (a) Microarray gene expression changes in CUTLL1 cells at 24 h treated with DMSO, CompE, dexamethasone and CompE plus dexamethasone. Relative expression levels are color coded as indicated at the bottom. (b) Quantitative RT-PCR analysis of the glucocorticoid receptor gene (NR3C1) and Western blot analysis and quantitation of glucocorticoid receptor protein levels in CUTLL1 cells treated with dexamethasone and CompE compared with vehicle only (DMSO). (c) Western blot analysis of NR3C1 levels and induction of apoptosis by dexamethasone and CompE in CUTLL1 cells infected with retroviruses expressing the glucocorticoid receptor (pMSCV NR3C1). (d) Analysis of apoptosis induction by dexamethasone plus CompE in CUTLL1 cells infected with shRNA lentiviruses targeting the glucocorticoid receptor (pGIPZ NR3C1) (e) Quantitative ChIP analysis of HES1 binding to NR3C1 promoter sequences. (f) Effects of HES1, MYB and dexamethasone (1 µM) in the activity of a human NR3C1 A1 reporter. Luciferase activity is shown relative to an internal control expressing Renilla luciferase. (g,h) NR3C1 expression (g) and analysis of apoptosis (h) in CUTLL1 cells treated with dexamethasone and CompE after lentiviral shRNA knockdown of HES1 (HES1 shRNA). A shRNA targeting the luciferase gene (shRNA LUC) was used as control. Drug concentrations in a–c were CompE 100 nM and dexamethasone 1 µM. Bars represent means ± SD of triplicate experiments. Statistical significance was assessed with Student’s t-test. HES1 and NR3C1 relative protein levels are indicated at the bottom of corresponding lanes in the Western blot.