Figure 5.

NOTCH-HES1 signaling regulates KLF4 expression in the gut. (a,b) Immunohistochemistry analysis of Klf4 expression in small intestine of mice treated with dexamethasone, DBZ or the combination of dexamethasone plus DBZ for 5 days. Scale bars represent 100 µm. (c) Real-time PCR analysis of Klf4 transcript levels in small intestine of mice treated with dexamethasone, DBZ or the combination of dexamethasone plus DBZ for 10 days. Gapdh levels were used as a reference control. Data are means ± SD of three animals per group. (d–e) Effects of ICN1 and HES1 expression in human KLF4 promoter activity. Luciferase reporter assays were performed in AGS cells with reporter constructs encompassing 2,006 bp (d, e), 994, 495 and 181 nucleotides (e) of the KLF4 promoter. Promoter activity is shown relative to an internal control expressing Renilla luciferase. Western blot shows expression of ICN1-HA and HA-HES1 proteins in AGS cells transfected with ICN1 and HES1 expression plasmids. (f) Quantitative ChIP analysis of HES1 binding to KLF4 promoter sequences. (g) Lentiviral shRNA knockdown of HES1 in AGS cells induces transcriptional upregulation of KLF4. (h) Expression of HA-HES1 protein in AGS cells induces transcriptional downregulation of KLF4. Expression of a control shRNA targeting the luciferase gene (shRNA LUC) was used as control. Bars represent means ± SD of triplicate experiments. TIS: transcription initiation site. HES1 protein levels normalized to the loading control are shown at the bottom of corresponding lanes in the Western blot.