Figure 1.

Vorinostat increases the mRNA expression of Notch receptors, Dll/Jagged ligands and Hey/Hes downstream target genes. Ovarian clear cell carcinoma ES2 cells were grown in the absence and presence of vorinostat (5 µM) for 6, 18 and 30 h, following starvation, with medium supplemented with 1% fetal bovine serum. (A) Notch receptor expression levels in ES2 cells. RT-qPCR revealed that Notch2, Notch3 and Notch4 mRNA expression was increased following vorinostat exposure, whereas Notch1 was not differentially expressed. (B) Representative staining of Notch receptors by immunofluorescence. It was observed that there was an increase of Notch2 and Notch4 protein following exposure to vorinostat (magnification, ×200). Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue) and Notch receptors with fluorescein isothiocyanate (green). (C) Notch ligand expression levels in ES2 cells. RT-qPCR indicated that Dll1, Dll3, Dll4 mRNA expression levels in cells treated with vorinostat were increased compared with cells without vorinostat treatment (magnification, ×200). Jagged1 and Jagged2 mRNA expression levels were not significantly different. (D) Notch downstream target gene expression levels. RT-qPCR revealed that Hes1, Hes5, Hey1 and Hey2 mRNA expression levels in cells treated with vorinostat were increased compared with cells without vorinostat treatment. Hes6 had a similar expression under all conditions. RT-qPCR was normalized to the hypoxanthine phosphoribosyltransferase gene. Data are presented as the mean ± standard deviation of triplicate experiments. *P<0.05; **P<0.01; ***P≤0.001 vs. control cells. Dll, Delta-like; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Hes, hairy enhancer of split; Hey, Hes-related proteins.