Figure 2.

Vorinostat increases the mRNA expression of Notch receptors, ligands and downstream targets in ovarian serous carcinoma OVCAR3 cell line. The 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 OVCAR3 cells. RT-qPCR indicated that Notch2, Notch3 and Notch4 mRNA levels were increased following vorinostat exposure. (B) Representative staining of Notch receptors by immunofluorescence. Vorinostat slightly increased the protein expression of Notch receptors 2 and 4, and decreased Notch1 expression 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 OVCAR3 cell line. RT-qPCR revealed that Dll1, Jagged1 and Jagged2 mRNA expression levels in cells treated with vorinostat were increased compared with cells not treated with vorinostat (magnification, ×200). Dll3 and Dll4 mRNA expression levels were not differentially expressed. (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 mRNA expression levels were not affected by vorinostat treatment. RT-qPCR was normalized to hypoxanthine phosphoribosyltransferase. 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 transpiration-quantitative polymerase chain reaction; Hes, hairy enhancer of split; Hey, Hes-related proteins.