Figure 5

Simultaneous inhibition of two miRNAs reduces entinostat-mediated downregulation of erbB2 and erbB3 in breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells were transfected with combinations of either the two control miRNA inhibitors no. 1 and no. 2 or the two indicated miRNA inhibitors (60 nmol/l each). After 24 h, the cells were then untreated or treated with entinostat (1.5 μmol/l) for another 24 h. (a) Half of the cells were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. RNU6B was used as an internal control. (b) Another half of the cells were collected and subjected to western blot analyses with specific antibody directed against erbB2, erbB3, or β-actin. The bar graph underneath was obtained by densitometry analysis. The relative signal intensities of erbB2 or erbB3 were measured by the Bio-Rad Gel Documentation System. Bars, S.D. The data are representative of three independent experiments