Figure 6

Panobinostat up-regulates CDH1 expression and initiates EMT reversal in MDA-MB-231 Cells. MDA-MB-231 cells were plated overnight and treated with panobinostat (100 nM) or vehicle (DMSO) for 24 hours. The expression of CDH1 was examined by (A) flow cytometry and (B) ELISA. Data are represented as mean ± SEM of two independent experiments, (***, P < 0.001). (C-E) MDA-MB-231 morphology changes were assessed in vehicle- and panobinostat- (100 n M) treated cells with three-color fluorescence staining on a BD Pathway 855 Bioimager. (C) Control and (D-E) Panobinostat treated cells were stained red (rhodamine phalloidin) for actin filaments, green (Alexa Fluor^® 488) for acetyl-histone H3 (Lys9), and blue (DAPI) nuclear counter stain. Partial reversal of EMT in treated cells is indicated by the presence of cuboidal/spherical cells (arrows). (E) Two-fold magnification of field with normal untransformed mesenchymal cell and transformed spherical cells. Original magnification is 400× with scale bars at 20 microns. DAPI, 4',6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial-to-mesenchymal transition; SEM, standard error of the mean.