Figure 6.
E6AP Enhances TGFβ-induced Mesenchymal Phenotype in BPH Cells
(A) BPH-E6AP or the parental control cells were cultured for two days in the presence or absence of Doxy, serum-starved overnight and then stimulated with 10ng/mL TGFβ in media containing 0.05% FBS for a further two days in the presence or absence of Doxy. Actin was then visualized with rhodamine-conjugated phalloidin using a fluorescent microscope. Representative images are shown. Scale bar represents 50μm. Each experiment was performed in triplicates and repeated three independent times. A total of 10 colonies were counted per condition.
(B) BPH-E6AP cells or the parental controls were plated at low density and allowed to form colonies for two days in the presence or absence of Doxy, serum-starved overnight and then stimulated with 10ng/mL TGFβ in media with 0.05% FBS for an additional two days in the presence or absence of Doxy. Representative phase-contrast images of the colonies formed are shown. Scale bar represents 100μm. Each experiment was performed in triplicates and repeated two independent times.
(C) RT-PCR analysis of the expression of E6AP, Slug and Snail mRNA in BPH-E6AP cells grown for two days in the presence or absence of Doxy, serum-starved overnight and then stimulated with 10ng/mL TGFβ in media with 0.05% FBS for an extra two days (in the presence or absence of Doxy). Data are ΔΔCt±SD of technical triplicates of one of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t test.