Skip to main content
. 2013 Jun 7;8(6):e65556. doi: 10.1371/journal.pone.0065556

Figure 3. AIB1 regulates E-cadherin expression through E-box-dependent transcription.

Figure 3

(A) Western blot analysis of E-cadherin and N-cadherin levels in T47D and MCF-7 cells that overexpressed AIB1 or had AIB1 knockdown. Cells were transfected with either empty vector (pcDNA) or AIB1, or with the negative control scrambled shRNA (NC) or shAIB1#1 (in the case of AIB1 knockdown). Western blot analysis was carried out using cell extract and antibody against AIB1, E-cadherin, N-cadherin or β-actin. (B) Immunofluorescence study showing the regulation of AIB1 on the expression of E-cadherin and N-cadherin. Cells overexpressed AIB1 and empty vector or AIB1 knockdown and negative control scrambled shRNA (NC) were cultured in the chamber slide and fixed, immunostained with anti-E-cadherin and anti-N-cadherin antibodies followed by the secondary antibody, Texas-red Fluor 589 anti-Rabbit. Nuclear protein was stained with DAPI. (C) Fluorescence microscopic images showing the morphology of T47D and MCF-7 cells that overexpressed AIB1 and the empty vector pEGFPC1. (D) RT-PCR analysis showing the regulation of AIB1 on the transcription of E-cadherin. The mRNA level of E-cadherin was expressed relative to GAPDH transcript level. (E) AIB1-regulated E-cadherin luciferase reporter activity. T47D cells were co-transfected with the E-cadherin-Luc reporter vector (pGL3-Ecad) and AIB1 or empty vector. Cells were harvested 48 h after transfection and subjected to luciferase activity assay. (F) Dependence of AIB1-suppressed E-cadherin transcription on E-box as shown by luciferase reporter activity. T47D cells were cotransfected with empty vector (pcDNA) or vector plus AIB1 insert (pcDNA-AIB1) together with the E-box wild-type (pGL3-Ecad) or E-box mutant (pGL3-Ecad-mut) E-cadherin-Luc reporter. Cells were harvested 48 h after transfection and subjected to luciferase activity. Relative luciferase activity was normalized to β-galactosidase activity, used as control to monitor the transfection efficiency. Each experiment was performed in triplicates and repeated at least three times. Data are the means ± SDs. Statistically significant differences (P<0.05) in paired Student’s t-test are marked with an asterisk.