Uously and immunohistochemical Solvent Yellow 14 web staining of E-cadherin, ErbB2 and Slug were also performed on the adjacent sections as described previously [34]. Briefly, tissues were fixed in 10 buffered formalin and embedded in paraffin. Four-micrometer-thick sections were deparaffinized in xylene and rehydrated in graded alcohols and distilled water. After antigen retrieval, endogenous peroxidase activity was blocked with 0.3 hydrogen peroxide in methanol for 30 minutes followed by rehydration in PBS, and incubation with 5 goat serum for 60 minutes to bind nonspecific antigens. Sections were incubated overnight at 4uC with the primary antibodies. The immuno-signals were detected with the ABC Kits at room temperature. After rinsing, the sections were incubated with DAB, counterstained with hematoxylin, dehydrated, and then mounted. As a negative control, the primary antibody was replaced with normal mouse IgG. The sections were then analyzed by standard light microscopy.or cell nucleus [35]. Cytoplasmic staining was scored based on the percentage of positive tumor cells and staining intensity. The positive cell percentage was determined by calculating the percentage of positive tumor cells in total observed cells: 0, ,10 ; 1, 10 ?0 ; 2, .20 . The intensity was decided as: 0, no staining or ambiguous staining; 1, medium staining; 2, strong staining. The two scores were multiplied to evaluate staining: 0 to 1, negative; 2 to 4, positive (+). Nuclear staining was scored using a 4006 magnification, and 100 nuclei were counted according to the percentage of nuclei showing positive immunoreactivity, which was graded on an arbitrary scale ranging from 0 to 2: 0, ,10 ; 1, 10 ?0 ; 2, .20 . A score of 2 was classified as high-level expression (+) and 0 to 1 as low-level expression (2). Membranous and cytoplasmic staining of E-cadherin was examined immunohistochemically using the same tissue as Twist2. Positive Ecadherin immunoreactivity was scored similarly to that of Imai T et al [36]. Weak or no expression of E-cadherin was regarded as aberrant E-cadherin expression, moderate to strong membranous and cytoplasmic staining of E-cadherin was classified as normal expression.Isolation of Subcellular Fractions, and Western Blot 
AnalysisDetails of cell fractionation and Western blot analysis were performed as our previous publications [37,38]. MedChemExpress Vitamin D2 protein Assay Kit for protein quantity analysis was purchased from Bio-Rad (CA,USA). The enhanced chemiluminescence (ECL) detection system was purchased from Amersham (IL, USA). All antibodies were described as materials.Confocal Immunofluorescent StainingTwist2- and vector-transfected MCF7 cells were grown on sterilized cover slips for 20 hr, then fixed in 4 paraformaldehyde for 30 min at 4uC and permeabilized in 0.2 TritonX-100 in PBS at room 
temperature (RT) for 15 min. Then, cells were incubated with 2 BSA for 1 hr at RT to block nonspecific binding before the primary antibody reaction. Cover slips were incubated with the primary antibodies to Twist2 and E-cadherin overnight at 4uC, then washed with PBS and incubated with Alexa Flour 555, FITC-conjugated secondary antibody (Dako) for 1 hr in the dark at RT. Cover slips were washed three times in PBS, mounted with Vectashield (Vector Laboratories) containing 4, 6 diamidino-2phenylindoledihydrocloride (DAPI) for DNA staining and analyzed using the laser scanning confocal microscopy (Olympus).Statistical analysisThe Chi-square test and Fisher’s exact test were used to a.Uously and immunohistochemical staining of E-cadherin, ErbB2 and Slug were also performed on the adjacent sections as described previously [34]. Briefly, tissues were fixed in 10 buffered formalin and embedded in paraffin. Four-micrometer-thick sections were deparaffinized in xylene and rehydrated in graded alcohols and distilled water. After antigen retrieval, endogenous peroxidase activity was blocked with 0.3 hydrogen peroxide in methanol for 30 minutes followed by rehydration in PBS, and incubation with 5 goat serum for 60 minutes to bind nonspecific antigens. Sections were incubated overnight at 4uC with the primary antibodies. The immuno-signals were detected with the ABC Kits at room temperature. After rinsing, the sections were incubated with DAB, counterstained with hematoxylin, dehydrated, and then mounted. As a negative control, the primary antibody was replaced with normal mouse IgG. The sections were then analyzed by standard light microscopy.or cell nucleus [35]. Cytoplasmic staining was scored based on the percentage of positive tumor cells and staining intensity. The positive cell percentage was determined by calculating the percentage of positive tumor cells in total observed cells: 0, ,10 ; 1, 10 ?0 ; 2, .20 . The intensity was decided as: 0, no staining or ambiguous staining; 1, medium staining; 2, strong staining. The two scores were multiplied to evaluate staining: 0 to 1, negative; 2 to 4, positive (+). Nuclear staining was scored using a 4006 magnification, and 100 nuclei were counted according to the percentage of nuclei showing positive immunoreactivity, which was graded on an arbitrary scale ranging from 0 to 2: 0, ,10 ; 1, 10 ?0 ; 2, .20 . A score of 2 was classified as high-level expression (+) and 0 to 1 as low-level expression (2). Membranous and cytoplasmic staining of E-cadherin was examined immunohistochemically using the same tissue as Twist2. Positive Ecadherin immunoreactivity was scored similarly to that of Imai T et al [36]. Weak or no expression of E-cadherin was regarded as aberrant E-cadherin expression, moderate to strong membranous and cytoplasmic staining of E-cadherin was classified as normal expression.Isolation of Subcellular Fractions, and Western Blot AnalysisDetails of cell fractionation and Western blot analysis were performed as our previous publications [37,38]. Protein Assay Kit for protein quantity analysis was purchased from Bio-Rad (CA,USA). The enhanced chemiluminescence (ECL) detection system was purchased from Amersham (IL, USA). All antibodies were described as materials.Confocal Immunofluorescent StainingTwist2- and vector-transfected MCF7 cells were grown on sterilized cover slips for 20 hr, then fixed in 4 paraformaldehyde for 30 min at 4uC and permeabilized in 0.2 TritonX-100 in PBS at room temperature (RT) for 15 min. Then, cells were incubated with 2 BSA for 1 hr at RT to block nonspecific binding before the primary antibody reaction. Cover slips were incubated with the primary antibodies to Twist2 and E-cadherin overnight at 4uC, then washed with PBS and incubated with Alexa Flour 555, FITC-conjugated secondary antibody (Dako) for 1 hr in the dark at RT. Cover slips were washed three times in PBS, mounted with Vectashield (Vector Laboratories) containing 4, 6 diamidino-2phenylindoledihydrocloride (DAPI) for DNA staining and analyzed using the laser scanning confocal microscopy (Olympus).Statistical analysisThe Chi-square test and Fisher’s exact test were used to a.