Sue microarray that includes 7 casesHeterogeneous Twist2 Expression in Breast CancersFigure 1. Twist2 is up-regulated in breast cancer. A. Immuboblot analysis of Twist2 expression 
in fresh breast cancer tissues. Twist2 was upregulated in breast carcinomas relative to matched normal breast tissues. B. MedChemExpress 11089-65-9 Immunohistochemical (IHC) staining of Twist2 on sections of paraffinembedded breast carcinomas and normal breast tissues. Twist2 was localized in the cytoplasm (Tumor 10) or nucleus (Tumor 9), depending on the tumor specimen (magnification:6400). doi:10.1371/journal.pone.0048178.gTable 1. Clinical pathological characteristics of Twist2associated breast carcinomas.CasesTwist2 2 +X46.P,0.Tissue type Normal breast tissues Fibroadenoma Mammaryadenosis Mastitis Malignant tumor Tumor histological type Ductal Lobular Squamous cell TNM clinical stage 0 I/II III/IV Metastasis Non-metastasis Regional lymph nodes metastasis Distant lymph nodes metastasis Age(years) 50 ,50 61 80 15 21 46 59 90 20 31 29 7 0 61 13 31 22 73 46 11 23 2 11 50 44 115 22 4 29 7 0 86 15 4 7 6 13 16 141 7 5 11 12 36 0 1 2 41..0.19.,0.13.,0.clearly polarized in the differentiated gland-like or tubular structures areas in both primary tumor center and metastases (Figure 2). In addition, the cancer cells were marked with high ErbB2 which indicated strong proliferation of epithelial cancer cells. In contrast, tumor cells at IF lost their polar orientation and displayed fibroblast-like shape, and were negative for ErbB2 expression. This morphological change is accompanied by nuclear accumulation of Twist2. Consistently, tumor cells in the areas of TC and LM expressed E-cadherin on cell membrane (Figure 2). Disseminating tumor cells at IF with nuclear Twist2 completely lost E-cadherin. Notably, a significant amount of nuclear Twist2expressing cells at the tumor invasion front (IF) SMER28 chemical information showed loss of Ecadherin in those IDC samples with surrounding lymph metastasis (76.47 , 13 of 17 cases), while in TC of the same cases, cells with Twist2 in the cytoplasm only retained E-cadherin staining on membrane or cytoplasmic E-cadherin staining (92.86 , 13 of 14 cases, Figure 3). Since Twist2 is a member of b-HLH family transcription factors, it must be in nuclei to activate transcription of downstream signal molecules. Twist2 in the nucleus was related to the abnormal expression of E-cadherin in IF, while cytoplasm Twist2 was related to cytoplasm or membrane E-cadherin expression in TC. As Table 4 shows, there’s a correlation between nuclear Twist2 and loss of E-cadherin. Thus, EMT at the IF, as evidenced by the loss of E-cadherin and fibroblastic morphology, correlated well with the presence of Twist2 in the nucleus. Cytoplasm Twist2 of cancer cells both in primary TC and the LM expressed E-cadherin and maintain their 1527786 epithelial morphology, suggesting that the cancer cells in LM may have reacquired this morphology by a reversion of EMT (i.e. performing a mesenchymal to epithelial transition) [24].0..0.Ectopic expression of nuclear Twist2 in MCF7 cells with down-regulates E-cadherinTo further investigate the regulation of E-cadherin expression by Twist2, we established Twist2 exogenous over-expression in MCF7 cell line. In this epithelial tumor cell line, no obvious downregulation of E-cadherin was detected when Twist2 was stably over-expressed (Figure 4A). Subcellular fraction analysis and immunofluorescent staining showed that stably expressed TwistTNM clinical stage of breast cancer is accordin.Sue microarray that includes 7 casesHeterogeneous Twist2 Expression in Breast CancersFigure 1. Twist2 is up-regulated in breast cancer. A. Immuboblot analysis of Twist2 expression in fresh breast cancer tissues. Twist2 was upregulated in breast carcinomas relative to matched normal breast tissues. B. Immunohistochemical (IHC) staining of Twist2 on sections of paraffinembedded breast carcinomas and normal breast tissues. Twist2 was localized in the cytoplasm (Tumor 10) or nucleus (Tumor 9), depending on the tumor specimen (magnification:6400). doi:10.1371/journal.pone.0048178.gTable 1. Clinical pathological characteristics of Twist2associated breast carcinomas.CasesTwist2 2 +X46.P,0.Tissue type Normal breast tissues Fibroadenoma Mammaryadenosis Mastitis Malignant tumor Tumor histological type Ductal Lobular Squamous cell TNM clinical stage 0 I/II III/IV Metastasis Non-metastasis Regional lymph nodes metastasis Distant lymph nodes metastasis Age(years) 50 ,50 61 80 15 21 46 59 90 20 31 29 7 0 61 13 31 22 73 46 11 23 2 11 50 44 115 22 4 29 7 0 86 15 4 7 6 13 16 141 7 5 11 12 36 0 1 2 41..0.19.,0.13.,0.clearly polarized in the differentiated gland-like or tubular structures areas in both primary tumor center and metastases (Figure 2). In addition, the cancer cells were marked with high ErbB2 which indicated strong proliferation of epithelial cancer cells. In contrast, tumor cells at IF lost their polar orientation and displayed fibroblast-like shape, and were negative for ErbB2 expression. This morphological change is accompanied by nuclear accumulation of Twist2. Consistently, tumor cells in the areas of TC and LM expressed E-cadherin on cell membrane (Figure 2). Disseminating tumor cells at IF with nuclear Twist2 completely lost E-cadherin. Notably, a significant amount of nuclear Twist2expressing cells at the tumor invasion front (IF) showed loss of Ecadherin in those IDC samples with surrounding lymph metastasis (76.47 , 13 of 17 cases), while in TC of the same cases, 
cells with Twist2 in the cytoplasm only retained E-cadherin staining on membrane or cytoplasmic E-cadherin staining (92.86 , 13 of 14 cases, Figure 3). Since Twist2 is a member of b-HLH family transcription factors, it must be in nuclei to activate transcription of downstream signal molecules. Twist2 in the nucleus was related to the abnormal expression of E-cadherin in IF, while cytoplasm Twist2 was related to cytoplasm or membrane E-cadherin expression in TC. As Table 4 shows, there’s a correlation between nuclear Twist2 and loss of E-cadherin. Thus, EMT at the IF, as evidenced by the loss of E-cadherin and fibroblastic morphology, correlated well with the presence of Twist2 in the nucleus. Cytoplasm Twist2 of cancer cells both in primary TC and the LM expressed E-cadherin and maintain their 1527786 epithelial morphology, suggesting that the cancer cells in LM may have reacquired this morphology by a reversion of EMT (i.e. performing a mesenchymal to epithelial transition) [24].0..0.Ectopic expression of nuclear Twist2 in MCF7 cells with down-regulates E-cadherinTo further investigate the regulation of E-cadherin expression by Twist2, we established Twist2 exogenous over-expression in MCF7 cell line. In this epithelial tumor cell line, no obvious downregulation of E-cadherin was detected when Twist2 was stably over-expressed (Figure 4A). Subcellular fraction analysis and immunofluorescent staining showed that stably expressed TwistTNM clinical stage of breast cancer is accordin.
