The protein level of galectin-3 was reduced after shRNA treatment.

<p>Three independent shRNAs against galectin-3 were used to construct stable cell lines.</p

Multivariate analysis of the factors related to post-operative distant metastasis.

<p>CI = confidence interval.</p><p>Multivariate analysis of the factors related to post-operative distant metastasis.</p

Correlations between Galectin-3 expression and chemotherapeutic resistance in breast cancers (n = 135).

<p>CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease.</p><p>Correlations between Galectin-3 expression and chemotherapeutic resistance in breast cancers (n = 135).</p

ATO treatment (2.5 µM) significantly increased endogenous galectin-3 expression in MDA-MB-231 cells.

<p>Cells were treated with ATO and anti-galectin-3 antibody (1∶1000) was used to detect endogenous galectin-3 proteins. GAPDH was used as loading control. The results shown are the mean of at least 3 independent experiments. *P<0.01.</p

Galectin-3 knockdown sensitized MDA-MB-231 cells to ATO-induced apoptosis.

<p>Cells were labeled with annexin V (x-axis) and PI (y-axis), and apoptosis was analyzed using a flow cytometer.</p

Immunohistochemical analysis revealed that galectin-3 was located in the cytoplasm and membrane of breast cancer cells (A). Galectin-3 protein is expressed at a significantly higher level in breast cancer tissues compared to paracancerous tissue (B).

<p>Immunohistochemical analysis revealed that galectin-3 was located in the cytoplasm and membrane of breast cancer cells (A). Galectin-3 protein is expressed at a significantly higher level in breast cancer tissues compared to paracancerous tissue (B).</p

Cell viability was reduced by combined galectin-3 knockdown and ATO treatment.

<p>Cell viability was reduced by combined galectin-3 knockdown and ATO treatment.</p

Antitumor activity of CDAK in breast cancer xenograft in vivo.

<p>(<b>A</b>) Tumor–growth curve. MDA-MB-231 cell lines were implanted into the right flank of female athymic mice. When the tumor size reached about 60 mm³, mice received an intravenous injection of CDAK (4 mg/kg,) and CRLK (4 mg/kg), or saline used as control (three times a week). The volumes of the tumors were measure three times a week. The tumor volume of CDAK group was smaller than control and CRLK, <i>P</i><0.01. (<b>B</b>) The athymic mice were sacrificed as 25 d after being injected with CDAK and CRLK and the weight of each tumor was measured. The CDAK group had low weight compared to the control and the CRLK groups. <i>P</i> = 0.003. (<b>C</b>) The inhibition of angiogenesis was evaluated in tumor sections using immunohistochemistry with anti-CD105 antibody. Angiogenesis was quantified by image analysis of Microvessel density (MVD) (400× magnification). CDAK caused significant inhibition of angiogenesis. <i>P</i> = 0.003. (D) Images (200× magnification) of the lung and liver from athymic mice were obtained by staining with hemaetoxylin and eosin (HE). No significant damage was observed. (E) Representative photographs of the tumor section examined by TUNEL assay, light microscope (×400). The number of apoptotic cells was counter 5 random field in blinded manner. There differences of apoptotic cell were not statistic significant in the three groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053491#pone-0053491-t002" target="_blank">table 2</a>) (ANOVA assay). The images were analyzed by pro plus 5.0. Each group had five nude mice. Data are shown as mean ± SD. *<i>P</i><0.05 versus control, ** <i>P</i><0.05 versus CRLK.</p

ATO treatment (2.5 µM) induces limited apoptosis in breast cancer cells.

<p>Untreated cells (top panel) and cells treated with ATO (bottom panel) were then analyzed by staining with PI and annexin V, followed by flow cytometry. The proportion of cells in apoptosis is shown in the figure.</p

Galectin-3 expression and clinicopathological features (n = 1187).

<p>DCIS = ductal carcinoma in situ, IDC = invasive ductal carcinoma.</p><p>χ²-test was used to assess the relationships between tumor marker and other parameters.</p><p>P<0.05 was considered statistically significant.</p><p>Galectin-3 expression and clinicopathological features (n = 1187).</p