HSA/TIMP-2 does not inhibit MMP-2 activity.

<p>(A) <i>In vitro</i> MMP-2 inhibitory activity was measured by co-incubating with a MMP-2 NIRF probe and activated MMP-2 in the presence of TIMP-2, MMP-2 inhibitors, or HSA/TIMP-2, followed by fluorescence imaging with an IVIS-200 imaging system (<i>top</i>). The corresponding fluorescence intensity (p/s) was quantified in each sample from 3 independent experiments (<i>bottom</i>). A reaction containing NIRF and MMP-2 was used as a positive control in this experiment (lane 4). (B) Representative whole-body NIRF images. NIRF imaging of MLL tumor-bearing mice was performed at 2 h after injection of the MMP-2 NIRF probe in the presence of MMP I (<i>n</i> = 4), HSA/TIMP-2 (<i>n</i> = 5), or control PBS (<i>n</i> = 6). (C) The quantitation of fluorescent signal in the tumor. The fluorescence intensity (p/s) was quantified based on the total photon count determined from regions of interest. Each data point represents the mean ± SEM; ^#<i>P</i><0.05 <i>vs.</i> control group by two-way ANOVA followed by Bonferroni post-hoc test.</p

HSA/TIMP-2 inhibits angiogenesis in vivo.

<p>(A) Representative images of blood vessels in PBS (control, upper panel) or 1 mg/dose HSA/TIMP-2-treated CAM (lower panel). (B) Quantification of CAM angiogenesis. Blood vessel number was quantified in a circular perimeter surrounding the filter disk (<i>n</i> = 10 per group). (C) Representative image of PECAM-1 blood vessel visualized by immunofluorescence staining. The vascular density of control and HSA/TIMP-2-treated tumors was determined by staining frozen sections with anti-PECAM-1 antibody (green) followed by Hoechst nuclear counterstaining (blue). Scale bar = 50 µm. (D) Quantitative measurement of vascular density in the tissue sections determined by calculating the mean pixel intensity of PECAM-1 fluorescence staining per that of Hoechst nuclear staining. (E) <i>In vivo</i> PECAM-1 protein expression detected by western blot analysis of tumor lysates. (F) Quantification of band intensity normalized by ß-actin. All quantitative data except for CAM assay represents the mean ± SEM (<i>n</i> = 4 per group). Significant difference compared to the control group: ^**<i>P</i><0.01 by Student's <i>t</i> test.</p

HSA/TIMP-2 inhibits extracellular proteolytic activity of MMP-2.

<p>(A) Representative images of the gelatin zymographic analysis. Culture media from HUVECs treated with TIMP-2 or HSA/TIMP-2 for 72 h were analyzed on a gelatin gel. Two forms of MMP-2 (latent form, 72 kDa; activated form, 62 kDa) are presented. (B) Quantification of band intensity. Data are expressed as the percentage of band intensity normalized with respect to the proliferation level (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035710#pone.0035710.s004" target="_blank">Fig. S4</a>). The quantitative data represent the mean ± SEM of duplicate samples from three independent experiments.</p

HSA/TIMP-2 inhibits MMP-2 expression independent of MT1-MMP expression.

<p>(A) Representative immunohistochemical images of MMP-2. The images were taken at an original magnification of 200×. (B) Quantification of MMP-2 expression determined by the percentage of brown pixels per field. (C) Representative RT-PCR images of MMP-2. HUVECs were treated with the indicated doses of HSA/TIMP-2 (lanes 2–4), and RNA was isolated 48 h after treatment. (D) Quantification of band intensity normalized by GAPDH. The quantitative data represent the mean ± SEM of duplicate samples from three independent experiments. (E) Representative images of PECAM-1, MMP-2 and MT1-MMP detected by immunofluorescence staining. Frozen sections of tumor tissues were triple-immunostained with anti-PECAM-1, anti-MMP-2 and anti-MT1-MMP followed by Hoechst nuclear (DAPI, blue) counterstaining. Scale bar = 20 µm. (F) Quantitative measurement determined by calculating the mean pixel intensity of MMP-2 (red) or MT1-MMP (green) fluorescence staining per that of Hoechst nuclear staining (blue). All quantitative data represents the mean ± SEM (<i>n</i> = 4 per group). Significant difference compared to the control group: ^**<i>P</i><0.01, ^*<i>P</i><0.05 by Student's <i>t</i> test. <i>ns</i> = non-significant.</p

HSA/TIMP-2 inhibits tumor cell proliferation but not apoptosis in prostate tumors.

<p>(A) Representative immunohistochemical images of Ki-67 and active caspase-3 immunohistochemistry. Tumor sections were obtained at 15 days after initial injection of 80 mg/kg HSA/TIMP-2 (<i>n</i> = 4 per group). The representative images were taken at an original magnification of 200×. (B) Quantification of Ki67- and active caspase-3-positive cells as expressed by the percentage of brown pixels per field. The quantitative data represent the mean ± SEM (<i>n</i> = 4 per group). Significant difference compared to the control group: ^*<i>P</i><0.05 by Student's <i>t</i> test. <i>ns</i> = non-significant.</p