Release of intracellular ROS in MDA-MB-231 (A) and MCF-7 (B) cells after temporin-1CEa treatment.

<p>ROS production was measured by FACS analysis using a sensitive free-radical indicator, 2′,7′-dichlorofluorescin-diacetate (DCFH-DA). Each bar represents the mean value from three determinations with the standard deviation (SD). Data (mean ± SD) with asterisk significantly differ (*p<0.05; **p<0.01) between treatments.</p

Transmembrane potential of breast cancer cells after peptides treatment.

<p>The transmembrane potential depolarization of cancer cells (A: MDA-MB-231 and B: MCF-7) were determined using the anionic dye, DiBAC₄(3).</p

Disruption of mitochondrial membrane potential in MDA-MB-231 (A) and MCF-7 cells (B) after temporin-1CEa exposure.

<p>Mitochondrial membrane potential was measured using the cell-permeable fluorescent cationic dye rhodamine 123 by flow cytometry. Each bar represents the mean value from three determinations with the standard deviation (SD). Data (mean ± SD) with asterisk significantly differ (*p<0.05; **p<0.01) between treatments.</p

Laser scanning confocal microscopy images of MDA-MB-231 and MCF-7 cells treated with FITC-labeled temporin-1CEa for 5 min (A: MDA-MB-231; D: MCF-7), 10 min (B: MDA-MB-231; E: MCF-7) and 60 min (C: MDA-MB-231; F: MCF-7).

<p>Laser scanning confocal microscopy images of MDA-MB-231 and MCF-7 cells treated with FITC-labeled temporin-1CEa for 5 min (A: MDA-MB-231; D: MCF-7), 10 min (B: MDA-MB-231; E: MCF-7) and 60 min (C: MDA-MB-231; F: MCF-7).</p

Elevation of intracellular calcium concentration in MDA-MB-231 or MCF-7 cells following temporin-1CEa treatment in either a calcium-containing solution (A-B) or calcium-free solution (C–D).

<p>Fluo3-AM green fluorescence was used for evaluation of the cytosolic calcium levels. Each bar represents the mean value from three determinations with the standard deviation (SD). Data (mean ± SD) with asterisk significantly differ (*p<0.05; **p<0.01) between treatments.</p

Effects of chronic exposure to Cd to genomic DNA methylation in rat livers.

<p>DNA methylation in the livers of rats with and without low-dose Cd treatment was initially profiled with MeDIP-chip, followed by GO analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033853#s4" target="_blank"><i>Materials and Methods</i></a>. The brief summary of these analyses is outlined (A) with the numbers of total genes with promoter CGI methylation changes (1,629) and the genes, for which all CGIs in the promoter were either hypermethylated (675) or hypomethylated (899). In addition, there were also some genes (55) with mixed hyper- and hypo-methylated CGIs in each promoter. After general analysis for the DNA methylation changes in the livers of rats with and without low-dose Cd treatment as described in A, the methylation profiles of the promoter CGI of CASP8 (B) and TNF (C) genes were selectively analyzed. The NimbleScan software was employed based on the raw data described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033853#pone.0033853.s001" target="_blank">Table S1</a>, to generate scaled log₂-ratio data for IP/input values and <i>p</i>-value enrichment data for each probe. Peaks (methylated regions) are then generated based on the <i>p</i>-value data. Therefore, the methylation signal levels (Y axis) are represented as either p-value of peaks (top track in each panel) or log₂-ratio (bottom track in each panel) along with different regions of the promoter gene. For each gene there are only positive numbers relative to base line (0) in term of <i>p</i>-value of peaks for each gene, but there are both negative and positive numbers for log₂-ratio. Conclusion was made by comparing the overall values between control and Cd-treated groups.</p

Effects of chronic exposure to Cd on CK 8/18 expression in mouse livers.

<p>Expression of hepatic CK8/18 as a new marker of mouse liver preneoplastic change was examined by immunochemical staining (A) and Western blotting (B). In panel A, series of sections of same tissues were conventionally stained with H&E (A-a, c, e, g, i, k) and immunochemically stained with anti-CK8/18 antibodies (A-b, d, f, h, j, l). The results indicated that there was a significant increase in CK8/18 staining foci with little notable change in the same locations using H&E staining. Bar = 50 µm. Data was presented as mean ± SD (n = 10). * <i>p</i><0.05 vs control; # <i>p</i><0.05 vs Cd group; ^&<i>p</i><0.05 vs 5-aza group.</p

Apoptosis and DNA double-strand break in rat livers.

<p>Hepatic tissues were collected from the rats at the 48^th week after 4-week exposure to Cd at 20 nmol/kg for TUNEL staining (A, left panel) with a semi-quantitative analysis (A, right panel). Cleaved caspase-3 (c-CASP3) was examined by double immunofluorescent staining for c-CASP3 as red and nuclei by DAPI as blue (B). The sections from these rats were also stained for DNA double-strand breaks (C) with double immunofluorescent stains of β-actin (green, a, b) and γ-H2AX (red, c, d). Bar = 50 µm. Data was presented as mean ± SD. *<i>P</i><0.05 vs control.</p

Apoptotic and cell proliferation effects of chronic exposure to Cd on mouse livers.

<p>Liver samples were collected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033853#pone-0033853-g003" target="_blank">Figure 3</a> for TUNEL staining with semi-quantitative evaluation (A). Apoptotic effect was further confirmed with cleaved-CASP3 (c-CASP3), detected by Western blot (B). Ki-67 measurement with immunohistochemical staining (C) and Western blot (D). The images for each group are the representative ones. Bar = 50 µm. Data was presented as mean ± SD (n = 10). * <i>p</i><0.05 vs control; # <i>p</i><0.05 vs Cd group.</p

CASP8 gene promoter CGI methylation and TNF-<i>α</i> expression in mouse livers.

<p>Mice were treated with Cd at 20 nmol/kg for 4 weeks and then were sacrificed at the 56^th week after Cd exposure. The Cd-treated and the age-matched mice were given with or without the methylation inhibitor 5-aza for 6 weeks (i.e.: 4 weeks during and 2 weeks after 4-week Cd-treatment). Liver tissues of these four groups of mice were collected for the analysis of CGI methylation status for CASP8 gene promoter with EpiTect Methyl qPCR assay (A), c-CASP8 expression by Western blotting (B), and TNF-<i>α</i> expression by Western blot (C). Data was presented as mean ± SD (n = 10). * <i>p</i><0.05 vs control; # <i>p</i><0.05 vs Cd group.</p