Cd treatment triggered liver apoptosis in purse red common carp.

<p>(A) White arrows indicate the brown TUNEL-positive cells (hepatocytes). Representative photographs are shown at 200 × magnification. (B) Quantitative analysis of the percentage of apoptotic cells. Data shown represent mean ± SD from 4 fish in each group, error bars indicate standard deviation. Statistical significance was analyzed using a factorial ANOVA (* <i>P</i> < 0. 05).</p

Primers used for caspase-3A gene cloning and expression analysis.

<p>Primers used for caspase-3A gene cloning and expression analysis.</p

Immunolocalization of caspase-3A in liver sections.

<p>NC represents negative control (substitution normal non-immune serum from the same host animal as the primary antibody). All the negative controls show no positive staining [Fig. 7(A-C), 7(G-I)]. Both control groups [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083423#pone-0083423-g007" target="_blank">Fig 7(D and J)</a>] and experimental groups [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083423#pone-0083423-g007" target="_blank">Fig 7(E, F, K and L)</a>] reveal weak immunolabeling. Arrows indicate the cytoplasm of hepatocytes. The representative photographs are shown at 200 × magnification.</p

Multiple alignment of caspase-3 amino acid sequences.

<p>Identical amino acids are indicated by asterisks, whereas those with high or low similarities are indicated by semicolons and dots. The putative cleavage sites at aspartic acid residues (D), which are shaded in gray, separate the pro-domain (<b><—></b>), the large subunit (<b><·····></b>) and the small subunit (<b><-----></b>) respectively. The caspase family signature and the pentapeptide active-site motif (QACRG) were boxed in a continuous and discontinuous line, respectively. The protein binding domain (GSWFI) and integrin recognition motif (RGD) are indicated by solid circles. The GenBank accession numbers of caspase-3 amino acid sequences used here are as follows: zebrafish caspase-3A NP_571952.1, zebrafish caspase-3B NP_001041531.1, Medaka caspase-3A NP_001098140.1, Medaka caspase-3B NP_001098168.1, human NP_116786.1, mouse NP_056548.2, chicken (<i>Gallus gallus</i>) NP_990056.1 and frog (<i>Xenopus laevis</i>) NP_001081225.1.</p

Dissolvability analyses of fusion protein and protein expression analysis

<p>(A): common carp caspase-3A clones expressed in <i>E. coli.</i> Lane M: protein molecular standard; Lane 1: total cellular proteins of PGEX-GST/BL21 after induction; Lane 2: supernate of purified recombinant PGEX-CSP3A; Lane 3: precipitate of purified recombinant PGEX-CSP3A. (B): Western blots showing procaspase-3A/ (Pro-csp3) and cleaved fragments (p17 and p12) after Cd treatment. (C): Protein band density was analyzed with the Gel-Pro Analyzer. GAPDH was used as a loading control. Each value was expressed as the ratio of caspase-3A proenzyme and its activated form (the sum of expression levels of p17 and p12 forms) to GAPDH level, which represents the mean ± SD. of 4 independent samples performed in triplicate. Statistical analysis was performed using a factorial ANOVA. Different upper case letters represent significant differences of proenzyme levels between exposure concentrations (<i>P</i> < 0.05), while different lower case letters represent significant differences of activated forms between exposure concentrations (<i>P</i> < 0.05) and asterisk (*) shows the significant difference between the two exposure time points for the same Cd-induced concentration (<i>P</i> < 0.05)</p

Effect of Cd treatment on mRNA transcript level of caspase-3A.

<p>The changes of mRNA levels were determined by real-time quantitative PCR. β-actin was used as a reference gene to normalize caspase-3A. The relative expression ratio was calculated with the 2^−ΔΔCT method. Error bars indicate standard deviation. Values represent the mean ± SD. of 4 independent samples. Statistical significance (<i>P</i> < 0.05) was analyzed using a factorial ANOVA. The mRNA levels of caspase-3A in carp were not significantly increased after exposure to Cd (<i>P</i> > 0.05).</p

Effects of Cd treatment on caspase-3A activity in liver from purse red common carp.

<p>The extracts were incubated with the specific substrates of caspase-3 and release of <i>p</i>-nitroanilide was measured at 405 nm. Values represent the mean ± SD; error bars indicate standard deviation. Statistical significance was analyzed using a factorial ANOVA (* <i>P</i> < 0. 05).</p

18 h cold ischemia-reperfusion caused I/R injury in heart grafts.

<p>Donor hearts were isolated from C57BL/6 mice, infused with UW solution and preserved in UW solution at 4°C for 18 h. After 18 h preservation, donor hearts were implanted into syngeneic recipient C57BL/6 mice. At day 2 after transplantation, the heart grafts were harvested and fixed in 10% formalin. The paraffin heart sections were subjected to HE staining, TUNEL assay, and MPO assay. (<b>A</b>) HE staining. (<b>B</b>) TUNEL assay for apoptosis. (<b>C</b>) MPO assay. Representative images were from experiments.</p

Enriched signalling pathways regulated in heart transplantation.

<p>note: ^a% genes in pathway that are present;</p>b<p>numbers of genes in list and in the pathway;</p>c<p>numbers of genes in list, not in pathway;</p>d<p>numbers of genes not in list, not in pathway.</p

Gene expression in heart grafts detected by microarray assays.

<p>Donor hearts were treated and transplanted into syngeneic C57BL/6 recipient mice. At day 2 post transplantation, total RNAs were extracted from grafts and gene expression in grafts were detected by microarray assays. (A) A Poisson distribution of gene expression. (B) Fluorescence intensities of altered genes detected by microarray assays. (C) Altered genes with two fold changes. (D) Angiopoietin 1 expression by Western Blotting.</p