Supplement

Supplemental Tables 1-3 for the paper

Protein Detection Based on Small Molecule-Linked DNA

Based on small molecule-linked DNA and the nicking endonuclease-assisted amplification (NEA) strategy, a novel electrochemical method for protein detection is proposed in this work. Specifically, the small molecule-linked DNA (probe 1) can be protected from exonuclease-catalyzed digestion upon binding to the protein target of the small molecule, so the DNA strand may hybridize with another DNA strand (probe 2) that is previously immobilized onto an electrode surface. Consequently, the NEA process is triggered, resulting in continuous removal of the DNA strands from the electrode surface, and the blocking effect against the electrochemical species [Fe­(CN)₆]^3–/4– becomes increasingly lower; thus, increased electrochemical waves can be achieved. Because the whole process is activated by the target protein, an electrochemical method for protein quantification is developed. Taking folate receptor (FR) as an example in this work, we can determine the protein in a linear range from 0.3 to 15 ng/mL with a detection limit of 0.19 ng/mL. Furthermore, because the method can be used for the assay of FR in serum samples and for the detection of other proteins such as streptavidin by simply changing the small molecule moiety of the DNA probes, this novel method is expected to have great potential applications in the future

Exon 11 deletion in male hemizygous <i>Atp7a^fl/Y</i>; <i>Clnd6Cre^+/−</i> embryos.

<p>A) PCR analysis with P1 and P2 primers of yolk sac DNA of E10.5 embryos generated by crossing heterozygous floxed female mice (<i>Atp7a^fl/+</i>) with <i>Cldn6Cre</i> mice (<i>Atp7a^+/Y; Cldn6Cre^+/+</i>). Cre-mediated deletion of the floxed allele (floxed) was detected by the presence of a 647 bp PCR product (Deleted). Sex was determined by PCR amplification of yolk sac DNA to simultaneously detect the X-chromosome-specific <i>Jarid1c</i> gene (331 bp) and the Y-chromosome-specific <i>Jarid1d</i> gene (302 bp). B) Immunoblot detection of Atp7a protein in E10.5 embryos. Blots were probed with anti-Atp7a antibody (1∶5000) followed by HRP-conjugated anti-rabbit antibody (1∶5000). Actin protein was detected on the same blot as a loading control.</p

Synergistic Antitumor Activities of Docetaxel and Octreotide Associated with Apoptotic-Upregulation in Castration-Resistant Prostate Cancer

<div><p>Androgen deprivation therapy has become the fist-line treatment of metastatic prostate cancer; however, progression to castrate resistance disease occurs in the majority of patients. Thus, there is an urgent need for improvements in therapy for castration-resistant prostate cancer. The aims of the present study were to determine the efficacy somatostatin analogue octreotide (OCT) combined with a low dose of docetaxel (DTX) using castration resistant prostate cancer cells and to investigate the involved molecular mechanisms in vitro. The anti-proliferative and synergism potential effects were determined by MTT assay. Induction of apoptosis was analyzed employing annexing V and propidium iodide staining and flow cytometry. VEGFA, CASP9, CASP3 and ABCB1 gene expression was evaluated by RT-PCR and Q-RT-PCR analysis. OCT in combination with DTX treatments on DU145 cell migration was also evaluated. Investigation revealed that combined administration of DTX and OCT had significant, synergistically greater cytotoxicity than DTX or OCT treatment alone. The combination of the two drugs caused a more marked increase in apoptosis and resulted in greater suppression of invasive potential than either individual agent. There was obvious increase in caspase 3 expression in the OCT alone and two-drug combined treatment groups, however, VEGFA expression was markedly suppressed in them. These results support the conclusion that somatostatin analogues combined with docetaxel may enhance the chemotherapy efficacies through multiple mechanisms in castration-resistant PCa cell line. This work provides a preclinical rationale for the therapeutic strategies to improve the treatment in castrate resistance disease.</p></div

Characterization of <i>Atp7a^fl/+</i>; <i>Clnd6Cre^+/−</i> heterozygous female mice.

<p>A) Patches of hypopigmentation are observed in the coats of weanling heterozygous <i>Atp7a^fl/+; Clnd6Cre^+/−</i> females. B) Kaplan-Meier plot showing postnatal survival of heterozygous <i>Atp7a^fl/+; Clnd6Cre^+/−</i> female (n = 16) and littermate control <i>Atp7a^+/+; Clnd6Cre^+/−</i> (n = 34). C) Pili torti of the whiskers in surviving <i>Atp7a^fl/+; Clnd6Cre^+/−</i> females is shown (arrow).</p

Frequency of offspring with global deletion of the <i>Atp7a</i> gene.

<p>The numbers of live-born offspring or embryos at day 10.5 post coitus (E10.5) are shown for each genotype resulting from a cross between <i>Atp7a^fl/+</i> females and <i>Atp7a^+/Y; Cldn6Cre^+/+</i> males.</p

Characterization of MEF7a⁺ and MEF7a⁻ cells.

<p>A) Immunoblot analysis of Atp7a protein in MEF7a⁺ and MEF7a⁻ cells. Blots were probed with anti-Atp7a antibody (1∶5000) followed by HRP-conjugated anti-rabbit antibody (1∶5000). B) Defective copper delivery to tyrosinase MEF7a⁻ cells. MEF7a⁺ and MEF7a⁻ cells were transiently transfected with a plasmid encoding tyrosinase (pcTYR) alone, or in combination with a plasmid encoding ATP7A (pATP7A). Cells were then fixed in methanol and incubated with L-DOPA to allow the formation of the brown pigment, DOPAchrome. Note the absence of tyrosinase activity in MEF7a⁻ cells transfected with pcTYR alone (left), and complementation of this defect by co-transfection of pcTYR and pATP7A (right). C) MEF7a⁺ and MEF7a⁻ cells were transiently transfected with a Metal Responsive Element (MRE) reporter plasmid and a Renilla expression plasmid (pRL) as an internal control. After 24 hours, MRE luciferase activities of cell lysates were measured and normalized for Renilla luciferase activities. Experiments were repeated at least three times and values are presented as fold induction ± SEM; *p<0.05. D). Immunoblot analysis of Ccs protein in MEF7a⁺ and MEF7a⁻ cells. Blots were probed with anti-CCs antibody (1∶500) followed by HRP-conjugated anti-rabbit antibody (1∶5000).</p

Schematic representation of the Atp7a gene-targeting strategy.

<p>Exons 8–13 of the wild type and recombinant <i>Atp7a</i> locus are shown. The targeting construct included LoxP sites (black triangles) that flanked exon 11 and a PGKneo gene cassette flanked by FRT sites (grey triangles). The PGKneo gene cassette was removed by crossbreeding with the ACT-FLPe deleter strain expressing FLP1 recombinase to generate mice with the desired floxed allele (<i>Atp7a^fl</i>). The exon 11-deleted <i>Atp7a</i> allele (Deleted) is depicted following excision by Cre recombinase, which was detected using PCR primers P1 and P2.</p

Morphological and vascular abnormalities in male hemizygous <i>Atp7a^fl/Y</i>; <i>Clnd6Cre^+/−</i> embryos.

<p>A) Developmental anomalies particularly in the frontonasal region and tail bud orientation were apparent in the <i>Atp7a^fl/Y; Cldn6Cre^+/−</i> embryos at E10.5. B) The blood-filled vascular network observed in wild type embryos and yolk sacs (arrowheads) was lacking for the <i>Atp7a^fl/Y; Cldn6Cre^+/−</i>.</p

Effect of DTX, OCT alone and two-drug combination treatment on DU145 cells apoptosis.

<p>Data are mean ± SEM of two independent experiments performed in triplicate. ***<i>p</i><0.000 vs. control.</p