Additional file 2: Table S1. of The common stress responsive transcription factor ATF3 binds genomic sites enriched with p300 and H3K27ac for transcriptional regulation

A list of unique genes containing overlapped ATF3 and p53 peaks where both ATF3 and p53 motifs are present. (XLSX 32 kb

hERG Potassium Channel Blockage by Scorpion Toxin BmKKx2 Enhances Erythroid Differentiation of Human Leukemia Cells K562

<div><p>Background</p><p>The hERG potassium channel can modulate the proliferation of the chronic myelogenous leukemic K562 cells, and its role in the erythroid differentiation of K562 cells still remains unclear.</p> <p>Principal Findings</p><p>The hERG potassium channel blockage by a new 36-residue scorpion toxin BmKKx2, a potent hERG channel blocker with IC₅₀ of 6.7±1.7 nM, enhanced the erythroid differentiation of K562 cells. The mean values of GPA (CD235a) fluorescence intensity in the group of K562 cells pretreated by the toxin for 24 h and followed by cytosine arabinoside (Ara-C) treatment for 72 h were about 2-fold stronger than those of K562 cells induced by Ara-C alone. Such unique role of hERG potassium channel was also supported by the evidence that the effect of the toxin BmKKx2 on cell differentiation was nullified in hERG-deficient cell lines. During the K562 cell differentiation, BmKKx2 could also suppress the expression of hERG channels at both mRNA and protein levels. Besides the function of differentiation enhancement, BmKKx2 was also found to promote the differentiation-dependent apoptosis during the differentiation process of K562 cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2 was able to decrease the intracellular Ca²⁺ concentration during the K562 cell differentiation, providing an insight into the mechanism of hERG potassium channel regulating this cellular process. </p> <p>Conclusions/Significance</p><p>Our results revealed scorpion toxin BmKKx2 could enhance the erythroid differentiation of leukemic K562 cells via inhibiting hERG potassium channel currents. These findings would not only accelerate the functional research of hERG channel in different leukemic cells, but also present the prospects of natural scorpion toxins as anti-leukemic drugs.</p> </div

GATA-1 binds to the <i>ZNF268</i> promoter <i>in vivo</i>.

<p>ChIP assays were performed with K562 cells using the indicated antibodies, with IgG serving as a negative control. The precipitated DNA was amplified by PCR, electrophoresed, and stained with ethidium bromide. For all antibodies, primers C-s/C-a (−1166 to −962) served as a negative control. Input lanes show products after PCR amplification and before immunoprecipitation. (<b>A</b>) PCR amplification of DNA precipitated with anti-GATA-1 or anti-FOG antibodies using the primers G1-s/G1-a, which flank the GATA-binding sites contained within −1406 to −1266. (<b>B</b>) PCR amplification of DNA precipitated with anti-RNA polymerase II (pol II) or anti-TFIID antibodies using primers flanking the transcription start site (PES1/PE12). (<b>C</b>) As a positive control, ChIP assays were conducted using anti-CREB-2 antibody and primers flanking the CRE binding site in the <i>ZNF268</i> promoter (+594 to +925).</p

Stable silencing of <i>ZNF268</i> suppresses apoptosis and promotes tumor formation in nude mice.

<p>(<b>A</b>) Apoptosis in of <i>ZNF268</i>-silenced (shh-268) and control (shh-c) cells, as assessed by FACS analysis of PI or Annexin V staining. **<i>p</i><0.01 (standard <i>t</i> test). (<b>B</b>, <b>C</b>). <i>ZNF268</i>-silenced or control K562 cells (∼1×10⁷ cells) were subcutaneously implanted into male athymic nude mice. Tumor-bearing mice were then sacrificed 30 days later. Representative mice and excised tumors are shown (<b>B</b>), along with a comparison of tumor weight between the groups (<b>C</b>).</p

Scorpion toxin BmKKx2 primary structure and its pharmacological effect on the hERG channel.

<p>(A) the sequence alignments between scorpion toxins BmKKx2 and BeKm-1 [11,19]. (B) The pharmacological effect of BmKKx2 on hERG channels. The hERG channels were transfected in HEK 293 cells, and their current traces were shown in the absence (control) or presence 10 nM BmKKx2. (C) Dose-dependence curve of BmKKx2 on hERG channels expressed in HEK 293 cells. Symbols and associated error bars represent means ± SD for several cells (n=5). (D) The current trace of hERG potassium channels in absence of BmKKx2 in wild type K562 cells; (E) The pharmacological effect of BmKKx2 on hERG channels in wild type K562 cells. Current traces were shown in the absence (control) or presence 1 μM BmKKx2 .</p

hERG channel expression suppressed by BmKKx2 in erythroid differentiation of K562 cells.

<p>(A) γ-globin expression in K562 cells during the differentiation with or without BmKKx2 shown by quantitative real-time PCR. (B) hERG channel expression in K562 cells during the differentiation process with or without BmKKx2 shown by quantitative real-time PCR. **<i>p</i><0.01 (Student’s <i>t</i>-test). Symbols and associated error bars represent means ± SD for three independent experiments (C) hERG and γ-globin expression in K562 cells with or without BmKKx2 tested by the western blotting analysis. Both HERG1 and HERG1B isoforms were detected [24]. </p

Stable silencing of <i>ZNF268</i> accelerates the proliferation of K562 cells.

<p>K562 cells were transfected with recombinant lentiviral particles containing ZNF268 short hairpin RNA (shRNA; shh-268) or control lentiviral vector (shh-control). Transfected cells were then sorted according to GFP expression to generate stably transfected cell lines. (<b>A</b>) Quantitative real-time PCR analysis of <i>ZNF268</i> mRNA in <i>ZNF268</i>-silenced and control cells. (<b>B</b>) Cellular proliferation, as determined by cell counting. Values are derived from an average of three independent experiments. (<b>C</b>, <b>D</b>) Cell cycle profiles, as assessed by DNA content in PI-stained cells. (<b>E</b>, <b>F</b>) EdU labeling showing proliferation of <i>ZNF268</i>-silenced and control cells. The percentage of positive cells was derived from triplicate samples. (<b>G</b>) Western blot analysis of c-myc, p53, and cyclin D1. ZNF268 and β-actin levels were also analyzed. *<i>p</i><0.05 and **<i>p</i><0.01 (standard <i>t</i> test).</p

GATA-1 selectively binds to the GATA binding site in the <i>ZNF268</i> promoter <i>in vitro</i>.

<p>(<b>A</b>) Schematic diagram of the 11 GATA sites (G1–G11) in the <i>ZNF268</i> promoter. (<b>B</b>) EMSAs using K562 nuclear extract and biotin-labeled probes corresponding to the GATA binding sites in the human <i>ZNF268</i> promoter. Nuclear extract was omitted from the binding reaction as a negative control. (<b>C</b>) Competitive EMSAs and supershift assays showing the binding of a GATA-1 complex to the G1 site (−1412 to −1388). Labeled wild type G1 probe or labeled mutant probe was added to the reaction (<i>lanes 2</i> and <i>3</i>). Unlabeled competitors were added prior to G1 probe addition (<i>lanes 4</i> and <i>5</i>). For supershift experiments, anti-GATA-1 antibody was incubated with nuclear extracts before addition to the reaction mixture (<i>lane 6</i>).</p

Oligonucleotides used in this study.

a<p>Underlined nucleotides represent GATA binding sites, and lowercase letters indicate mutated residues.</p>b<p>Shown are the oligonucleotide positions, where +1 is the transcription start site of the <i>ZNF268</i> gene.</p>c<p>Data are from Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029518#pone.0029518-Guo1" target="_blank">[8]</a>.</p>d<p>Data are from Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029518#pone.0029518-Guo1" target="_blank">[8]</a>.</p

BmKKx2 binding causing the Ca²⁺ concentration decrease during the erythroid differentiation of K562 cells.

<p>(A) Intracellular Ca²⁺ was stained by Fluo-8, and the Ca²⁺ concentration was measured by flow cytometric analysis. The mean values were mean ± SD from three independent experiments. **<i>p</i><0.01 (Student’s <i>t</i>-test). (B) Flow cytometric analysis of Fluo-8 fluorescence intensity in K562 cells with or without BmKKx2 after Ara-C induced for 24 h.</p