Histology of MDA-MB-231 xenografts.

<p>Tumors dissected from animals were sectioned and stained with (A) H&E, and (B) mitotic figures (arrowheads), and (C) apoptotic bodies (arrows) evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>.</p

NAHA inhibits invasive behavior of breast cancer cells and capillary morphogenesis of endothelial cells.

<p>(A) Cell adhesion. MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours and cell adhesion to vitronectin determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (B) Cell migration. Cell migration of MDA-MB-231 cells was determined after 5 hours of incubation in the presence of NAHA (0–50 µM), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (C) Cell invasion. Invasion of MDA-MB-231 cells through Matrigel was determined after 24 hours of incubation in the presence of NAHA (0–50 µM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (D) uPA secretion. MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours, and the expression of uPA detected in conditioned media from the same amount of cells with anti-uPA antibody by Western blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. The results are representative of three independent experiments. (E) MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours, media collected and secretion of VEGF determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (F) HAECs were treated with NAHA (0–50 µM) for 16 hours. Capillary morphogenesis was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05.</p

Expression of CD31 and VEGF in tumors.

<p>The expression of (A) CD31, and (B) VEGF were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Data are mean ± SD (n = 6–10), * significantly different from control (p<0.05) by ANOVA.</p

NAHA inhibits growth of breast cancer cells <i>in vivo</i>.

<p>(A) Tumor volume. MDA-MB-231 cells were inoculated s.c. in the right flank of each mouse. Seven days after inoculation, mice were randomly divided into 3 groups (n = 10) and treated with NAHA (i.p., 10 mg or 50 mg/kg of body weight/3 times per week) or vehicle (n = 11) for 32 days. Tumor volume was measured three times a week. Data are mean ± SD, * significantly different from control (p<0.05) using a two-sample Student t-test. (B) Tumor weight, * p<0.05. (C) Body weight of mice.</p

NAHA inhibits growth of breast cancer cells.

<p>(A) Structure of NAHA, <i>2-[Benzyl-(2-nitro-benzenesulfonyl)-amino]-N-hydroxy-3-methyl-N-propyl-butyramide</i>. (B) MDA-MB-231, (C) MCF-7, (D) MCF-10A, (E) HMEC cells were treated with NAHA (0–50 µM). Cell proliferation was determined by the tetrazolium salt method as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Cell viability was determined by trypan blue staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Data are the means ± SD. Similar results were obtained in at least two additional experiments. * p<0.05 for cell proliferation, # p<0.05 for cell viability. (F) Anchorage-independent growth (colony formation) of MDA-MB-231 cells was assessed on 1% agarose after incubation with NAHA (a – 0, b – 10 µM, c -25 µM, d – 50 µM) for 14 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. (G) MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours and whole cell extracts were subjected to Western blot analysis with anti-Cdk2 and anti-CDC20 antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. The equal protein loading was verified with anti β-actin antibodies. The rfesults are representative of three independent experiments.</p

The hERG current in stably transfected HEK293 cells is reduced by the Src-family tyrosine kinase inhibitor, PP1.

<p><b>A. i.</b> Representative current traces in response to the voltage-clamp protocol shown in the inset. From the holding potential of −80 mV, 3-s long depolarizing steps were applied between −70 and +60 mV (10-mV increments). This opens the channels, but at positive potentials, some inactivation is seen (c→o→i). The voltage was then stepped to −40 mV to remove inactivation (i→o) and monitor outward tail currents. <b>ii.</b> The current amplitude at the end of each 3-sec long depolarizing step (open arrow in Ai) was used to construct a current-versus-voltage (I–V curve) before and after perfusing the hERG blocker, 1 µM E-4031, into the bath. <b>iii.</b> Summary of instantaneous I–V relations from tail currents (closed arrow in Ai) before and after adding E-4031. In all graphs, the values are mean±SEM for the number of cells in parentheses. <b>B.</b> Representative whole-cell currents are shown before and 25 min after adding 10 µM PP1 to the bath. From a holding potential of −80 mV, a 1 s long pulse to +60 mV was used to activate and then inactivate the channels (c→o→i). Then, inactivation was rapidly removed by a 16 ms long pulse to −120 mV (i→o). Instantaneous tail currents (arrow) during test pulses between −110 and +20 mV (10-mV increments) were used to measure the open-channel current-<i>vs</i>-voltage (I–V) relationship (summarized in D) and inactivation kinetics (summarized in E). <b>C.</b> PP1 reduces the hERG current. To monitor time-dependent changes, the instantaneous tail current at +20 mV was repeatedly measured in control cells and in separate cells exposed to 10 µM PP1. Each current was normalized to its initial value (measured at 3–4 min after starting the recording). Significant differences between control and PP1-treated cells are indicated as *<i>p</i><0.05; **<i>p</i><0.01. <b>D.</b> Open-channel I–V relations from instantaneous tail currents (as in panel B) were recorded at 25 min in control cells and in separate cells exposed to 10 µM PP1. For each cell, the current was normalized to cell capacitance to yield current density (pA/pF). <b>E.</b> The time constant of inactivation (τ) was measured after 25 min in control cells or with 10 µM PP1 in the bath. That is, for test pulses between +20 and −20 mV, the decay of the tail current (o→i; as in panel B) was fitted to a mono-exponential function: <i>I_t</i> = <i>AMP</i>*exp(−<i>t/τ</i>), where <i>I_t</i> is the outward current at time <i>t</i> and <i>AMP</i> is the initial current amplitude. Each fit was begun at the time indicated by the arrow in panel B.</p

The hEAG1/Kv10.1/KCNH1 current is regulated by a Src-family kinase.

<p>Whole-cell recordings of hEAG1 currents in transiently transfected HEK293 cells. The same voltage-clamp protocol (inset) was used for all recordings; i.e., from a holding potential of −80 mV, 1 s-long voltage steps were applied between −70 and +60 mV (in 10-mV increments). <b>A.</b> Typical recordings from a non-transfected HEK293 cell, and a hEAG1-transfected cell before and 5 min after adding the EAG blocker, astemizole. Note that the currents are plotted on the same scale. <b>B.</b> Representative hEAG1 currents from a transfected cell recorded 3–4 min after establishing a whole cell recording; and 25 min after bath addition of the Src-family kinase inhibitor, PP1. <b>C.</b> Summary of time-dependent inhibition of hEAG1 currents by PP1. The voltage protocol was applied every 5 min, and the peak current measured at +60 mV was normalized to its initial value. Significant differences are indicated: **<i>p</i><0.01. <b>D.</b> To obtain activation -<i>vs</i>-voltage curves, the peak current during each voltage step was divided by the driving force (<i>V_m</i>–E_K) to calculate conductance, and then normalized to the largest conductance (i.e., at +60 mV). Each conductance-<i>vs</i>-voltage curve at 25 min was fitted to a Boltzmann equation: <i>G/G_max</i> = 1/(1+exp[(<i>V_m</i>−<i>V_1/2</i>)/<i>k</i>]), where <i>V_m</i> is membrane potential, <i>V_1/2</i> is the potential at which <i>G/G_max</i> = 0.5, and <i>k</i> is the slope factor. The summarized curves are for control cells and separate PP1-treated cells. <b>E.</b> Time constants (τ) for hEAG1 current activation were measured for each test pulse by fitting a mono-exponential function: <i>I_t</i> = <i>AMP</i>*exp(−<i>t/τ</i>), as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090024#pone-0090024-g001" target="_blank">Fig. 1E</a>.</p

Src tyrosine kinase regulates the hERG current.

<p>Currents were compared between cells recorded with pipette solutions containing saline (to control for spontaneous changes), 100 µg/mL of a Src-inhibitory peptide (Src40-58), or 100 µg/mL of a scrambled version of the same peptide (Src40-58s). For each cell, the voltage protocol was repeated at 0, 10, 20 and 30 min of recording; and the representative current traces (upper panels) are from 2 control cells and 2 treated cells, all at 30 min. <b>A.</b> The voltage protocol was similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090024#pone-0090024-g001" target="_blank">Fig. 1B</a>; i.e., from a holding potential of −80 mV, a 1 s long pulse to +60 mV was applied to activate the channels. Then inactivation was removed by a 16 ms long pulse to −120 mV, and the maximal outward tail current was measured during a test pulse to +20 mV. The lower panel summarizes the time course, measured from the maximal tail current, normalized to its initial value at 3–4 min after establishing a recording. <b>B.</b> Following a 6 s long pre-pulse to +20 mV to activate the channels, the maximal inward tail current was measured during the test pulse to −120 mV (similar protocol to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090024#pone-0090024-g002" target="_blank">Fig. 2A</a>). The summarized time course in the lower panel was constructed as in panel A. Significant differences for Src40-58 are shown compared with controls (*<i>p</i><0.05) or the scrambled peptide (^#<i>p</i><0.05).</p

EAG-family members contain a conserved ITIM motif.

<p>Sequence alignments of C-termini for the EAG family of K⁺ channels show each immuno-receptor tyrosine inhibitory motif (ITIM) in bold, with its potentially phosphorylated tyrosine residue in parentheses beside the channel name. Also shown for hERG/Kv11.1/KCHN2 is the sequence of the 15 amino acid peptide made for use in pull-down assays or deleted in the ΔITIM mutant channel (line above the sequence).</p

The hEAG1/KCNH1 current is regulated by SHP-1 tyrosine phosphatase.

<p>The voltage protocols and normalization procedures were the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090024#pone-0090024-g004" target="_blank">Fig. 4</a> (only traces at +60 mV are shown). <b>A.</b> Time-dependent effects of adding 100 µg/mL recombinant wild-type SHP-1 protein to the pipette solution. Inset: representative currents from two cells, recorded 30 min after establishing the whole-cell configuration. For each cell, the peak current at +60 mV was repeatedly measured and normalized to its initial value (3–4 min after establishing a recording) for the number of cells indicated (*<i>p</i><0.05). <b>B.</b> Comparison of cells recorded with control pipette solution with those containing a 3∶1 mixture of the inactive substrate-trapping SHP-1 mutant (SHP-1 C453S; 150 µg/mL) and active wild-type SHP-1 (50 µg/mL). Inset: representative currents recorded 30 min after establishing the whole-cell configuration. The normalized peak currents at +60 mV are shown; *<i>p</i><0.05.</p