Comprehensive analysis of Ano1 protein expression in normal tissues.

<p>#Mucosa and mucous glands were negative.</p><p>Summary of normal tissues with detectable Ano1 protein expression. Most normal tissues do not express Ano1. Only gall bladder, stomach and duodenum express Ano1 (A). Apical positivity (B) and Ano1-positive Cajal cells (C) were detected in several types of tissues. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043265#pone.0043265.s005" target="_blank">Table S5</a> for complete listing.).</p

Survival analysis of patients with HNSCC.

<p>A) Kaplan-Meier curve analysis illustrating a worse overall survival for HNSCC patients with detectable Ano1 protein expression (solid line: Ano1 protein negative, dashed line: Ano1 protein positive). B) Kaplan-Meier curve analysis demonstrating a worse outcome for patients with 11q13 (<i>Ano1)</i> amplified HNSCC. (Solid line: normal <i>Ano1</i> gene copy status, dashed line: 11q13 amplified).</p

Expression of Ano1 and Ca²⁺ activated Cl⁻ currents in HNSCC cells.

<p>A) Western blots of Ano1 expressed in the three different human cell lines CAL-27, CAL-33, and BHY. B) Current/voltage relationships of whole cell currents activated by 100 µM ATP (filled circles) in CAL-27, CAL-33, and BHY cells. Application of the K⁺ channel inhibitors Ba²⁺ (5 mM) and TEA⁺ (10 mM) (grey circles) did not significantly change currents, suggesting a dominant role Cl⁻ currents. C) Summary of the calculated ATP-activated whole cell conductances. Mean ± SEM, (number of experiments).</p

Analysis of the 11q13 (Ano1 and CCND1) genomic amplification and protein expression in 365 HNSCCs.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043265#pone-0043265-t001" target="_blank"><b>Table 1</b></a><b> Legend.</b> A) Overview of the prevalence of genomic amplification of <i>Ano1</i> and Ano1 expression in HNSCCs from different tumor locations.</p><p>B)Genomic amplification of the 11q13 locus is associated with the presence of Ano1 protein expression.</p><p>C)Strong correlation between the genomic amplification of <i>Ano1</i> and <i>CCND1</i>.</p

<i>Ano1 causes cell migration.</i>

<p>A) Summary of BrdU incorporation in BHY cells after various treatments, shown as % of BrdU incorporation, when compared to cells treated with scrambled RNA (red dashed line). B) Representative images of wound healing in a scratch assay with BHY cells, treated with scrambled RNA (upper panels) or after Ano1-knockdown with siRNA-Ano1 (lower panels). Yellow arrowheads indicate edges of the damage. C) Time course for the wound healing process (closure of the scratch) for control cells (scrambled RNA) and after Ano1-knockdown (si-Ano1). D) Migration of BHY and CAL-33 cells measured continuously as cell index using the xCELLigence system. Inhibition of migration by blockade of Ano1 with AO1 (10 µM). E) Cell Proliferation measured continuously as cell index using the xCELLigence system. AO1 (10 µM) has no effect on proliferation. Experiments were performed at least in triplicates. Mean ± SEM, (number of experiments). ^#indicates significant difference (p<0.05, ANOVA).</p

Comprehensive analysis of Ano1 protein expression in human tumors.

*<p>These tumors only showed unspecific cytoplasmic Ano1 staining and were thus classified as negative.</p><p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043265#pone-0043265-t002" target="_blank"><b>Table 2</b></a><b> Legend.</b> Summary of human tumors with detectable Ano1 protein expression. Most tumors do not express Ano1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043265#pone.0043265.s004" target="_blank">Table S4</a> for complete listing).</p

Ano1 supports regulatory volume decrease.

<p>A) Original recordings of the calcein fluorescence in BHY cells, treated with scrambled RNA or siRNA-Ano1, before and after stimulation with 100 µM ATP. ATP induces transient cell shrinkage that was not affected by siRNA-knockdown of Ano1. B) Summary of the ATP-induced changes in calcein fluorescence in BHY and CAL-33 cells treated with scrambled RNA or siRNA-Ano1. C) Original recordings of calcein fluorescence in BHY and CAL-33 cells treated with scrambled RNA (blue) or siRNA-Ano1 (red) and effect of a hypotonic (Hypo; 200 mosmol/l) bath solution. D) Summary of the hypotonicity-induced changes in calcein fluorescence in BHY and CAL-33 cells treated with scrambled RNA or siRNA-Ano1. Mean ± SEM, (number of experiments). *indicates significant difference (p<0.05, paired t-test). ^#indicates significant difference (p<0.05, paired t-test).</p

Typical images of Ano1 gene amplification and protein expression in human tissues.

<p>A–B) FISH analysis of <i>Ano1</i> amplified (A) and non-amplified (B) HNSCC from the oral cavity. C–E) Immunohistochemical analysis of Ano1 expression in different tissue types. C) HNSCC of the oral cavity: strong membranous staining of the tumor cells. D) Invasive urothelial bladder cancer: strong membranous staining of the tumor cells. E) Invasive lobular breast cancer: cytoplasmic and membranous staining of the tumor cells. F) Normal muscular wall of the appendix: Ano1 protein positivity was detected in the interstitial cells of Cajal. Green signals: <i>Ano1</i> gene. Red signals: Centromere 11. Green arrows point towards Ano1 genes.</p