SOX18 binds to the proximal site in the <i>MMP7</i> promoter <i>in vitro</i> and <i>in vivo</i>.

<p>A, Electrophoretic mobility shift assay (EMSA). Binding of SOX18 to its proximal site in the <i>MMP7</i> promoter was analyzed using nuclear extracts from HUVEC. Specificity of binding was demonstrated by competition experiments using the same (wt), a mutated (mut) or an unrelated (unrel) oligonucleotide as indicated, the specific complex is indicated by an arrow. B, The same site was analyzed by chromatin immunoprecipitation in HUVEC. Lane 1–3: different α-Sox18 antibodies (SantaCruz, Thermo, Chemicon, respectively) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030982#s2" target="_blank">Materials and Methods</a>, Ig: IgG control antibody; inp: input, M: 100 bp marker. The 120 bp PCR fragments obtained after amplification ofDNA precipitated by the anti-SOX18 antibodies and input DNA are marked by an arrow.</p

Alpha-Catulin Contributes to Drug-Resistance of Melanoma by Activating NF-κB and AP-1

<div><p>Melanoma is the most dangerous type of skin cancer accounting for 48,000 deaths worldwide each year and an average survival rate of about 6-10 months with conventional treatment. Tumor metastasis and chemoresistance of melanoma cells are reported as the main reasons for the insufficiency of currently available treatments for late stage melanoma. The cytoskeletal linker protein α-catulin (CTNNAL1) has been shown to be important in inflammation, apoptosis and cytoskeletal reorganization. Recently, we found an elevated expression of α-catulin in melanoma cells. Ectopic expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. In the current study we showed that α-catulin knockdown reduced NF-κB and AP-1 activity in malignant melanoma cells. Further, downregulation of α-catulin diminished ERK phosphorylation in malignant melanoma cells and sensitized them to treatment with chemotherapeutic drugs. In particular, cisplatin treatment led to decreased ERK-, JNK- and c-Jun phosphorylation in α-catulin knockdown melanoma cells, which was accompanied by enhanced apoptosis compared to control cells. Altogether, these results suggest that targeted inhibition of α-catulin may be used as a viable therapeutic strategy to chemosensitize melanoma cells to cisplatin by down-regulation of NF-κB and MAPK pathways.</p></div

Real-time PCR analysis of SOX18 target genes.

<p>RNA was isolated from non-transduced (mock), control virus (AdvEGFP) and SOX18 adenovirus (AdvSOX18) transduced HUVEC and analyzed for the expression of matrix metalloproteinase 7 (<i>MMP7</i>), ephrinB2 <i>(EFNB2), EPHA7</i>, semaphorin 3G <i>(SEMA3G)</i>, interleukin 7 receptor (<i>IL-7R</i>), and cannabinoid receptor 1 (<i>CNR1</i>). Values were normalized for ß2 microglobulin expression, and are expressed as fold induction compared to control cells.</p

Top SOX18 regulated genes.

<p>*verified by real-time PCR;</p>†<p>induction by real-time PCR was 1.5-fold;</p>‡<p>no change in real-time PCR.</p

Knock-down of SOX18 diminishes the expression of target genes.

<p>HUVEC were transfected with siRNAs directed against <i>SOX17</i> or <i>SOX18</i>, or with a scrambled control (Scr), and expression of target genes analyzed by real-time PCR. Values were normalized for ß2 microglobulin levels, and are expressed as fold induction compared to control cells.</p

α-Catulin promotes NF-κB activation in human primary melanocytes and melanoma cells.

<p>(<b>A</b>) A 5x NF-B-luc reporter gene (0.25μg) was co-transfected into melanocytes together with different concentrations of α-catulin (1 or 1.5 μg) with or without IKK-β(0.5μg) or p65 (0.5μg). 24 h later cells were non-stimulated or stimulated with TNF-α or LPS. Luciferase levels were normalized for a co-transfected RFP control (0.25μg). (<b>B</b>) Mel.7, Mel.15 and Mel.17 cells were stable infected with lentiviral particles containing a vector-based mirRNA construct directed against α-catulin (sh-catu 1 or sh-catu2; Materials and Methods), and α-catulin mRNA levels were analyzed by real-time PCR. (<b>C</b>) Mel.7, Mel.15 and Mel.17 cells were analyzed by Western blot with antibodies against α-catulin. (<b>D</b>) A NF-κB-luc reporter gene was tranfected into melanocytes and different melanoma cells containing stable integrated α-catulin mirRNA constructs (α-catulin-knockdown), and luciferase values were determined 24 h later and normalized for co-transfected JRED values. (<b>E</b>) Melanoma 7 cells as in (<b>D</b>) except that the cells were stimulated with TNFα, LPS, HGF and 10% FCS for further 8 h. (<b>F</b>) Mel.7 cells (n.s., sh-catu1/2) were transfected with NF-κB reporter plasmid and with or without si-RNA construct directed against E-cadherin and luciferase values were determined. (<b>G</b>) NF-κB-luc reporter assay with A375 melanoma cells transfected with mock, myc-α-catulin or sh-catu2 plasmids together with or without E-cadherin si-RNA. *Indicates P>0.005, **P>0.001, ***P>0.0001, Student´s <i>t</i> test.</p

α-Catulin knockdown reduces phosphorylation of ERK, JNK and c-Jun in cisplatin treated melanoma cells.

<p>Stable infected Mel.7 cells (n.s., sh-catu2) were treated with 0, 5, 10 and 20 μg/ml cisplatin for 24h and analyzed by Western blot with antibodies against (<b>A</b>) p-ERK, total ERK, (<b>B</b>) p-JNK, total JNK, (<b>C</b>) p-c-Jun (<b>D</b>) Mcl-1 and CBP. GAPDH or Tubulin were used as loading control.</p

Cell proliferation is reduced in a dose- and time dependent manner in cisplatin treated melanoma cells when α-catulin is knocked down.

<p>(<b>A</b>) Stable infected Mel.7 cells (n.s., sh-catu2) were treated with 0, 5, 10 and 20 μg/ml cisplatin for 24 h and analyzed by Western blot with antibody against Ki67. α-Tubulin was used as a loading control and quantification was performed with BioRad software. (<b>B</b>) Mel.7 cells (n.s., sh-catu2) were treated with 20, 10, 5, or 0 μg/ml cisplatin for 48 h and BrdU assay was performed. Therefore, cells were stained with BrdU solution and antibodies against BrdU and HRP conjugated secondary antibody was detected at 450 nm using a multiplate reader. (<b>C</b>) Mel.7 cells (n.s., sh-catu2) were treated with 0 or 10 μg/ml cisplatin for 18 hours, fixed, stained with propidium-iodide solution and analysed for cell cycle distribution using flow cytometry. (<b>D</b>) Cells as in (<b>C</b>) were analysed using western blot with antibodies against p21^cip/waf and p53.</p

α-Catulin knockdown enhances apoptosis in cisplatin-treated melanoma cells.

<p>(<b>A</b>) Stable infected Mel.7 cells (n.s., sh-catu1, sh-catu2) were treated with 0 and 10 μg/ml cisplatin for 48 h and stained with Annexin V and PI and analysed using flow cytometry (<b>B</b>) Stable infected Mel.7 cells (n.s., sh-catu2) were treated with 0 and 2.5μg/ml cisplatin for 6 h. For cytochrome c release assay, cells were treated with permeabilization buffer, fixed with formaldehyde, stained with antibody against cytochrome c and analyzed by Flow Cytometry. (<b>C</b>) Mel.7 cells (n.s., sh-catu2) were seeded in a 96 well plate and treated with 0, 2.5, 5, 10 or 20 μg/ml cisplatin for 6 h and mitochondrial membrane potential was determined using JC-1 assay (<b>D</b>) Mel.7 cells (n.s., sh-catu2) were treated with 0, 2.5, 5 or 10 μg/ml cisplatin for 6 h and analyzed for caspase 9 activity using caspase glo luminescence assay. (<b>E</b>) Cells as in (<b>D</b>) were analyzed for caspase 3/7 and (<b>F</b>) caspase 8.</p

α-Catulin knockdown enhances susceptibility of melanoma cells to cisplatin.

<p>(<b>A-F</b>) Stable infected (n.s., sh-catu2) (<b>A</b>) Mel.7, (<b>B</b>) Mel.17, (<b>C</b>) Mel.15, (<b>D</b>) Mel.7 spheroids, (<b>E</b>) Mel.7 (n.s., sh-catu1) or (<b>F</b>) Melanocytes (mock, myc-α-catulin) were treated with different concentrations of cisplatin for 48h and cell survival normalized to untreated cells (pos. contr.). Viability was analyzed by CellTiter-Blue Assay. (<b>G</b>) Stable infected Mel.7 spheroids were treated with 200 μg/ml cisplatin for 96 hours and diameter of the spheroids determined before and after treatment and statistically evaluated. Observations (•) mean (-) n = 14, (<b>H</b>) Microscopic images from Mel.7 spheroids.</p