Active 3C protease is sufficient to degrade nuclear proteins/nucleoporins in transfected cells.

<p>(<b>Ai</b>) COS-7 cells transfected to express either GFP-3C or GFP-3Cinac were trypsinized 18 h after transfection, harvested in ice-cold PBS and FACS sorted to collect GFP-expressing cells. Cells were then lysed using RIPA buffer plus protease and phosphatase inhibitors and lysates were subjected to Western analysis as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g001" target="_blank">Figure 1A</a>; untransfected cells were lysed similarly and used as control. The antibodies used are shown on the left of the figure. The arrow in the nucleolin blot denotes a clear cleavage product, with the approximate molecular weight (kDa) indicated on the right. (<b>Aii</b>) Results for densitometric analysis of nucleolin and Nup153 protein bands such as those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g003" target="_blank">Figure 3A</a>i, where data were normalised to the corresponding value of tubulin, relative to the corresponding value for the control sample. Densitometric analyses were performed using ImageJ; values represent the mean (+ SD) from two independent experiments (<b>Bi</b>) Ohio-Hela whole cell lysates were incubated with 4 units of HRV14 3C protease at 37°C for the indicated incubation times, subsequent to SDS-PAGE on 10% gels and Western analysis as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g001" target="_blank">Figure 1A</a>. (<b>Bii</b>) Plot of densitometric analysis of protein bands in (Bi), where data were normalised to the corresponding values for tubulin, relative to 0 h samples. Densitometric analyses were performed using Image J and values were the mean of two different experiments (± SD). (<b>C</b>) COS-7 cells transfected to express either GFP-3C or GFP-3Cinac were fixed and permeabilized 18 h post-transfection, and immunostained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g002" target="_blank">Figure 2</a> with the indicated primary and Alexa-568 conjugated secondary antibodies. Fluorescence was imaged by CLSM (see Materials and Methods). In each panel, images on the left depict localisation of HRV16 proteins (green channel) and the images in the middle depict localisation of cellular proteins (red channel), with the merged image on the right.</p

Specific nucleoporins are degraded in HRV16-infected cells.

<p>(<b>A</b>) Ohio-HeLa cells were infected without (mock) or with HRV16 (MOI of 1) and cells lysed using RIPA buffer containing protease and phosphatase inhibitors at the time points shown. Cell lysates were subjected to SDS-PAGE on 4–20% gradient gels and Western analysis using the indicated primary antibodies/horseradish peroxidise-conjugated secondary antibodies and enhanced chemiluminescence (Perkin Elmer). The specificity of the antibodies is indicated on the left. Bands corresponding to 3C, 3CD’ and 3CD are indicated on the right. p.i. - post-infection. (<b>B</b>) Results for densitometric analysis of FG-Nup protein bands (left) and non-FG-Nups (right) such as those shown in (A), where data were normalised to the corresponding values for tubulin, relative to the corresponding values for the mock sample. Densitometric analyses were performed using Image J; values represent the mean (± SD) from two independent experiments.</p

HRV16 infection leads to mislocalisation of nuclear proteins.

<p>Ohio-HeLa cells grown on coverslips were infected without (mock) or with HRV16 as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g001" target="_blank">Figure 1</a>; cells were fixed at the indicated times and permeabilized, and then probed with the indicated pairs of primary antibodies, followed by Alexa 488 and Alexa-568 conjugated secondary antibodies. Fluorescence was imaged by CLSM (see Materials and Methods). In each panel, images on the left depict localisation of HRV16 proteins (green channel) and the images in the middle depict localisation of cellular proteins (red channel), with the merged image on the right.</p

DUSP4 regulates p300 phosphorylation status.

<p>Confocal laser scanning microscopy was performed on either MDA-MB-231 cells or MCF-7 cells treated with vehicle alone or PMA for 60 h (MCF-7/PMA^AD or MCF-7/PMA^SUS cells) and subsequently transfected with either MOCK siRNA or DUSP4 siRNA. Cells were fixed and probed with primary rabbit antibodies to p300 (<b>A</b>), p300-1834p (<b>B</b>), or p300-89p (<b>C</b>) and primary mouse antibodies to DUSP4 followed by the corresponding secondary antibody conjugated to Alexa-Fluor 568 or Alexa-Fluor 488. Representative images for each antibody dataset pair are shown: green = p300; red = DUSP4; yellow = overlap between DUSP4 and p300 fluorescence signals. The total nuclear intensities of p300 (<b>A</b>), p300-1834p (<b>B</b>), p300-89p (<b>C</b>), DUSP4 (<b>D</b>) and H3K27ac were also measured using ImageJ software to select the nucleus of each cell and measure the total NFI signal minus background for at least 20 individual cells ± SE plotted in the bar graphs.</p

DUSP6 is moderately expressed in malignant HER2⁺ breast cancers.

<p>DUSP6 expression in a breast cancer cell line and breast cancer tissues were examined by immunohistochemistry. (<b>A</b>) Table summarising DUSP6 expression in the indicated breast tissues. (<b>B</b>) Cell block of MDA-MB-231 cells (x630). (<b>C</b>) Grade 2 ER⁺/PR⁺/HER2^- breast cancer (x400). (<b>D</b>) Grade 3 triple-negative (ER^-/PR^-/HER2^-) breast cancer (x400). (<b>E</b>) Grade 3 ER⁺/PR⁺/HER2⁺ breast cancer (x400) with benign breast duct. (<b>F</b>) Grade 3 ER^-/PR^-/HER2⁺ breast cancer (x400).</p

DUSPs are induced during epithelial-to-mesenchymal transition.

<p><i>DUSP1</i>, <i>DUSP4</i>, and <i>DUSP6</i> transcript levels measured by real-time PCR in (<b>A</b>) MCF-7 and MDA-MB-231 cells. Data are expressed as arbitrary copy numbers normalised to <i>PPIA</i> and are representative of the mean ± SE. (<b>B</b>) MCF-7 cells after incubation with PMA for various periods of time. Spline curves represent mean arbitrary copy numbers normalised to <i>PPIA</i> of two independent experiments. (<b>C</b>) MCF-7 cells after incubation with vehicle alone or in the presence of either PMA or PMA+TGF-β for 60 h. Data are expressed as relative fold change to MCF-7 normalised to <i>PPIA</i> and are representative of the mean ± SE of four independent experiments. MCF-7 cells were incubated with vehicle alone or in the presence of PMA for 60 h. Transcript levels measured by real-time PCR of: (<b>D</b>) <i>CDH1</i>, <i>VIM</i>, <i>FN1</i>, <i>SNAI1</i>, <i>SNAI2</i>, <i>CD44</i>, and <i>PLAUR</i> and (<b>E</b>) <i>DUSP1</i>, <i>DUSP4</i>, and <i>DUSP6</i>. Data are presented as relative fold change to MCF-7 normalised to <i>PPIA</i> and are representative of the mean ± SE. (<b>F</b>) <i>DUSP1</i>, <i>DUSP4</i>, and <i>DUSP6</i> transcript levels in PKC-θ siRNA transfected cells. Data are expressed as relative fold change to MCF-7 MOCK siRNA normalised to <i>PPIA</i> and are representative of the mean ± SE for four experiments. (<b>G</b>) PKC-θ ChIP-seq across <i>DUSP1</i>, <i>DUSP4</i>, and <i>DUSP6</i> transcripts in MCF-7 and MCF-7/PMA cells. Data are shown in the UCSC Genome Browser. The scale in all UCSC images is indicated on the y-axis as numbers in reads per million mapped reads.</p

PKC-θ regulates DUSP4, p300 and H3K27ac in mesenchymal MCF-7/PMA^SUS cells.

<p>Confocal laser scanning microscopy was performed on MCF-7/PMA^SUS cells and treated with either BIM or C27 PKC-θ catalytic inhibitors. Cells were fixed and probed with primary mouse antibodies to DUSP4 and either primary rabbit antibodies to <b>(A)</b> p300 or <b>(B)</b> H3K27ac followed by the corresponding secondary antibody conjugated to Alexa-Fluor 568 or Alexa-Fluor 488. Representative images for each antibody dataset pair are shown: green = p300 or H3K27ac; red = DUSP4; yellow = overlap between DUSP4 and p300 fluorescence signals. The PCC was determined as described in methods for both MOCK control and cells treated with either BIM or C27 PKC-θ catalytic inhibitors. PCC indicates the strength of relation between the two fluorochrome signals for at least 20 individual cells ± SE. <b>(C)</b>. Bar graphs indicate the total NFI of DUSP4, p300, or H3K27ac as measured using ImageJ software to select the nucleus of each cell and measure the total NFI signal minus background for at least 20 individual cells ± SE.</p

DUSPs show distinct localisation in the mesenchymal state.

<p>Confocal laser scanning microscopy was performed on either MDA-MB-231 cells or MCF-7 cells treated with vehicle alone or PMA for 60 h. Cells were fixed and probed with primary rabbit, mouse, or goat antibodies to DUSP1, DUSP4, and DUSP6, respectively, followed by the corresponding secondary antibody conjugated to Alexa-Fluor 568 or Alexa-Fluor 488. Fn/c values for: (<b>A</b>) MCF-7 and MCF-7/PMA cells; (<b>B</b>) MDA-MB-231 cells. Data shown represent the mean ± SE. Value 1 = equal cytoplasmic and nuclear fractions; >1 = nuclear bias; <1 = cytoplasmic bias. MCF-7 cells were probed with either: (<b>C</b>) primary anti-rabbit-DUSP1 and anti-mouse-H3K9me1; or (<b>D</b>) primary anti-mouse-DUSP4 and anti-rabbit-H3K27ac or anti-rabbit-H3K4me1. All antibodies were conjugated to Alexa-Fluor 568 or Alexa-Fluor 488. MDA-MB-231 cells probed with either: (<b>E</b>) primary anti-rabbit-DUSP1 and anti-mouse-H3K4me3 or anti-mouse-H3K9me1 or (<b>F</b>) primary anti-mouse-DUSP4 and anti-rabbit-H3K4me1 or anti-rabbit-H3K27ac. All antibodies were conjugated to Alexa-Fluor 568 or Alexa-Fluor 488. Representative images for each dataset are shown: green = DUSPs; red = histone marks; yellow = overlap between DUSPs and histone mark fluorescence signals. The plot-profile feature of ImageJ was used to plot the fluorescence signal intensity along a single line spanning the nucleus (n = 5 lines per nucleus, 5 individual cells). The average fluorescence signal intensity for the indicated pair of antibodies was plotted for each point on the line ±SE. Signal was plotted to compare how the signals for each antibody varied compared to the opposite antibody. The PCC was determined for each plot profile. PCC indicates the strength of relation between the two fluorochrome signals for at least 20 individual cells ± SE. Colours from representative images correspond to plot profiles.</p

DUSPs are involved in breast CSC regulation.

<p>MCF-7 cells were transfected with MOCK, DUSP1, DUSP4, or DUSP6 siRNAs and subsequently incubated with either vehicle or in the presence of PMA for 60 h. (<b>A</b>) Percentage of CD44^hi/CD24^lo/EpCAM⁺ CSC-like cells as measured by flow cytometry after transfection ± SE. Numbers above each column represent percentage inhibition (-) or promotion (+) relative to MOCK siRNA for three independent experiments. MCF-7 and MDA-MB-231 were inhibited with either NSC 95397 or triptolide for 24 h. (<b>B</b>) MCF-7 cells were then incubated with either vehicle alone or PMA for 60 h. Bar graph indicates percentage inhibition of CD44^hi/CD24^lo/EpCAM⁺ CSC-like MCF-7, MCF-7/PMA^AD, and MCF-7/PMA^SUS cells after inhibition ± SE for two independent experiments. (<b>C</b>) Bar graph indicates CD44^hi/CD24^lo/EpCAM⁺ CSC-like MDA-MB-231 cells after inhibition ± SE for two independent experiments. (<b>D</b>) mRNA levels <i>CDH1</i>, <i>VIM</i>, and <i>FN1</i> in MDA-MB-231 cells after inhibition. Data represent the mean relative fold change to untreated cells normalised to <i>PPIA</i> ± SE for two independent experiments.</p

DUSP1 and DUSP4 directly tether to the promoters of mesenchymal genes.

<p>MCF-7 cells were incubated with either vehicle alone or with PMA for 60 h. ChIP was performed with DUSP1 and DUSP4 antibodies. ChIP DNA was analysed by SYBR Green real-time PCR. Enrichment across the promoter regions of <i>FN1</i>, <i>PLAUR</i>, and <i>IL6</i> are shown for: (<b>A</b>) DUSP1 and (<b>B</b>) DUSP4. Data are expressed as percentage enrichment relative to total input control and represent the mean ± SE of three independent experiments. (<b>C</b>) Nuclear extracts were obtained from MCF-7 cells stimulated as previously described and subjected to half-ChIP using DUSP4 pull down or a no antibody control. Immunoblots of the samples were probed with primary rabbit antibodies to RNA-Pol-II-serine2p or RNA-Pol-II-serine5p and detected as described in the methods. Representative images of the immunoblots are depicted along with the Novex loading control (LC).</p