Additional file 1: of The role of human rights litigation in improving access to reproductive health care and achieving reductions in maternal mortality

Open peer review. (PDF 165 kb

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

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

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

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

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