Silencing of endogenous enhances oxytocin receptor signalling via p44/42 MAPK in Hs578T cells

<p><b>Copyright information:</b></p><p>Taken from "Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers"</p><p>http://breast-cancer-research.com/content/9/6/R85</p><p>Breast Cancer Research : BCR 2007;9(6):R85-R85.</p><p>Published online 8 Dec 2007</p><p>PMCID:PMC2246188.</p><p></p> Hs578T cells transfected with either a scrambled control small interfering (si)RNA (SiCON) or an siRNA targeting (siRGS) were serum and insulin starved for 2 hours then stimulated with 1 × 10mol/l oxytocin. Cells were harvested at timepoints up to 120 minutes after oxytocin addition and lysates analysed for levels of phospho- and total p44/42 mitogen-activated protein kinase (MAPK) by immunoblotting. Quantitation of siRNA knockdown from two independent transfections. The upper plot is the quantitation of the blots shown in panel a. siRGS2 caused a significant (< 0.001) increase in phosphorylation in response to oxytocin. Quantitative real-time PCR analysis of expression in Hs578T cells transfected with the siRGS2. Each bar represents the mean ± 95% confidence limits of the fold difference in expression compared with the mean expression in the siCON transfected cells. Data from four samples harvested from two independent transfections is shown (one of which was also used in the lower oxytocin response experiment shown in panel b). Significant differences are indicated (**< 0.01)

ANXA8 expression arrests Kim2A8 cells in G₀.

<p>(<b>A</b>) Kim2A8 and Kim2RTS cells were grown in 6-well plates with or without 100ng/ml of dox for 48 hours. The graph shows the average percentage numbers of cells in G₀/G₁, S and G₂/M as quantified by FACS from three independent experiments. (<b>B</b>) Kim2A8 and Kim2RTS cells grown in chamber slides with or without 100ng/ml dox for six days were fixed and stained for Ki67 antigen. EGFP was used as a reporter of ANXA8 expression. (<b>C</b>) Graph showing the percentage of Ki67-positivity in the EGFP positive and negative populations of Kim2A8 cells grown with or without 100ng/ml dox. At least 1000 cells were analyzed in each population. (<b>D</b>) Western blot showing Ki67 and ANXA8 protein expression in cells after six days in culture. Actin was used as a loading control. Numbers show the relative intensities of ANXA8 and Ki67 bands respectively (normalised to actin) determined by measuring area pixel intensities using AIDA Image Analyzer software. The reduction of Ki67 levels (∼50%) is consistent with the reduced number of Ki67+ve Dox-treated Kim2A8 cells seen in (<b>B</b>).</p

ANXA8 expression inhibits proliferation of Kim2A8 cells.

<p>(<b>A</b>) Kim2A8 and Kim2RTS cells were seeded in 24-well plates, allowed to attach and grown in the presence or absence of 100ng/ml dox (first treatment at time point 0). At each time point protein extracts were prepared (in duplicates). The graph shows the amount of protein as determined by BCA assay against time. This assay was performed in triplicate and the graph shows a representative result from one experiment. (<b>B</b>) Cells were seeded in 96-well plates and treated with dox for 48 hours, labelled with BrdU and the incorporation of BrdU was quantified and plotted for each condition (six wells per condition). ***p< 0.001 (<b>C</b>) Equal amount of cells (250–300) were grown in the presence or absence of 100ng/ml dox. After 14 days cells were fixed, stained and the plates photographed. A representative plate per condition is shown. The experiment was carried out in triplicate. (<b>D</b>) Graph showing the number of colonies per plate from the experiment (<b>C</b>) as quantified by Image J (**p<0.003).</p

ANXA8 expression induces morphological changes in Kim-2 cells.

<p>Kim2A8 and Kim2RTS cells were grown in the presence or absence of 100ng/ml dox. Pictures were taken after 48 hours (<b>A</b>) or six days (<b>B</b>) of treatment. EGFP was used as a reporter of ANXA8 expression. Both proteins are expressed from opposite sides of a bidirectional promoter. (<b>C</b>) Nuclear sizes were analysed after 6 days by measuring the nuclear area (stained with DAPI) of at least 90 individual cells from each dox-treated and untreated populations using ImageJ. There was a significant difference between Kim2A8 cells expressing and not expressing ANXA8 (ANOVA: p<0.05).</p

Is highly expressed in CD24Sca-133A10mouse mammary basal/myoepithelial and human breast myoepithelial cells

<p><b>Copyright information:</b></p><p>Taken from "Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers"</p><p>http://breast-cancer-research.com/content/9/6/R85</p><p>Breast Cancer Research : BCR 2007;9(6):R85-R85.</p><p>Published online 8 Dec 2007</p><p>PMCID:PMC2246188.</p><p></p> Flow cytometric staining profile of mouse mammary cell preparations stained with anti-Sca-1 and anti-CD24 antibodies together with either a nonspecific rat IgG and anti-rat-FITC or 33A10 and anti-rat FITC. The nonspecific IgG and 33A10 staining profiles of the CD24Sca-1basal/myoepithelial cells (33A10), CD24Sca-1(Esr1) luminal cells (33A10) and CD24Sca-1(Esr1) luminal cells (33A10) [14] are indicated. Mean fold differences ± 95% confidence limits in RNA abundance for the gene in CD24Sca-1basal/myoepithelial, CD24Sca-1(Esr1) luminal epithelial and CD24Sca-1(Esr1) luminal epithelial mouse mammary cells (= 3 for all samples) [14]. The dotted lines indicate the 95% confidence limits of the comparator sample. All samples show a significant difference to the comparator (**< 0.01). The basal myoepithelial cells also have a significantly higher level of expression than either of the two luminal populations. Mean fold differences ± 95% confidence limits in expression levels for the gene in myoepithelial and luminal epithelial human breast cells compared with human breast fibroblasts (comparator). See Materials and methods for details of the samples. The dotted lines indicate the 95% confidence limits of the comparator sample. Both samples show a significant difference to the comparator (**< 0.01) and the myoepithelial cells have a significantly higher level of expression than the luminal cells

ANXA8 is expressed strongest during pre-puberty.

<p>(<b>A</b>) Microarray results from a previous microarray study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119718#pone.0119718.ref041" target="_blank">41</a>], using RNA extracted from 3-, 4-, 5-, 6- and 7-week old CD1 mice, show a reduction in <i>AnxA8</i> mRNA at the onset of puberty. Signal intensities for two independent probes targeting <i>AnxA8</i> were normalised to cytokeratin 18 (Krt18) to eliminate changes due to differences in epithelial content. (<b>B</b>) qRT-PCR results for <i>AnxA8</i> normalised to Krt18 expression from RNA extracted from mammary glands of 3-, 4-, 6-, and 12-week-old mice. (<b>C-D</b>) Immunohistochemical analysis of ANXA8 expression using the E2R6.2 antibody on mammary glands from 3- (C) and 6-week old (<b>D</b>) mice showing staining for ANXA8 in the pre-pubertal rudiment and in ducts, but not TEB, of pubertal mice. Negative control (−ve ctrl): no primary antibody. Bars represent 100μm.</p

ANXA8 positive cells are ERα and Ki67 negative.

<p>Co-immunofluorescence staining for ANXA8 and Ki67 (<b>A</b>) or ERα (<b>C</b>) in 6-week old mice shows that those cells strongly positive for ANXA8 are negative for ERα and Ki67. Graphs represent the mean percentage of ANXA8 positive and negative cells that are positive or negative for Ki67 (B) or ERα (D) based on 1,000 cells per developmental time point (<b>B</b>) and at least 500 cells (<b>D</b>). V6: virgin 6 weeks; P4.5: pregnancy day 4.5; P12.5: pregnancy day 12.5. Error bars denote standard error of the mean.</p

Isolation and characterisation of mammary epithelial cell subpopulations

<p><b>Copyright information:</b></p><p>Taken from "Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers"</p><p>http://breast-cancer-research.com/content/9/6/R85</p><p>Breast Cancer Research : BCR 2007;9(6):R85-R85.</p><p>Published online 8 Dec 2007</p><p>PMCID:PMC2246188.</p><p></p> Flow cytometric staining profiles (dead and CD45cells excluded) of anti-Sca-1 and 33A10 stained, freshly isolated mouse mammary cell preparations together with nonspecific IgG-stained control. Graphical space representation of principal component analysis of mammary fibroblasts, Sca-133A10, Sca-133A10and Sca-133A10cells. Mean fold differences ± 95% confidence limits in RNA abundance measured by quantitative real-time PCR for estrogen receptor () and prolactin receptor () transcripts in Sca-133A10(= 5 samples) and Sca-133A10(= 3 samples) mammary subpopulations compared with bulk mammary cell preparations depleted for CD45cells (comparator; = 3 samples). The dotted lines indicate the 95% confidence limits of the comparator sample. All samples show a significant difference to the comparator (**< 0.01) [35]

RGS2 is expressed in human myoepithelial and luminal cells and in breast cancers

<p><b>Copyright information:</b></p><p>Taken from "Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers"</p><p>http://breast-cancer-research.com/content/9/6/R85</p><p>Breast Cancer Research : BCR 2007;9(6):R85-R85.</p><p>Published online 8 Dec 2007</p><p>PMCID:PMC2246188.</p><p></p> RNA isolated from normal primary breast cells, normal and breast cancer cell lines and primary breast cancers was analysed by semi-quantitative RT-PCR for expression at the transcriptional level of and a housekeeping gene . Quantitative real-time PCR analysis of RGS2 expression levels in a selection of primary human cells, human breast cancer cell lines, solid breast cancers and F19-depleted cancers. Data are mean relative expression levels ± 95% confidence limits (= 3 analyses of each sample). For comparison, the primary myoepithelial and primary luminal cell data from Figure 2c have been included on this graph. Note that the y-axis is a log10 scale

ANXA8 is expressed in ERα−ve/c-kit+ve luminal progenitor cells.

<p>(<b>A</b>) Primary mammary epithelial cells were sorted and RNA extracted as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119718#pone.0119718.ref039" target="_blank">39</a>]. qRT-PCR was used to measure <i>AnxA8</i> mRNA abundance in four different populations. <i>AnxA8</i> was not detected in either mammary stem cells (MaSC) or myoepithelial cells. Although very low levels were detected in differentiated ERα+ve luminal epithelial cells, ERα−ve luminal epithelial progenitor cells showed a 17-fold higher abundance. The graph shows the abundance relative to the levels of expression in differentiated cells and 95% confidence limits. (<b>B</b>) Bar graph showing the proportion of c-kit+ve/AnxA8+ve and c-kit+ve/AnxA8−ve cells during puberty (V6), early (P4.5) and late (P12.5) pregnancy. 1,000 cells were assessed per developmental time point. Error Bars denote standard error of the mean. (<b>C</b>) Co-immunofluorescence staining for ANXA8 and c-kit in mouse mammary gland from an early-pregnant (day 4.5) mouse.</p