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

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 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

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

Comparison of <i>Grb10KO</i> and <i>Grb10Δ2-4</i> mice.

<p>(A) Structure of <i>Grb10</i>, according to UCSC annotation, showing numbered exons (boxes) and translated regions (filled boxes). The integrated gene-trap cassettes include splice acceptor (SA) and polyadenylation (pA) signals, and a <i>LacZ</i> reporter. (B) Comparative <i>LacZ</i> staining of bisected embryos at e14.5 inheriting the <i>Grb10KO</i> and <i>Grb10Δ2-4</i> alleles through each of the parental lines. CNS expression observed in <i>Grb10KO</i>^+/p embryos is not detected in <i>Grb10Δ2-4</i>^+/p embryos. (C) Comparative <i>LacZ</i> staining of adult mammary glands at days 7.5 and 12.5 of gestation (G7.5, G12.5) and day 6 of lactation (L6), showing pregnancy-dependent reporter expression in <i>Grb10KO</i>^m/+ but not <i>Grb10Δ2-4</i>^m/+ females. WT (+/+) glands were stained with carmine alum to illustrate morphological changes.</p

Functional <i>Grb10</i> is required in mother and pup for WT offspring body proportions.

<p>(A) Lean/fat mass ratio in a subset of cross-fostered pups indicating that <i>Grb10</i> ablation in either nurse or pup increases the lean/fat mass ratio relative to WT pups raised by WT nurses. (B) Total lean mass. (C) Total fat mass. (D) Lean/fat mass ratio in a subset of cross-fostered pups, indicating that the body composition of <i>Grb10KO</i>^m/+ pups raised by <i>Grb10KO</i>^m/+ nurses is similar to that of WT pups raised by WT or <i>Grb10KO</i>^m/+ nurses. Data points represent individual animals; mean values are represented by horizontal lines. Datasets in (B) and (C) were analysed using one-way ANOVA with Tukey's post hoc test. **<i>p</i><0.01. ns, not significant.</p

Characterisation of CRM1 and STAT5-mediated expression of <i>Grb10</i>.

<p>(A) <i>In silico</i> identification of conserved elements among selected vertebrate sequences. Conserved intronic sequences between <i>Grb10</i> homologs are plotted against annotated mouse transcripts. The PReMod track shows the position of the single regulatory module (CRM1). This site aligns with a sequence highly conserved between mouse, human, chimpanzee, cow, and chicken (highlighted). (B) Assay for DNase I hypersensitivity at CRM1, using probe A. A 6 kb <i>Bam</i>HI fragment was detected in all samples. A 3.8 kb DNase I digestion fragment was detected in brain, but not liver, chromatin exposed to 200 U DNase I (arrow). The label “B” indicates a <i>Bam</i>HI site. (C) <i>In situ</i> hybridisation autoradiographs showing examples of overlapping sites of <i>Grb10</i> and <i>Stat5b</i> mRNA expression in adult mouse brain, including the arcuate nucleus of the hypothalamus (ARC), dorsomedial nucleus of the hypothalamus (DMH), lateral septal nucleus (LSV), medial amygdaloid nucleus (posteroventral part) (MePV), medial habenular nucleus (MHb), medial preoptic nucleus (MPA), median preoptic nucleus (medial part) (MPOM), periaqueductal grey (PAG), paraventricular thalamic nucleus (PVA), paraventricular nucleus of the hypothalamus (PVH), supraoptic nucleus (SON), ventromedial nucleus of the hypothalamus (VMH), and ventraltegmental area (VTA). (D) <i>In vitro</i> transfection assay of the enhancer capability of CRM1. Luciferase activity was measured in cells transfected with a minimal promoter driving luciferase (pGL3-Pro) or with CRM1 cloned upstream of the minimal promoter (pGL3-Pro-CRM1). Only pGL3-Pro-CRM1 responded to increasing doses of constitutively active STAT5b (STAT5b1*6). ***<i>p</i><0.001 (one-way ANOVA).</p

Overview of complementary <i>Grb10</i> functions in mother and pup.

<p><i>Grb10</i> expressed in the mother promotes postnatal nutrient supply through the mammary gland, while offspring <i>Grb10</i> suppresses nutrient demand. Together, this regulation of nutrient acquisition ensures offspring achieve an optimal body size. Body proportions are also influenced by both <i>Grb10</i> expressed in the mother and in the offspring. Offspring <i>Grb10</i> suppresses the development of lean mass, while offspring fat mass is promoted by <i>Grb10</i> expressed in the mother and acting on postnatal nutrient supply, jointly promoting optimal offspring body proportions.</p