PIKE-A is required for prolactin-mediated STAT5a activation in mammary gland development
2010; Springer Nature; Volume: 29; Issue: 5 Linguagem: Inglês
10.1038/emboj.2009.406
ISSN1460-2075
AutoresChi-Bun Chan, Xia Liu, Michael A. Ensslin, Dirck L. Dillehay, Christopher J. Ormandy, Philip Sohn, Rosa Serra, Keqiang Ye,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoArticle14 January 2010free access PIKE-A is required for prolactin-mediated STAT5a activation in mammary gland development Chi-Bun Chan Chi-Bun Chan Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Xia Liu Xia Liu Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Michael A Ensslin Michael A Ensslin Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Dirck L Dillehay Dirck L Dillehay Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Division of Animal Resources, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Christopher J Ormandy Christopher J Ormandy Cancer Research Program, Department of Medicine, Garvan Institute of Medical Research, Sydney, Australia Search for more papers by this author Philip Sohn Philip Sohn Department of Cell Biology, University of Alabama at Birmingham, University Boulevard, MCLM, Birmingham, AL, USA Search for more papers by this author Rosa Serra Rosa Serra Department of Cell Biology, University of Alabama at Birmingham, University Boulevard, MCLM, Birmingham, AL, USA Search for more papers by this author Keqiang Ye Corresponding Author Keqiang Ye Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Chi-Bun Chan Chi-Bun Chan Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Xia Liu Xia Liu Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Michael A Ensslin Michael A Ensslin Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Dirck L Dillehay Dirck L Dillehay Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Division of Animal Resources, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Christopher J Ormandy Christopher J Ormandy Cancer Research Program, Department of Medicine, Garvan Institute of Medical Research, Sydney, Australia Search for more papers by this author Philip Sohn Philip Sohn Department of Cell Biology, University of Alabama at Birmingham, University Boulevard, MCLM, Birmingham, AL, USA Search for more papers by this author Rosa Serra Rosa Serra Department of Cell Biology, University of Alabama at Birmingham, University Boulevard, MCLM, Birmingham, AL, USA Search for more papers by this author Keqiang Ye Corresponding Author Keqiang Ye Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Author Information Chi-Bun Chan1, Xia Liu1, Michael A Ensslin2, Dirck L Dillehay1,3, Christopher J Ormandy4, Philip Sohn5, Rosa Serra5 and Keqiang Ye 1 1Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA 2Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA 3Division of Animal Resources, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA 4Cancer Research Program, Department of Medicine, Garvan Institute of Medical Research, Sydney, Australia 5Department of Cell Biology, University of Alabama at Birmingham, University Boulevard, MCLM, Birmingham, AL, USA *Corresponding author. Department of Pathology and Laboratory Medicine, Emory University, 615 Michael Street, Atlanta, GA 30322, USA. Tel.: +1 404 712 2814; Fax: +1 404 712 2979; E-mail: [email protected] The EMBO Journal (2010)29:956-968https://doi.org/10.1038/emboj.2009.406 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info PI 3-kinase enhancer A (PIKE-A) is critical for the activation of Akt signalling, and has an essential function in promoting cancer cell survival. However, its physiological functions are poorly understood. Here, we show that PIKE-A directly associates with both signal transducer and activator of transcription 5a (STAT5a) and prolactin (PRL) receptor, which is essential for PRL-provoked STAT5a activation and the subsequent gene transcription. Depletion of PIKE-A in HC11 epithelial cells diminished PRL-induced STAT5 activation and cyclin D1 expression, resulting in profoundly impaired cell proliferation in vitro. To confirm the function of PIKE-A in PRL signalling in vivo, we generated PIKE knockout (PIKE−/−) mice. PIKE−/− mice displayed a severe lactation defect that was characterized by enhanced apoptosis and impaired proliferation of mammary epithelial cells. At parturition, STAT5 activation and cyclin D1 expression were substantially reduced in the mammary epithelium of PIKE−/− mice. The defective mammary gland development in PIKE−/− mice was rescued by overexpression of a mammary-specific cyclin D1 transgene. These data establish a critical function for PIKE-A in mediating PRL functions. Introduction PI 3-kinase enhancer (PIKE) is a family of GTPases that regulate PI 3-kinase (PI3K)/Akt signalling pathway. It has three family members (PIKE-S, PIKE-L and PIKE-A), which are generated by differential transcription and alternative splicing of the gene CENTG1 (Chan and Ye, 2007). PIKE-S is a nuclear protein that enhances the kinase activity of PI3K and executes the anti-apoptotic function of NGF (Ye et al, 2000, 2002). In hippocampal neurons, PIKE-L binds to Homer, an adaptor protein known to link mGluR I to multiple intracellular targets including IP3 receptor. Activation of mGluR I enhances the formation of an mGluR I-Homer-PIKE-L complex, leading to activation of PI3K activity and prevention of neuronal apoptosis (Rong et al, 2003). Recently, we showed that PIKE-L exerted its neuroprotective action through inhibition of SET proteolytic cleavage by AEP, an asparagine endopeptidase (Liu et al, 2008). PIKE-L also binds to Unc5B and mediates the prosurvival effect of netrin 1 in neurons (Tang et al, 2008). Although PIKE-S and -L associates with PI3K, PIKE-A interacts with Akt, but not PI3K (Ahn et al, 2004b). PIKE-A is co-amplified with CDK4 in human glioblastomas, which specifically binds to active Akt in a guanine nucleotide-dependent manner, and stimulates Akt-kinase activity (Ahn et al, 2004a). Human cancer cells with PIKE amplification are more resistant to apoptosis than those with normal PIKE copy number. Knockdown of PIKE-A diminishes Akt activity and, therefore, enhances apoptosis (Ahn et al, 2004a, 2004c). We also reported that PIKE-A acted as a proto-oncogene, and promoted cell transformation through Akt activation (Liu et al, 2007). Indeed, overexpression of PIKE-A has been observed in a variety of tumours including prostate and breast cancers (Liu et al, 2007; Cai et al, 2009). These observations indicate that PIKE-A amplification promotes cancer cell growth by inhibiting apoptosis through stimulation of Akt. Prolactin (PRL) is a pituitary hormone with numerous physiological functions (Ben-Jonathan et al, 2008). It provokes multiple signalling cascades including the Janus-kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) (Hennighausen et al, 1997) and the Ras-MAPK (Erwin et al, 1995). Binding of PRL to its receptor (PRLR) leads to receptor dimerization and autophosphorylation of the receptor-associated JAK2 (Rui et al, 1994). JAK2 phosphorylates the PRLR, thereby creating docking sites for Src homology 2-domain proteins such as STAT5, Src, Fyn and Tec. Subsequently, Jak2 phosphorylates both STAT5 forms (STAT5a and STAT5b) and triggers the formation of STAT5 dimers, which translocate into the nucleus, tether to specific DNA sequence motifs and activate the transcription of target genes such as WAP, β-casein and cyclin D1 (Liu et al, 1997; Miyoshi et al, 2001; Brockman et al, 2002; Brockman and Schuler, 2005; Sakamoto et al, 2007). Among the many functions ascribed to PRL, its involvement in mammary gland development has been best characterized. Mammary gland development is a highly regulated process (Hennighausen and Robinson, 2001). The tissue begins to develop during embryogenesis as a rudimentary ductal system. Under the influence of systemic hormones at puberty, the ducts begin to expand into the surrounding fat pad. With repeated estrous cycles and during pregnancy, the complexity of the ductal system increases through the addition of side branches (Brisken, 2002). The last stage of mammary gland morphogenesis, alveologenesis, is closely intertwined with the functional differentiation of the mammary epithelium called lactogenesis (Brisken and Rajaram, 2006). Lactogenesis contains two stages. Stage 1 begins at mid-pregnancy and involves increased expression of genes involved in the synthesis of different milk proteins such as β-casein, lactalbumin and WAP (whey acidic protein) (Neville et al, 2002), which are also transiently increased during the estrous cycles (Robinson et al, 1998). The second stage of lactogenesis occurs around parturition; expressions of milk protein genes are further increased and cytoplasmic lipid droplets and casein are moved to the alveolar lumen (Jensen et al, 2001; Neville and Morton, 2001). PRL exerts only minor effects on morphological changes that occur in the mammary gland during peripubertal life, but it is heavily involved in lobuloalveolar differentiation, lactogenesis galactopoiesis (maintenance of milk secretion) and involution (a return to a nonlactating state) (Oakes et al, 2006). In this report, we show that PIKE-A forms a complex with STAT5 and couples it to PRLR, which is regulated by PRL. Ablation of the PIKE gene cripples PRL/JAK2/STAT5 and Akt signalling leading to substantial apoptosis and defective epithelial cell proliferation in mammary gland at postpartum, resulting in underdeveloped lobuloalveolar network and failed lactation. Cyclin D1 expression is decreased in vitro and in vivo and forced Cyclin D1 expression in vivo can rescue these defects. Thus, PIKE-A is a critical factor in mediating PRL function during lactation by promoting mammary epithelial cell proliferation and differentiation. Results PIKE-A specifically interacts with STAT5a and PRLR PIKE-A is overexpressed in breast cancer (Liu et al, 2007). Aberrant STAT activities highly correlate with breast cancer progression (Clevenger, 2004; Liu et al, 2007). These observations lead us to test whether PIKE-A directly interacts with STAT proteins. We conducted an immunoprecipitation assay from HEK293 cells that were co-transfected with mammalian GST-PIKE-A and various GFP-STAT proteins. Immunoblotting analysis revealed that GST-PIKE-A selectively interacted with STAT5a, but not STAT5b, STAT1 or GFP alone (Figure 1A). To determine which portion of PIKE-A associated with STAT5a, we conducted truncation assays. This deletion mapping experiment demonstrated that the N-terminal 1–72 residues in PIKE-A were necessary and sufficient for the interaction between PIKE-A and STAT5a (Figure 1B and C). We also examined the PIKE-A interaction domain in STAT5a by co-transfecting HEK293 cells with GFP-PIKE-A and different myc-tagged STAT5a truncations mutants (Figure 1D). PIKE-A strongly interacted with the C-terminal DNA-binding domain of STAT5a (Figure 1E). Weak but detectable interaction was also found between the trans-activation domain of STAT5a and PIKE-A. In contrast, we did not observe any interaction between PIKE-A and JAK2 (data not shown). Figure 1.PIKE-A interacts with STAT5a and PRLR. (A) PIKE-A specifically binds STAT5a. HEK293 cells were co-transfected with various GFP-STAT plasmids and mGST-PIKE-A. The GFP proteins were immunoprecipitated and the bound PIKE-A proteins were detected using anti-GST-HRP antibody (top panel). The expression of mGST-PIKE-A (middle panel) and different GFP-STAT proteins (bottom panels) were verified. (B) Diagrammatic representation of GST-PIKE truncation constructs. (C) PIKE-A N-terminus associates with STAT5a. In vitro mapping of PIKE-A domains that associate with STAT5a. Purified GST-tagged PIKE-A proteins were incubated with lysates of HEK293 cells transfected with GFP-STAT5a. The N-terminal end (1–72 a.a.) of PIKE-A, but not the C-terminal domain associates with STAT5a (middle panel). The GST-fused PIKE-A fragments (as indicated with asterisk) used in the in vitro binding were detected using anti-GST-HRP antibody (bottom panel). (D) Diagrammatic representation of myc-STAT5a truncation constructs. (E) STAT5a C-terminal associates with PIKE-A. HEK293 cells were co-transfected with GFP-PIKE-A and various deletion mutants of myc-STAT5a as shown in (D). The expressed STAT5a mutants were immunoprecipitated and the bound GFP-PIKE-A was detected using anti-GFP antibody (second panel). The expression of GFP-PIKE-A (third panel) and myc-STAT5a mutants (fourth panel) were also examined. (F) Diagrammatic representation of GFP-PRLR truncation constructs. (G) PIKE-A binds C-terminal PRLR intracellular-domain. HEK293 cells were co-transfected with mGST-PIKE-A and various deletion mutants of GFP-PRLR intracellular domains. The expressed PRLR mutants were immunoprecipitated and the bound mGST-PIKE-A was detected using anti-GST-HRP antibody (top panel). The expression of mGST-PIKE-A (bottom panel) and GFP-PRLR mutants (middle panel, indicated with asterisk) were also examined. (H) PIKE-A does not interact with other STAT5a-associated cytokine receptor. HEK293 cells were transfected with mouse EPO receptor (EPOR) plasmid and various GFP constructs as indicated. After 24 h of serum starvation, the transfected cells were stimulated with 3 ng/ml EPO for 15 min. The GFP-tagged proteins were immunoprecipitated and the associated EPOR was examined by using anti-EPOR antibody (top panel). The expression of EPOR (middle panel) and GFP-tagged proteins (bottom panel) were also verified. Download figure Download PowerPoint PRLR is one of the well-studied receptors that trigger STAT5a activation. We thus tested whether PIKE-A associated with the PRLR. We co-transfected HEK293 cells with GST-PIKE-A and different truncated constructs of mouse GFP-tagged PRLR intracellular domains (Figure 1F). We found that PIKE-A interacted with the C-terminus of the PRLR intracellular domain between a.a. 486–608 (Figure 1G). Moreover, this interaction is receptor specific, as PIKE-A does not associate with other STAT5-activating receptors such as the erythropoietin (EPO) receptor (Figure 1H). PRL provokes the formation of PIKE-A/STAT5/PRLR complex Binding of PRL to PRLR induces receptor dimerization and activation of JAK2 (Darnell et al, 1994; Schindler and Darnell, 1995), which subsequently phosphorylates STAT5 and triggers its dimerization and nuclear translocation (Ihle and Kerr, 1995). To determine whether the binding of STAT5 to PIKE-A is regulated by PRL, we treated mammary epithelial HC11 cells with PRL and monitored the interaction at various time points. Coimmunoprecipitation assays revealed that the tight association between PIKE-A and STAT5 was gradually disrupted by PRL in a time-dependent manner (Figure 2A, first panel). Concomitantly, the PIKE-A/PRLR association was initially increased (Figure 2A, second panel) and returned to basal level after 2 h (data not shown). Accordingly, phosphorylation of STAT5 was evident at 15 min, reached a maximal value at 30 min and partially decayed by 60 min after PRL stimulation. The PIKE-A/STAT5/PRLR binding was specific, as PIKE-A antibody, but not control IgG selectively pulled down STAT5 and PRLR from HC11 cells (Figure 2A, first and second panels). To determine whether phosphorylation of PRLR is necessary for PIKE-A/PRLR interaction, we pre-treated the PIKE-A-expressing HC11 cells with tyrosine-kinase inhibitor genistein, which blocked the PRLR phosphorylation by JAK2 on PRL treatment. The strong association of PIKE-A and PRLR in control cells was abolished in genistein-treated cells (Figure 2B), suggesting phosphorylation of PRLR is a prerequisite to PIKE-A docking. The PRLR/PIKE-A association was also abolished when HC11 cells were pre-treated with JAK2-specific inhibitor AG490 (Figure 2C, second panel), which further support the notion that PRLR phosphorylation is required for their interaction. Figure 2.PRL stimulation provokes the formation of PRLR/PIKE-A/STAT5a complex. (A) PRL stimulation interferes with the PIKE-A and Stat5 interaction, but enhances the formation of PRLR/PIKE-A complex. HC11 cells infected with adenovirus-expressing wild-type PIKE-A were stimulated with 10 nM recombinant mouse PRL for various time intervals as indicated. PIKE-A was then immunoprecipitated by control IgG or anti-PIKE-A antibody and the bound STAT5 and PRLR were detected using anti-Stat5 (first panel) and anti-PRLR antibodies (second panel). The expression of STAT5 (fourth panel) and PIKE-A (fifth panel) were examined. Phosphorylation of total STAT5 was also verified to confirm the activation of HC11 by PRL (third panel). (B) Phosphorylation of PRLR is essential for PIKE-A/PRLR interaction. HC11 cells were infected with adenovirus-expressing wild-type PIKE-A for 48 h followed by serum starvation for 24 h. Before 10 mM PRL stimulation, the cells were pre-treated with tyrosine-kinase inhibitor genistein (1 μM) for 45 min. The expressed PIKE-A was immunoprecipitated and the associated PRLR was detected using an anti-PRLR antibody (first panel). Phosphorylation of PRLR was examined using anti-phospho-tyrosine antibody on immunoprecipitated PRLR (second panel). The expression of PRLR (third panel) and PIKE-A (fourth panel) was also verified. (C) PIKE-A/PRLR interaction is JAK2-kinase dependent. HC11 cells were infected with adenovirus-expressing wild-type PIKE-A for 48 h followed by serum starvation for 24 h. Before 10 mM PRL stimulation for 15 min, the cells were pre-treated with JAK2-kinase inhibitor AG490 (50 μM) for 1 h. PIKE-A was immunoprecipitated and the associated STAT5 and PRLR was detected using specific antibody (first and second panels). Phosphorylation of JAK2 and STAT5 were determined to examine the effect of JAK2 inhibition (fourth and sixth panels). The expressions of PRLR, JAK2, STAT5 and PIKE were also verified (third, fifth, seventh and eighth panels). (D) PRL-provoked STAT5 phosphorylation is abolished in PIKE-A-depleted HC11 cells. HC11 cells were infected with either control adenovirus or adenovirus-expressing shPIKE. Two days after infection, the cells were serum-starved for 24 h and stimulated with 10 nM recombinant mouse PRL for 15 min. PRLR was immunoprecipitated and the associated STAT5 were determined by anti-STAT5 antibody (first panel). Phosphorylation of STAT5 was determined using specific antibody against phosphorylated STAT5 (third panel). Total PRLR (second panel), STAT5 (fourth panel) and PIKE-A (seventh panel) expressions was examined. Phosphorylation of ERK (fifth panel) by PRL was not affected in PIKE-depleted HC11 cells. Total ERK and tubulin were also determined (sixth and eighth panels). (E) PRL triggers nuclear translocation of STAT5a, but not PIKE-A. GFP-STAT5a and myc-PIKE-A were co-transfected in HC11 cells, serum-starved for 24 h and stimulated with 10 nM recombinant mouse PRL for 45 min. Cellular localization of PIKE-A and STAT5a was examined by confocal microscopy. Exclusive cytoplasmic localization of myc-PIKE-A was detected in both control and PRL-stimulated HC11 cells, whereas GFP-STAT5a accumulated in the nucleus after PRL stimulation. Download figure Download PowerPoint To test whether PIKE-A is essential for STAT5 activation induced by PRL, we depleted PIKE-A from HC11 epithelial cells using adenovirus-expressing-specific shRNAs. PRL elicited potent STAT5 activation in control adenovirus-infected cells, whereas STAT5 activation was completely abrogated in PIKE-A knockdown cells (Figure 2D, third panel). This reduction of STAT5 phosphorylation might be a result of disconnection between PRLR and STAT5 interaction as PRL failed to induce STAT5 docking to PRLR (Figure 2D, first panel). However, PRL-induced ERK phosphorylation was intact (Figure 2D, fifth panel), underscoring that PIKE-A selectively mediates STAT5 in PRL signalling. Immunofluorescent staining revealed that co-transfected PIKE-A and STAT5 co-localized in the cytoplasm. PRL stimulation-provoked STAT5 nuclear translocation, whereas PIKE-A remained in the cytoplasm (Figure 2E). These data suggest a model in which PRL triggers the PIKE-A/STAT5 complex to bind PRLR. STAT5 is subsequently phosphorylated by Jak2, which provokes STAT5 to dissociate from PIKE-A and leads to its nuclear translocation. Depletion of PIKE abolishes cyclin D1 expression and decreases epithelial cell growth PIKE-A is essential for PRL-induced STAT5 phosphorylation, conceivably, depletion of PIKE-A might abolish STAT5-induced gene transcription. Cyclin D1 is a downstream target of PRL signalling, and transcriptional expression and post-translational modification are mediated through PRL-provoked JAK2/STAT5, Ras/MAP kinase and PI3K/Akt signalling cascades (Fantl et al, 1995; Sicinski et al, 1995). PRL activates the cyclin D1 promoter via the JAK2/STAT pathway (Brockman et al, 2002). To explore whether PIKE-A regulates cyclin D1 transcription, we conducted a luciferase assay with construct containing GAS sites (γ-interferon activation sites) isolated from the cyclin D1 promoter (Brockman et al, 2002). PRL stimulated the activation of cyclin D1 promoter in control HC11 cells, but not PIKE-A-ablated cells (Figure 3A). Immunoblotting analysis further showed that expression of cyclin D1 was significantly diminished in PIKE-A-depleted HC11 cells (Figure 3B), confirming the observation made using the luciferase assay. As depletion of cyclin D1 in mice leads to impaired mammary epithelial proliferation (Sicinski et al, 1995), we assessed the effect of PIKE-A depletion on cell growth. Cell proliferation assays revealed that depletion of PIKE-A in HC11 cells substantially diminished cell growth (Figure 3C). These findings indicate that PIKE-A is indispensable for cyclin D1 expression and mammary epithelial cell proliferation. Figure 3.PIKE-A is essential for epithelial cell proliferation. (A) PIKE-A is required for PRL-stimulated cyclin D1 promoter activity. After transient transfection with the PRE3-luciferase vector, PIKE-A expression in HC11 cells was suppressed by infecting the cells with adenovirus-expressing sh-PIKE. After 48 h, the infected cells were then serum-starved for 24 h and treated with (solid bar) or without (open bar) 10 nM PRL in serum-free medium for another 24 h, and luciferase activity was then determined (**P<0.01, ***P<0.001, Student's t-test). (B) PIKE-A is critical for basal and PRL-induced cyclin D1 expression in HC11 cells. HC11 cells were infected with either control adenovirus or adenovirus-expressing sh-PIKE. Two days after infection, the cells were serum-starved for 24 h and stimulated with 10 nM recombinant mouse PRL for 24 h. Expression of cyclin D1 was examined by immunoblotting (top panel). PIKE-A expression in HC11 after shRNA infections was confirmed (middle panel). Tubulin level was also examined to show equal input of the proteins (bottom panel). (C) Diminished proliferation in PIKE-depleted mammary gland epithelial cells. HC11 cells were infected with control adenovirus or adenovirus-expressing sh-PIKE. Three days after infection, cell proliferations of the infected cells were examined by trypan blue exclusion assay (***P 30 alveoli in each animal) from each genotype. Scar bars represent 10 μm. (E) Alveoli density of pregnant and lactating mammary gland (1 day postpartum). The results are expressed as mean±s.e.m. of three mice (four individual fields in each sample) of each genotype. (F) Impaired protein expression in PIKE−/− mammary gland. Mammary tissues (fourth inguinal) from late gestation (18.5 dpc) and lactating (1 day postpartum) animals were collected and various milk proteins (β-casein and WAP), phosphorylation of signal transduction molecules (STAT5, Akt and ERK) and cyclin D1 levels were analysed by immunoblotting. Expression of β-tubulin was also performed to show equal loading. The results are representative blot of three individual animals from each genotype. Download figure Download PowerPoint PIKE depletion leads to impaired mammary epithelial cell proliferation and apoptosis To further explore the effect of PIKE ablation on epithelial proliferation from lactating mice, we conducted Ki67 staining. Compared with wild-type control, substantially less Ki67 positive staining was found in PIKE−/− mammary glands during lactation (Figure 5A). In contrast, no significant difference in cell proliferation, as revealed by positive Ki67 staining, was found between wild-type and PIKE−/− mice during pregnancy (Figure 5B). Similar resu
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