Confluent growth arrest is associated with increased FOXP2 expression.

<p>(A) Cell cycle analysis of 143B subjected to increasing confluence (approximate percentage confluence indicated top), numbers within plots from left to right indicate percentage of cells in G1/G0, S, and G2/M phase respectively, representative of three experiments; (B) Quantitation of apoptotic cell death in 143B cell populations by flow cytometric analysis of Annexin V positivity, in cultures either exponentially growing (Exp. Growth), subjected to overnight culture with 20μg/ml cyclohexmide as a positive control that induces apoptosis (cycloheximide), or subjected to 4 days growth arrest at confluence (late arrest, as per <i>A</i>). Numbers represent mean % annexin positive ± SD from three experiments; (C) Real-time PCR analyses of <i>FOXP</i> expression in MG-63, 143B and U2-OS cultured to increasing confluence, expressed as 2^-δβCT, relative to growing culture, <i>N</i> = 3 ± SD; (D) Immunoblot analyses of nuclear extracts from cells cultured as in <i>C</i>, including nucleophosmin (NPM) as a loading and transfer control, representative of two experiments, (E) Real-time PCR analyses of <i>p21</i>, <i>p27</i> and <i>IL-6</i> expression in 143B cultured to increasing confluence, expressed as 2^-δδCT, relative to growing culture, <i>N</i> = 3 ± SD.</p

Growth arrest-induced FOXP2 transcription is upstream of the cell cycle machinery.

<p>(A, C) Cell cycle analyses of 143B grown for 6 days in reduced serum or 1 day in the CDK4/6 inhibitor Palbociclib 100 nM or 1 μM as indicated; (B, D) Real-time PCR analyses of gene expression in 143B cultured as in <i>A</i> and <i>C</i>, expressed as 2^-δδCT, relative to growing or vehicle-treated culture, <i>N</i> = 3 ± SD; (E) Real-time PCR analyses of gene expression in 143B reduced serum experiments similar to <i>A</i> over a shorter timecourse, <i>N</i> = 3 ± SD; (F) Real-time PCR analysis of <i>FOXP2</i> expression in 143B cultured at subconfluence or confluence for 24hrs in the presence of inhibitors/vehicle as indicated, (PD-98059 ERK1/2 inhibitor and LY-294002 PI3K inhibitor at 50μM, IKK inhibitor 7, Bay117082 NFκB inhibitor, and DBZ Notch pathway inhibitor at 1μM), expressed as fold change induced by confluence, inhibitors had minimal effect on subconfluent <i>FOXP2</i> expression, <i>N</i> = 5 ± SD; (G) Immunoblot analysis of nuclear extracts from 143B treated at confluence as in <i>F</i>, including nucleophosmin (NPM) as a loading and transfer control.</p

FOXP2 induction is required for efficient 143B growth arrest and p21 ^CIP1/WAF1 control.

<p>(A) Immunoblot analysis of FOXP expression in nuclear extracts from confluent 143B cells 48hr after siRNA transfection, including TATA-binding protein (TBP) as a loading and transfer control, representative of three experiments; (B) Quantitation of changes in cell cycle fractions induced by confluence as per <i>A</i>, relative to dividing cells, <i>N</i> = 3 ± SD; (C) Phase contrast images of cells as in <i>A</i>, to show frequently increased saturation density following FOXP2 depletion;; (D) Real-time PCR analysis of <i>p21/CDKN1A</i> expression in 143B cultured as in <i>A</i>, expressed as 2^-δδCT, relative to subconfluent culture (T = 0), control is mean of four different control siRNAs. <i>N</i> = at least 4 ± SD; (E) Immunoblot analyses of whole cell lysates from cells cultured as in <i>A</i>; (F) Real-time PCR analysis of <i>p21CDKN1A</i> expression in cells transfected at subconfluence (T = 0) with siRNAs as shown and harvested still at subconfluence after 48 hr culture in 1% serum, <i>N</i> = 3 ± SD; (G) Real-time PCR analysis of <i>p21CDKN1A</i> expression in and phase contrast images of cells cultured as in <i>A</i>, in the presence of either 10ng/ml hIL-6 (+ IL-6) or PBS/BSA carrier alone (—IL-6); (H) Real-time PCR analyses of cells transfected at subconfluence with p53 cDNA plasmid or empty control plasmid (vector), then treated with siLGC or each FOXP2 siRNA and harvested still at subconfluence after 48hr culture in 10% serum. Expression is shown as percentage relative to highest (100%).</p

Foxp2/FOXP2 expression in murine bone and association with reduced growth of osteosarcoma cell lines.

<p>(A,B) Real-time PCR analysis of gene expression in primary murine tissues from 12-week old male C57Bl/6 mice using SyBr-green, specifically whole bone marrow (BM), spleen (Spl), thymus (Thy), whole long bones after flushing and 2 rounds of collagenase digestion (Bone) and bone-associated cells from the collagenase fraction (CF). Data represent mean +/- SD of three mice. While <i>Foxp1</i> and <i>Foxp4</i> are most expressed in <i>CD45</i>+ haematopoietic tissues, <i>Foxp2</i> expression is highest in bone as are the established osteoblast genes <i>Ibsp</i> and <i>Sp7</i>. Expression normalised to <i>Hprt</i>, expressed relative to highest sample (100%); (C) Immunoblot analysis of COS-1 fibroblast-like cells transiently transfected with CMV-driven mammalian expression plasmids containing murine full-length Foxp cDNAs or pCDNA4 empty vector (control) as indicated top. All antibodies exhibited specificity for appropriate ectopically-expressed proteins, low level endogenous FOXP4 expression was detectable in all lysates; (D) Immunohistochemical detection of Foxp2 protein in murine E17.5 long bone, detail (middle panels) showing variation in Foxp2 positivity along the periosteum, staining with the anti-rabbit murine monoclonal antibody MR12 was performed on serial sections as negative control (same regions, right panels)</p