Schematic representation of the p16-related regulation of AUF1,

<p>E2F1 and CyclinD1. See text for details.</p

Additional file 2: of Identification of a novel genetic locus underlying tremor and dystonia

Variants in candidate interval. (PDF 217 kb

Additional file 4: of Identification of a novel genetic locus underlying tremor and dystonia

CAMTA2 expression in brain. (PDF 1628 kb

p16 modulates apoptosis through E2F1.

<p>(A) Western blots showing the expression of the indicated pro-and anti-apoptotic proteins in U2OS and EHI. (B) Western blots showing the expression of the indicated proteins in U2OS, U2OS cells stably transfected with plasmids encoding either scrambled sequence (ctl) or E2F1 (+). The numbers below the bands indicate the corresponding expression levels. (C) U2OS, EH1 and E2F1-expressing U2OS cells were either mock-treated or challenged with doxorubicin (2 µM) and then re-incubated for 72 hrs. Cells were then divided into two groups; one was used to analyze cell death by annexinV/PI flow cytometry. The numbers in the charts indicate the proportions of early and late apoptosis. (D) Histogram showing the proportions of apoptosis (early+late) induced by different doses of doxorubicin. The error bars represent standard deviation of three different experiments. (E) The second group of cells was used to assess the level of the indicated proteins by immunoblotting. (F) Graph showing the Bax/Bcl-2 ratio in the indicated cells after treatment with doxorubicin. Error bars represent standard deviation of at least three different experiments.</p

p16 modulates E2F1 and cyclin D1 protein and mRNA levels in human and mouse cells.

<p>Whole cell extracts and total RNA were prepared from different human and mouse cell lines. (A–C) Western blots using the indicated antibodies. The histogram shows the expression levels of the indicated proteins. EH2 cells were treated with IPTG at 1 mM (D) Upper panel, ethidium bromide stained agarose gels showing RT-PCR products of the indicated genes. The numbers below the bands indicate the corresponding expression levels relative to β-actin. These experiments were repeated several times and representative ones are shown. The histogram shows data of real time RT-PCR of the indicated genes. Error bars indicate standard errors of 3 different experiments.</p

List of genes under the control of both p16 and E2F1.

<p>List of genes under the control of both p16 and E2F1.</p

List of genes under the control of both p16 and AUF1.

<p>*: The variation in the expression of these genes was less than 2 fold in microarray analysis data. However, the variation was significant when RT-PCR was used for assessment, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021111#pone-0021111-g001" target="_blank">figure 1D</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021111#pone-0021111-g007" target="_blank">figure 7</a>.</p

Involvement of ARE in the E2F1 3′UTR and response to AUF1 and p16.

<p>(A) Schematic diagram of the E2F1 3′UTR, ARE region sequences, and locations. (B) The E2F1 3′UTR sequences in different species (C) Sequences from the E2F1 3′UTR (<i>ARE regions 1 to 3</i>), IL-8 3′UTR (<i>ARE control</i>), and a control that lacks ARE were inserted in BamHI/XbaI sites in EGFP expression vector as shown. The Huh7 cell line (2.10⁴ cells per well) in 96-well black clear-bottom microplates were transfected with the different 3′UTR constructs. The reporter activity was assessed after 24 hr using BD bio-imaging apparatus and software. The non-ARE 3′UTR was used as control and its fluorescence activity was taken as 100%. Data are presented as Mean±SEM (n = 4) of % of the control. *** denote <i>p</i> values of <0.005 (student t- test) when compared to non-ARE control. (D) Huh7 cells (left panel) or U2OS (right panel) in 96-well microplates were co-transfected with siRNA against AUF1 or scrambled control (50 ng per well) and reporter constructs (25 ng per well) as indicated. Reporter activity was assessed at 48 hr post-transfection. Data (Mean±SEM, n = 4) were presented as % increase in reporter fluorescence due to AUF1 silencing when compared to the fluorescence in control-siRNA-treated cells. * and *** denote <i>p</i> <0.05 and <0.005, respectively (student t-test) when compared to non-ARE control. (E) U2OS and EH1 cells (2.10⁴ cells per well) were seeded in 96-well black clear-bottom microplates and then transfected with the different 3′UTR constructs. Reporter activity was assessed as described in (D). ANOVA was performed to compare between U2OS and EH1 data groups.</p

p16 controls the mRNA levels of <i>cyclin D1</i> and <i>E2F1</i> through AUF1.

<p>(A) HFSN1 cells were transfected with plasmids expressing either AUF1-siRNA (pSILENCER-AUF15) or control-siRNA (pSILENCER). 3 days after transfection total RNA was purified from both cells, and RT-PCR (top panel) as well as real time RT-PCR (histogram) using specific primers for the indicated genes were performed. Error bars indicate standard errors of 3 different experiments (B) RNAs bound to the AUF1 protein were isolated by immunoprecipitation from HFSN1 cells expressing either p16-siRNA or control-siRNA using anti-AUF1 antibody or anti-IgG (as control), and then target transcripts were amplified by RT-PCR visualized on Ethidium bromide stained 1% agarose gels. Amplification of the highly abundant GAPDH transcript, which bound IP materials at background levels, was used as loading control. (C) HFSN1 whole cell lysate was immunoprecipitated using the indicated antibodies and then used for immunostaining analysis. (D) Total RNA was purified from p16 proficient (HFSN1 and EH1) and p16-deficient (U2OS and HFSN1 expressing p16-siRNA) cells, expressing either <i>AUF1</i>-siRNA or control-siRNA. Transcripts for the indicated genes were detected by RT-PCR, and the corresponding products were visualized on ethidium bromide stained 1% agarose gels. The numbers below the bands indicate the corresponding expression levels. GAPDH was used as internal control. These experiments were repeated several times and representative ones are shown.</p