Effect of #22.23 mutation on cell growth and etoposide-induced cell death.

<p>(<b>A</b>) Five-hundred thousand p53^−/− cells in a dish were cultured for 24 hr. Cells were transfected with an expression plasmid for p53 (WT) or #22.23 (mut) together with a TLP expression plasmid. After 24 hr, 8×104 cells were replated and maintained. Cell numbers were counted every 24 hr (panels a–c). ctr: vacant plasmid. (<b>d</b>) Cell numbers at each time shown in panels a–c are displayed as ratios to the initial cell number. (<b>B</b>) Experiments were performed as described above, but replated cells were maintained in a medium containing 30 µM etoposide to examine the effect of TLP on apoptotic cell death (<b>a–c</b>). Numbers of remaining viable cells were counted. (<b>d</b>) Data are summarized as described above.</p

Function of p53 mutants in TLP-stimulated transcriptional activation.

<p>(<b>A</b>) Schematic representation of the structure of human p53. (<b>a</b>) Positions of TAD (transactivation domain), DBD (DNA-binding domain) and TD (tetramerization domain) are indicated with AA positions. Positions of mutation in the examined mutants are shown by vertical triangles. (<b>b</b>) AA residues of TAD1 in the TAD region. 22L and 23W have been reported to be critical for transactivation (TA), and 18T and 20S are phosphorylated (PH) amino acids (6). (<b>B</b>) Analysis of TLP-stimulated function for individual p53 mutants by an overexpression experiment. Cells were co-transfected with <i>p21</i> upstream promoter-carrying reporter plasmid and expression plasmid for p53/mutant alone or p53/mutant+TLP. Results are shown as relative luciferase activities (RLA). Ratio represents RLA of p53/mutant expression to RLA of p53/mutant+TLP expression. Some data were examined by statistical analysis. Since the control experiment (ctr) was performed with a vacant effector plasmid, ratios could not be obtained because measured faint luciferase activities are meaningless. (<b>C</b>) Analysis of TLP-stimulated function of representative p53 mutants by a knockdown experiment. TLP siRNA and scrambled (control) siRNA were used as depicted in the figure, and promoter activity was determined as described in panel B.</p

Examination of mutant TLPs on transcriptional activation and p53 binding.

<p>(<b>A</b>) Structural relationship between TBP and TLP. Amino acid numbers are indicated from N-termini. TLP covers the evolutionally conserved region of TBP. A putative p53-binding region in TBP deduced from deletion analyses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090190#pone.0090190-Liu1" target="_blank">[44]</a> and its TLP counterpart (from 63 to 115) are depicted as a gray area. Positions of AAs of the TLP mutants used in this study (R86S, F100E, and F114E) are indicated with vertical arrowheads. (<b>B</b>) Transcription activation function of wild-type (WT) and mutant TLPs were assayed in native (<b>a</b>) and p53^−/− (<b>b</b>) cells. (<b>C</b>) Binding of TLP and p53. Wild-type and F100E TLPs were analyzed for the p53-bidnding ability by two-hybrid assay.</p

Effect of #22.23 mutation on gene expression from endogenous <i>p21</i> promoters.

<p>(<b>A</b>) Two kinds of major <i>p21</i> transcripts produced from the human <i>p21</i> gene. Position of exons of <i>p21</i> alt-a and <i>p21</i> variant-1 transcripts and genomic DNA around the two <i>p21</i> promoters are schematically illustrated. Open and solid boxes represent non-coding and coding exons, respectively. Two primer sets indicated by thick arrows were used for RT-PCR to detect variant-1 and alt-a, respectively. (<b>B</b>) p53^−/− cells were transfected with expression vectors for wild-type and mutant (#22.23) p53, and two species of <i>p21</i> transcripts were determined by RT-PCR. Vector: vacant vector. RNAs of endogenous β-actin, p53 and TLP were also analyzed. (<b>C</b>) Assays for TLP-stimulated function of wild-type p53 and #22.23. (<b>a</b>) Experiments were performed as described in panel B. Cells were transfected with a TLP expression plasmid in addition to a p53 expression plasmid as indicated. ctr and vec: corresponding vacant vectors. (<b>b</b>) Amounts of intracellular p53 and #22.23 proteins were also detected by immunoblotting in addition to GAPDH and endogenous and exogenous TLPs. (<b>c</b>) Degree of increase in alt-a transcripts stimulated by exogenous TLP in p53-expressing cells. Ratios of band intensities of alt-a of panel (a) in vacant vector-introduced cells to that in TLP overexpressed cells were calculated for three kinds of cells.</p

Effect of F100E mutation of TLP on the expression of endogenous <i>p21</i> gene and cell growth.

<p>(<b>A</b>) Wild-type (a) and p53^−/− cells (b) were transfected with expression vectors of wild-type and mutant (F100E) TLPs, and two species of <i>p21</i> transcripts were determined by RT-PCR as described in a legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090190#pone-0090190-g004" target="_blank">Fig. 4</a>. (<b>B</b>) Wild-type and mutant TLP-transfected native (<b>a</b>) and p53^−/− (<b>b</b>) cells were cultured for 24 hr. Cells (1×10⁵) were replated and cell numbers were counted every 24 hr. ctr: vacant plasmid.</p

TLP binds to p53 in solution.

<p>(<b>A</b>) Detection of p53-biding ability of TLP. TLP and TBP were examined for p53 binding by a GST pull-down assay, and affinities of both proteins against p53 were roughly determined by a competitive pull-down assay. (<b>a</b>) FH-p53 was challenged to GTS-tagged TBP (lane 1) or TLP (lane 3) as indicated and a simple pull-down assay was performed. TBP/TLP and FH-p53 were detected by α-GST antibody and α-53 antibody, respectively. No signal was detected when only GST tag was used (data not shown). FH-TLP (lane 2) and FH-TBP (lane 4) were co-applied to the GST-fused protein-adsorbed beads together with FH-p53, respectively, as competitors for GST proteins. (<b>b</b>) Relative band intensities of lane 2 (TBP+TLP), lane 3 (TLP) and lane 4 (TLP+TBP) to that of lane 1 (TBP) of panel (a) are displayed. (<b>B</b>) Comparison of p53-binding affinities of TLP and TBP. GST pull-down assays of lane 1 and lane 2 of panel A-a were performed with increasing amounts of GST-TBP (a) and GST-TLP (b), respectively. input: input protein corresponding to experimental (pull-down) materials. (<b>c</b>) Relative band intensity for p53 protein of panel (a). Results of 0.05 and 0.1 pmole of GST proteins of panel (a) are shown again in the magnified graph.</p

Summary of the mutation analysis.

<p>* Activation of the mutants are displayed in multiple degrees such as +++ (very strong) ∼ ± (weak).</p><p>BTA: basal transactivation function.</p><p>TLP-SF: TLP-stimulated function examined by over-expression assay.</p><p>TLP-BA: TLP-binding activity.</p><p>NT: not tested.</p

TLP-binding ability of p53 mutants.

<p>(<b>A</b>) <i>In vitro</i> binding of various p53 mutants. A GST pull-down assay was performed as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090190#pone-0090190-g001" target="_blank">Fig. 1</a> by using several representative p53 mutants. (<b>B</b>) Binding between TLP and p53 or its mutants in cells was examined by a mammalian two-hybrid assay. Binding was monitored by luciferase reporter assay. Plasmids for TLP-containing bait (BIND) and p53/mutant-containing prey (ACT) were introduced into cells as indicated. Since TLP is a transcriptional activator with poor DNA-binding capacity, experiments with bait alone brought significant luciferase activity. (<b>C</b>) Immunoprecipitation to detect <i>in vivo</i> binding of TLP and p53. FH-TLP and HA-tagged p53 or its mutant (#22.23) were overexpressed in cells and immunoprecipitataied with M2 beads. Immunoprecipitates were analyzed for TLP-associating p53, TLP and GAPDH.</p

MOESM1 of Oxygen-radical pretreatment promotes cellulose degradation by cellulolytic enzymes

Additional file 1: Table S1. The content of cellulose, hemicellulose, and lignin in non-pretreated, oxygen-gas-pretreated, and oxygen-radical-pretreated wheat straw. Figure S1. Effects of oxygen-radical pretreatment on MCC hydrolysis by cellulolytic enzymes in culture supernatant. Reducing sugars released from (a) oxygen-gas- or (b) oxygen-radical-pretreated MCC by enzymatic hydrolysis using culture supernatant were assayed using the DNS method. Error bars represent the mean ¹ standard error of the mean of three independent experiments. Figure S2. Gas chromatography spectra of the washing water of oxygen-gas- and oxygen-radical-pretreated wheat straw. (a) Oxygen-gas- and (b) oxygen-radical-pretreated wheat-straw samples were extracted with water to wash out enzyme inhibitors. Each treatment sample was washed with 25°C Milli-Q water, followed lyophilization, trimethylsilylation, and analysis of the liquid fraction by gas chromatography. Figure S3. Reducing-sugar production from washed and unwashed oxygen-radical-pretreated wheat straw. Reducing sugars released from washed and unwashed oxygen-radical-pretreated wheat straw after enzymatic hydrolysis using the supernatant from Phanerochaete chrysosporium cultures were assayed using the DNS method. Data are presented as the mean ¹ standard deviation of three experiments

MOESM1 of Oxygen-radical pretreatment promotes cellulose degradation by cellulolytic enzymes

Additional file 1: Table S1. The content of cellulose, hemicellulose, and lignin in non-pretreated, oxygen-gas-pretreated, and oxygen-radical-pretreated wheat straw. Figure S1. Effects of oxygen-radical pretreatment on MCC hydrolysis by cellulolytic enzymes in culture supernatant. Reducing sugars released from (a) oxygen-gas- or (b) oxygen-radical-pretreated MCC by enzymatic hydrolysis using culture supernatant were assayed using the DNS method. Error bars represent the mean ¹ standard error of the mean of three independent experiments. Figure S2. Gas chromatography spectra of the washing water of oxygen-gas- and oxygen-radical-pretreated wheat straw. (a) Oxygen-gas- and (b) oxygen-radical-pretreated wheat-straw samples were extracted with water to wash out enzyme inhibitors. Each treatment sample was washed with 25°C Milli-Q water, followed lyophilization, trimethylsilylation, and analysis of the liquid fraction by gas chromatography. Figure S3. Reducing-sugar production from washed and unwashed oxygen-radical-pretreated wheat straw. Reducing sugars released from washed and unwashed oxygen-radical-pretreated wheat straw after enzymatic hydrolysis using the supernatant from Phanerochaete chrysosporium cultures were assayed using the DNS method. Data are presented as the mean ¹ standard deviation of three experiments