HPV E6-mediated p53 degradation.

<p>A) HA-tagged p53 levels were visualized by Western blot after co-transfection with HA-tagged HPV E6 from HPV11, HPV16 and HPV18 into C-33A cells. Lanes 1, 3, 5 and 7 show results from co-transfection of HA-p53 and vectors indicated at the top of the figure. Lanes 2, 4, 6 and 8 show the p53 levels after treatment with MG132, as indicated at the top of the figure. β-tubulin was visualized as a loading control. (B) Half life of HA-p53 in transfected 293T cells. 293T cells were transfected with HA-p53 and control (pQCXIN) or E6 ORFs (HPV11, HPV18, HPV53, HPV56 and HPV66). The band intensities were determined from the scanned Western blot using ImageQuant and the signals at time 0 were defined as 100%. The band intensities of the indicated time points were normalized to time 0.</p

Alignment of HPV alpha E6 ORFs.

<p>The alignment of all 27 E6 ORFs tested in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone-0012816-g002" target="_blank">Figure 2</a> is shown. HPV types degrading p53 are shown at the top of the alignment and those not degrading p53 are shown at the bottom. The shaded region represents a proposed E6-AP binding domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Liu1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Pim1" target="_blank">[26]</a>. The amino acid sequences of the E6 ORFs were aligned using Clustal X (version 1.81) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Thompson1" target="_blank">[36]</a>. The amino acid at position 31 (arrow, <b>bold</b>) was associated with p53 degradation (p<0.01). “_” indicate gaps, whereas “.” indicates identical residues with the HPV16 E6 amino acid sequence shown in the top row.</p

Phylogeny of alpha-HPVs and degradation of p53 by HPV E6 ORFs.

<p>HPV E6 activity on HA-p53 steady state levels were determined by Western blot using the assay shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone-0012816-g001" target="_blank">Figure 1A</a>. The phylogenetic tree at the left was constructed using the combined early gene sequences as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Narechania1" target="_blank">[35]</a>. Epidemiological carcinogenicity was extrapolated from recent reviews <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Schiffman1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Bouvard1" target="_blank">[24]</a> and is indicated in the column labeled “carcinogenic risk”: ++, highly oncogenic; +, oncogenic; +/−, probably oncogenic; −, not significantly associated with cervix cancer; NA, insufficient data. The p53 levels after co-transfection with E6 from each type indicated on the left are shown in the far right column labeled, “p53” (with and without MG132) and the results are summarized in the column labeled“↓p53”. Endogenous β-tubulin (far right column) represents a loading control. The alpha-HPV species groups are indicated by brackets with a number to the right. The empty vector control, pQCXIN is shown at the bottom.</p

HPV E6 mutagenesis and degradation of p53.

<p>(A) The steady state levels of HA-p53 co-transfected with wild type or mutant HPV71, 90 and 106 HA-E6 in C-33A cells are shown in the row labeled HA-p53. The unmodified E6 ORFs are indicated with “WT” and the mutated E6 ORFs are indicated with the replacement amino acid at the top of the figure. β-tubulin is shown as a loading control in the bottom row. (B) Representative images of double immuno-fluorescence experiments. HA-tagged HPV E6 constructs are green, whereas the endogenous p53 is labeled red. In the p53 image, arrows indicate the location of the E6 expressing cells. Absence of p53 signal (red) is the result of degradation. In the merged images, yellow signifies a non-degrader, whereas green only (HPV E6) is indicative of a p53 degrader. Cell nuclei are detected with DAPI and are shown in the bottom row. Each HPV E6 construct is indicated at the bottom of each column.</p

Additional file 1 of Targeting MYH9 represses USP14-mediated NAP1L1 deubiquitination and cell proliferation in glioma

Supplementary Material

Human Papillomavirus 16 Non-European Variants Are Preferentially Associated with High-Grade Cervical Lesions

<div><p>HPV16 accounts for 50–70% of cervical cancer cases worldwide. Characterization of HPV16 variants previously indicated that they differ in risks for viral persistence, progression to cervical precancer and malignant cancer. The aim of this study was to examine the association of severity of disease with HPV16 variants identified in specimens (n = 281) obtained from a Cervical Pathology and Colposcopy outpatient clinic in the University Hospital of Espírito Santo State, Southeastern Brazil, from April 2010 to November 2011. All cytologic and histologic diagnoses were determined prior to definitive treatment. The DNA was isolated using QIAamp DNA Mini Kit and HPV was detected by amplification with PGMY09/11 primers and positive samples were genotyped by RFLP analyses and reverse line blot. The genomes of the HPV16 positive samples were sequenced, from which variant lineages were determined. Chi² statistics was performed to test the association of HPV16 variants between case and control groups. The prevalence of HR-HPV types in </p></div

Tree topology.

<p>Phylogenetic tree was inferred from global alignment of complete and partial genome nucleotide sequences. Distinct variant lineages (i.e., termed A, B, and C) are classified according to the topology and nucleotide sequence differences from >1% to <10%; distinct sublineages (e.g., termed A1 and A2) were also inferred from the tree topology and nucleotide sequence differences in the >0.5% to <1% range <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100746#pone.0100746-Burk1" target="_blank">[22]</a>.</p

The microbiome associated with high <i>E. coli</i> inhibitory activity is significantly different than the microbiome in women with low activity (p = 0.042).

<p>The pairwise Kantorovich-Rubinstein (KR) distances between sample microbiomes were calculated and a PERMANOVA principal component analysis was performed with <i>E. coli</i> inhibitory activity (A) and pregnancy (B) as the factors. The two axes represent the first two principal components of the pairwise KR distance matrix. The red point demarks the group centroid, while the black open points represent the sample coordinates in the first two principal components. The p-value test statistic is displayed at the top of each plot area, indicating the statistical significance of the difference in variances when samples were grouped according to the factor (i.e., <i>E. coli</i> inhibitory activity or pregnancy). The Eigen values, or the amount of variation in the data accounted for by each principal component, are found in parentheses adjacent to PC1 and PC2.</p

<i>L. crispatus</i> culture supernatants inhibit <i>E. coli.</i>

<p>(a) Bar graphs depicting the <i>E. coli</i> cfu/ml after overnight incubation with 1∶10 dilution in normal saline of culture supernatants obtained from the indicated bacterial species or respective control media. The results were adjusted for differences in colony counts of the bacteria from which SCS were obtained and are presented as mean ± SD obtained from 3 independent experiments. (b) The spent culture supernatants obtained from three additional strains of <i>L. crispatus</i> (M35, SJ-3C, and 60) were normalized by diluting in culture media to that of the lowest growth (2×10⁷ cfu/ml) and then serial dilutions (neat, 1∶5, 1∶10 and 1∶25) were mixed with <i>E. coli</i> and tested for inhibitory activity. Results are mean ± SD from duplicate plates. The number symbol indicates that no bacterial colonies were observed on plates after incubation with undiluted <i>L. crispatus</i> 60 SCS. The asterisks represent a significant reduction in <i>E. coli</i> cfu relative to its control growth media (p<0.01).</p

HPV16 variant distribution by diagnostic category.

<p></p><p>CIN3+: case group, comprising the samples from CIN 3 or worse (cervical cancer in situ or invasive);</p><p>E: HPV16 European variant; NE: HPV16 non-European variant.</p