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

Metabolomic analysis of key intermediates in glycolysis, lactate production, and the TCA cycle in TRACK kidneys.

<p>A: Comparison of the levels of glycolysis and lactate production intermediates, glucose, pyruvate, and lactate in TG^<b>−</b> vs. TRACK kidney samples (n = 14 and 12). □ = TG^<b>−</b>; </p><p></p><p><mi>■</mi></p><p></p> = TRACK. B: Comparison of the levels of fumarate, α-ketoglutarate, and citrate/isocitrate in WT vs. TRACK kidney samples (n = 14 and 12). □ = TG^<b>−</b>; <p></p><p><mi>■</mi></p><p></p> = TRACK. C: Schematic overview of changes to glycolysis, lactate production, and the TCA cycle in TRACK kidneys. Error bars indicate the mean ± SD. * indicates a p value <0.05, ** indicates a p value <0.01, and *** indicates a p value <0.001.<p></p

Metabolomic analysis of amino acid levels in TRACK kidneys.

<p>A: Levels of amino acids in WT vs. TRACK mouse kidneys (n = 14 and 12). □ = TG^<b>−</b>; </p><p></p><p><mi>■</mi></p><p></p> = TRACK. B: Schematic overview of changes to amino acid levels and TCA cycle. Error bars indicate the mean ± SD. * indicates a p value <0.05, ** indicates a p value <0.01, and *** indicates a p value <0.001.<p></p

Comparison of TRACK kidney gene transcripts with human clear cell renal cell carcinoma.

<p>Selected genes that are most highly expressed in human ccRCC, and which show increased mRNA levels (fold change > 2) in the kidney tissue of the TRACK mice relative to kidneys from wild type littermates. Human data were collected from Oncomine, and TRACK data were obtained by RNAseq.</p><p>Comparison of TRACK kidney gene transcripts with human clear cell renal cell carcinoma.</p

Metabolomic analysis of key intermediates in glycolysis, lactate production, and the TCA cycle in ccRCC samples.

<p>A: Comparison of the levels of glycolysis and lactate production intermediates, glucose, pyruvate, and lactate in normal vs. ccRCC samples (n = 12). </p><p></p><p><mi>■</mi></p><p></p> = Normal; ■ = ccRCC. B: Comparison of the levels of fumarate, α-ketoglutarate, and citrate/isocitrate in normal vs. ccRCC samples (n = 12). <p></p><p><mi>■</mi></p><p></p> = Normal; ■ = ccRCC. C: Schematic overview of changes to glycolysis, lactate production, and the TCA cycle in ccRCC. Error bars indicate the mean ± SD. * indicates a p value <0.05, ** indicates a p value <0.01, and *** indicates a p value <0.001.<p></p