Pocket proteins expression in skin grafts.

<p>(<b>A</b>) Pocket protein and eGFP expression was analyzed by immunoblotting with specific antibodies using graft protein lysates from control vector, 5E7 and 16E7 samples. (<b>B</b>) Pocket protein and eGFP protein expression bands were quantified and relative (pocket protein/eGFP) values plotted. Horizontal green lines represents mean relative values. According to Student’s t-test, significant differences were only detected in pRb/eGFP expression values between vector and 16E7 samples (threshold p-val<0.05). *: p-val<0.05.</p

E7-grafts showed no significant apoptosis.

<p>Sections of paraffin-embedded grafts were processed for immunofluorescence staining with antibodies to the apoptosis markers caspase-3 active and p53. Caspase-3 active is not expressed in control vector samples (<b>A</b>), but some scattered positive cells are observed in HPV5 E7 (<b>B</b>) and HPV16 E7 grafts (<b>C</b> and <b>D</b>). No p53 staining was observed in control and HPV5 E7 samples (not shown), but eventual, p53-positive cells were observed in the HPV16 E7-transplants (<b>D</b>). Dotted line indicates the location of the basal membrane. Inserts in panels <b>B</b>–<b>D</b> show magnified areas of staining. DAPI was used to visualize cell nuclei.</p

HPV surrogate marker MCM7 is ectopically expressed in E7-transplants.

<p>Sections of paraffin-embedded grafts were processed for immunofluorescence staining with antibodies to MCM7. MCM7 is normally confined to the basal layer as in control vector transplants (<b>A</b>). Suprabasal expression is eventually observed in HPV5 E7-samples (<b>B</b>). Importantly, MCM7 is expressed in HPV16 E7-grafts in most suprabasal cells, reaching the uppermost spinous cells. Representative images of different 16E7-grafts are shown (<b>C, C’, C’’, C’’’</b>).</p

RNA expression quantification of oncogenic HPV biomarker genes and miRNAs.

<p>mRNA quantification of E2F1, CENPF, MELK and RFC4 genes was conducted by qRT-PCR using RNA purified from PHK cells or grafts, or obtained from reported microarray experiments for CC or VIN. Each dot represents an individual sample. Horizontal lines represent mean values in each sample group. (<b>A</b>) All genes were induced in E7-transduced PHKs, though not significantly (Student’s t-test threshold p-val<0.05). Significant overexpression was also observed for all genes in HPV16 E7-transplants, but only for E2F1 in HPV5 E7. Shown are log₂-based, z-values of expression relative to housekeeping GUSB in PHK cells and transplants (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041743#s2" target="_blank">Materials and Methods</a>). (<b>B</b>) Microarray expression patterns of each gene in CC and VIN infected with HR-HPVs are shown as log₂-based, z-values (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041743#s2" target="_blank">Materials and Methods</a>). All genes display significant overexpression in CC with respect to cervix uteri (N), and in VIN with respect to normal vulva (N). Horizontal lines represent mean values in each sample group. *: p-val <0.05, **: p-val <0.005, ***: p-val <0.0005, ****: p-val <0.00005. <b>V</b>: control vector; <b>5</b>: HPV5 E7; <b>16</b>: HPV16 E7. (<b>C</b>) miRNA was quantified by qRT-PCR for miR-146a, miR-21 and miR-200a, using RNA purified from skin grafts. Shown are log₂-based, z-values of expression relative to housekeeping U6B. Each dot represents an individual sample value. All miRNAs were overexpressed in HPV16 E7-grafts. Only miR-21 displayed significant deregulation in transplants expressing HPV5 E7. Horizontal lines represent mean values in each sample group. *: p-val <0.05, **: p-val <0.005, ***: p-val <0.0005, ****: p-val <0.00005. <b>V</b>: control vector; <b>5</b>: HPV5 E7; <b>16</b>: HPV16 E7.</p

Histopathology of E7-grafts and human HPV-infected pathology samples.

<p>H&E staining of representative samples of control vector, HPV5 E7 and HPV16 E7 transplants [<b>Skin grafts:</b> upper images (<b>A</b> to <b>F</b>)], as well as CIN1 and bowenoid papulosis (BP) human HR-HPV-infected samples [<b>Human pathology samples</b>: lower images (<b>G</b> to <b>J</b>)]. The histology of vector and 5E7 samples is similar to that of normal skin, with basal, spinous, granulous and cornified layers properly assembled in a mature, differentiated squamous epidermal epithelium of normal thickness (<b>A</b> and <b>B</b>). All 16E7-grafts display epidermal acanthosis (<b>C</b>). Features observed in human HR-HPV-infected lesions are present in HPV16 E7-grafts including suprabasal mitosis (white arrows in <b>D</b>), nuclear atypia (black arrowheads in <b>D, </b><b>G</b> and <b>H</b>), hyperkeratosis (black circles in <b>C</b>, <b>E</b>, <b>F</b>, and <b>J</b>), parakeratosis (black arrows in <b>E</b> and <b>I</b>), papillomatosis (<b>F</b>), hypergranulosis (asterisks in <b>C</b> and <b>D</b>) and capillaries (white arrowheads in <b>F</b> and <b>J</b>).</p

Histopathology of skin transplants.

<p>Histopathology of skin transplants.</p

Induction of p21 and cyclin A in E7-grafts.

<p>Sections of paraffin-embedded grafts were processed for immunohistochemistry staining with antibodies to cell cycle inhibitor p21 and cell cycle regulator cyclin A. With respect to control vector grafts (<b>A</b> and <b>D</b>), both p21 and cyclin A proteins are induced in suprabasal cells of HPV16 E7 transplants (<b>C</b> and <b>F</b>) and in focal HPV5 E7 areas (<b>B</b> and <b>E</b>). In the inserts, dotted lines indicate the location of the basal membrane.</p

Similarities between 16E7-grafts and HPV-infected anogenital neoplasias identified by immunohistochemistry.

<p>(<b>A</b>) Both PCNA and MCM7 displayed similar ectopic expression in most suprabasal cells of 16E7-grafts. In the basal layer, areas of expression coexisted with expression-free areas. (<b>B</b>) The same patterns were observed in tumor areas of cervical carcinoma samples where p16 was also present. PCNA and MCM7 expression expands progressively upwards in CIN1 and CIN3 (<b>C</b>), while most basal and subrabasal cells were stained in different cases of bowenoid papulosis (<b>D</b>). Middle panels in <b>C</b> and inserts in <b>D</b> highlight areas of basal cells showing negative staining for MCM7, PCNA and p16 (the latter, in CIN1). Dotted lines indicate the location of the basal membrane.</p

Cutaneous HPV5 E7 protein reduces pocket proteins levels.

<p>(<b>A</b>) Immunoblots of Saos2 lysates cotransfected with either pcDNA3.1(−)-FLAG (<b>Vector</b>), pcDNA3.1(−)-10E7FLAG (<b>10E7</b>), pcDNA3.1(−)-5E7FLAG (<b>5E7</b>), or pcDNA3.1(−)-16E7FLAG (<b>16E7</b>) plasmids. The effect on pRb expression levels was tested upon cotransfection with the pcDNA-mycRb vector in all cases. Transfection efficiencies were assesed using pcDNA-eGFP. Note that both 5E7 and 16E7, but not 10E7, induced a significant decrease in transiently expressed pRb protein. (<b>B</b>) Immunoblots of Saos2 lysates cotransfected with pcDNA3.1(−)-FLAG (<b>Vector</b>) or with increasing amounts of either pcDNA3.1(−)-5E7FLAG (<b>5E7</b>) or pcDNA3.1(−)-16E7FLAG (<b>16E7</b>) plasmids in the presence of similar amounts of pcDNA-mycRb. Lower panel shows the relative quantification of pRb with respect to eGFP. Note that both 5E7 and 16E7 reduced pRb levels in a dose-dependent manner. (<b>C</b>) Immunoblots of Saos2 lysates cotransfected with either pcDNA3.1(−)-FLAG (<b>Vector</b>), pcDNA3.1(−)-5E7FLAG (<b>5E7</b>), pcDNA3.1(−)-ΔDLFC_5E7FLAG (<b>mut_5E7</b>) pcDNA3.1(−)-ΔDLYC_16E7FLAG (<b>mut_16E7</b>) or pcDNA3.1(−)-16E7FLAG (<b>16E7</b>) plasmids in the presence of pcDNA-mycRb. (<b>D</b>) Immunoblots of Saos2 lysates cotransfected with either pcDNA3.1(−)-FLAG (<b>Vector</b>), pcDNA3.1(−)-5E7FLAG (<b>5E7</b>) or pcDNA3.1(−)-16E7FLAG (<b>16E7</b>) plasmids in the presence of pcDNA-mycRb and increasing concentrations of the proteasome inhibitor MG132. Lower panel shows the relative quantification of pRb with respect to eGFP. Note that MG132 inhibits pRb reduction mediated by 5E7 and 16E7. (<b>E</b>) Foreskin PHK cells were infected with retroviruses generated by pLZRS-E7-IRES-GFP vectors. Immunoblots prepared using specific antibodies showed reduced protein levels of pRb, p107 and p130 upon transduction with E7 from both HPV5 and 16. 5E7 was visualized with an anti-FLAG antibody, and 16E7 with an anti-16E7 antibody. E7 induced the expression of proliferation markers (PCNA and cyclin A), tumor suppressor p53, the proapoptotic effector Bax and the cell cycle inhibitor p21. No increase in the cell cycle inhibitor p16 was observed.</p