Antigen titration using the indicated amounts of DENV-1 (A), -2 (B), -3 (C) and -4 (D) Equad and DENV-2 Ewt (E) proteins with three different DENV, WNV and negative (NEG) human sera.

<p>Measurements were performed in duplicates in at least two independent experiments.</p

A: Expression and purification of DENV-2 Ewt and Equad from Drosophila S2 culture supernatants; supernatant before induction (b.ind.), after 7 days of expression culture (7 d expr.), concentrated via tangential flow (Conc.) and the two step purification with immobilized imidazole affinity (IMAC) and size exclusion chromatography (SEC) were separated on a 10% SDS-PAGE gel under reducing conditions. B: 6 μg of purified DENV1-4 (D1-D4) Equad and DENV-2 Ewt proteins were analyzed with SDS-PAGE. Proteins were stained with Coomassie blue. Size of molecular weight markers in kilo Daltons is indicated on the left.

<p>A: Expression and purification of DENV-2 Ewt and Equad from Drosophila S2 culture supernatants; supernatant before induction (b.ind.), after 7 days of expression culture (7 d expr.), concentrated via tangential flow (Conc.) and the two step purification with immobilized imidazole affinity (IMAC) and size exclusion chromatography (SEC) were separated on a 10% SDS-PAGE gel under reducing conditions. B: 6 μg of purified DENV1-4 (D1-D4) Equad and DENV-2 Ewt proteins were analyzed with SDS-PAGE. Proteins were stained with Coomassie blue. Size of molecular weight markers in kilo Daltons is indicated on the left.</p

Alignment of the amino acid sequences containing the fusion loop domain from E proteins of DENV 1–4, WNV, JEV, YFV, TBEV and the four point mutations of the QUAD proteins.

<p>Amino acids numbering: 1 is start of the E protein.</p

300 ng per well of DENV-2 Ewt (A) and Equad (B) and 160 ng per well of a DENV 1–4 Equad mixture (C) were tested with DENV- WNV- and TBEV- infected and YFV-vaccinated sera compared to negative (NEG) samples in an IgG-ELISA (n = number of individuals).

<p>Bottom and top of the boxes are the first and third quartiles. The median signal is depicted as a line inside the box. Whiskers represent the 9^th and the 91^st percentile. Outliers in B and C are marked with numbers (1–5). Measurements were performed in duplicates in at least two independent experiments.</p

Detection and localization of HPV in human sperm.

<p><b>a</b>. Fluorescence in situ hybridization (fluorescence microscope) for HPV DNA on sperm from a patient with HPV16 in semen. Infected and noninfected sperm are shown. Red: HPV DNA (Texas red); blue: nuclear staining (DAPI). <b>b</b>. Immunofluorescence (confocal fluorescence microscope) for HPV16 capsid protein L1 on sperm from a control (left) and a patient with HPV16 in semen (right). Upper panel, L1 antibody; central panel, L1 antibody and Pisum Sativum (acrosome); lower panel, L1 antibody and Pisum Sativum after induction of the acrosome reaction. Red: HPV16 L1; green: Pisum Sativum; blue: nuclear staining (DAPI). <b>c</b>. PCR for HPV E7 gene from sperm DNA. Lane M: DNA marker (100 bp); 1: negative control (no template); 2: positive control (sperm transfected with recombinant plasmid pIRES2-AcGFP1-E6E7); 3: sperm from a patient with HPV16 in semen; 4: sperm from a control subject.</p

Oocytes penetrated by sperm transfected with HPV E6/E7 express HPV E6/E7 genes.

<p><b>a</b>. PCR for E6 gene from single oocyte penetrated by transfected sperm. Lane M: DNA marker (100 bp); 1: positive control (pIRES2-AcGFP1-E6E7 plasmid); 2: single oocyte penetrated by sperm transfected with recombinant plasmid; 3: negative control (no template); 4: single oocyte penetrated by sperm transfected only with Lipofectamine 2000. <b>b</b>. GFP-E6 expression in oocyte penetrated by control sperm. <b>c</b>. GFP-E6 expression in oocyte penetrated by transfected sperm. <b>d</b>. RT-PCR for E6 gene from single oocyte penetrated by transfected sperm. Lane M: DNA markers (100 bp); 1: negative control (no template for Reverse Transcription); 2: negative control (no template for PCR); 3: single oocyte with green fluorescence penetrated by transfected sperm; 4: single oocyte without green fluorescence penetrated by control sperm.</p

Real-time RT-PCR for <i>CDC73</i> mRNA in transfected and nontransfected HEK293A cells.

<p>Histogram representing the <i>CDC73</i> mRNA fold change in HEK293A cells transfected with Ile60Asn-tGFP-fused parafibromin (<b>Ile60Asn</b>), wild-type-tGFP-fused parafibromin (<b>WT-CDC73</b>) or empty vector (<b>tGFP</b>). Nontransfected cells (<b>Neg ctrl</b>) have been used as a negative control.</p

Western blotting for cyclin D1 and c-myc 48 hours post-transfection in HEK293A cells.

<p>Immunoblotting for cyclin D1 and c-myc in HEK293A cells transfected with Ile60Asn-tGFP-fused parafibromin (<b>Ile60Asn</b>), wild-type-tGFP-fused parafibromin (<b>WT-CDC73</b>) or tGFP-vector (<b>tGFP</b>). Nontransfected HEK293A cells (<b>Neg ctrl</b>) have been used as a negative control.</p

Cell proliferation assay.

<p>Cell counts at three different time points (24, 48 and 72 hours) after transfection of HeLa cells with Ile60Asn-tGFP-fused parafibromin (<b>Ile60Asn</b>), wild-type-tGFP-fused parafibromin (<b>WT-CDC73</b>) or tGFP-vector (<b>tGFP</b>).</p

Western blotting for parafibromin and tGFP 48 hours post-transfection.

<p>Immunoblotting for parafibromin in HEK293A (<b>A</b>) and HeLa cells (<b>B</b>) transfected with Ile60Asn-tGFP-fused parafibromin <b>(Ile60Asn</b>), wild-type-tGFP-fused parafibromin (<b>WT-CDC73</b>) or empty vector (<b>tGFP</b>). Nontransfected cells (<b>Neg ctrl</b>) have been used as a negative control. <b>C</b>. Western blotting with an anti-tGFP antibody has been performed as a control on Ile60Asn and wild-type-parafibromin-transfected cells and on negative control cells. ∼61 kDa: band showing proteolytic degradation of the exogenous parafibromin-tGFP, which generated a protein fragment of approximately the same size of endogenous parafibromin. ∼26 kDa: band corresponding to unconjugated tGFP molecular weight.</p