Journal: mBio

Article Title: An mRNA-encoded scFv antibody targeting the helix-α3 of HPV18 E7 oncoprotein as a novel antiviral strategy

doi: 10.1128/mbio.02627-25

Figure Lengend Snippet: Characterization of EC₅₀ and binding properties of six anti-HPV18E7 mAbs. ( A ) Antibody against HPV18E7 binding ability analysis by ELISA. Recombinant HPV18 E7 protein (1 μg/mL) was coated onto plates. Commercial antibody F-7 as a positive control. ( B ) Competition matrix of six anti-E7 mAbs. Yellow box and black lines indicate mAbs that compete with one another. The value contained in each box is 1 − a given mAb/control across duplicate; a value <0.5 was taken to be negative binding of the detection mAb. Non-competing pairs are shown in green. ( C ) Kinetic parameters for the binding of each antibody to HPV18E7, as determined by surface plasmon resonance analysis. ( D ) Six Anti-E7 mAbs could recognize HPV18 E7 protein in the HPV18-positive HeLa cells by immunofluorescence. Scale bar = 20 um. Commercial antibody F-7 as a positive control. No primary antibody group as a negative control. The rectangular artifacts on the right side of F-7, 14E5, and 17F2 are the result of a technical issue that occurred during file conversion. ( E ) Quantitative analysis of panel D . The signal targeting E7 (the green signal in panel D) was quantitatively analyzed using ImageJ and normalized. Y normalization = mean density (experimental group)/average of mean density (negative control). A pairwise difference analysis was performed between the experimental and control groups using t -test, n = 3, * P < 0.05, ** P < 0.01, and *** P < 0.001. ( F ) Western blot showed that Anti-E7 mAbs could recognize endogenously expressed HPV18 E7 protein in the HPV18-positive HeLa cells. β-actin as the control. This figure represents spliced gel segments combined for imaging.

Article Snippet: The primary antibodies were monoclonal antibodies (2E8, 5C5, 8A7, 9A5, 14E5, and 17F2) and anti-HPV18E7 (Santa Cruz, no. sc-365035; working concentration, 4 μg/mL), anti-HA-HRP (Abcam, no. ab128131, 1:1,000), anti-β-actin-HRP (ProteinTech, HRP-60008, 1:5,000), anti-Rb (CST, no.9309S, 1:100), anti- phospho-Rb (CST, no.8516S, 1:100).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Positive Control, Control, SPR Assay, Immunofluorescence, Negative Control, Western Blot, Imaging

Journal: Oncology Reports

Article Title: Crosstalk between histone modification and DNA methylation orchestrates the epigenetic regulation of the costimulatory factors, Tim-3 and galectin-9, in cervical cancer

doi: 10.3892/or.2019.7388

Figure Lengend Snippet: Primer sequences.

Article Snippet: The antibodies used were as follows: Rabbit polyclonal antibody against human Tim-3 (1:500 dilution, ab185703; Abcam), rabbit polyclonal antibody against human galectin-9 (1:100 dilution, ab123712; Abcam), mouse monoclonal antibody against human DNMT3A (1:200 dilution, sc-365769; Santa Cruz Biotechnology), rabbit monoclonal antibody against human EZH2 (1:1,000 dilution, 5246; Cell Signaling Technology), rabbit monoclonal antibody against human H3K27me3 (1:1,000 dilution, 9733; Cell Signaling Technology), mouse monoclonal antibody against HPV18 E6 (1:250 dilution, NB100-2729; Novus Biologicals) and mouse monoclonal antibody against HPV18 E7 (1:250 dilution, NB110-17215; Novus Biologicals), β-actin mouse monoclonal antibody (1:500 dilution, 60008-1-lg; Proteintech).

Techniques: Sequencing, Control

Journal: Oncology Reports

Article Title: Crosstalk between histone modification and DNA methylation orchestrates the epigenetic regulation of the costimulatory factors, Tim-3 and galectin-9, in cervical cancer

doi: 10.3892/or.2019.7388

Figure Lengend Snippet: Function of HPV18 oncoproteins E6/E7 in the regulation of Tim-3/galectin-9 and DNMT3A expression in human cervical cancer cells. (A) and (B) mRNA levels in of HPV18 E6/E7 -overexpressing C33A cells and of HPV18 E6/E7 -knockdown HeLa cells. (C) and (D) C33A cells were transfected with 18E6/E7/E6/7 lentiviral vector, and HeLa cells were transfected with 18E6/E7/E6/7-specific siRNAs. Western blot analysis was then used to detect DNMT3A expression. (E) Co-IP assay was used to analyzed the incorporation between DNMT3A and HPV18 E6/E7. (F and G) The methylation level of the HAVCR2/LGALS9 promoters was monitored by MS-PCR. Gray level analysis of HAVCR2/LGALS9 methylation levels in cervical cancer cells (M, methylated; U, unmethylated); the methylated and unmethylated levels were quantified as M/M+U% and U/M+U%, respectively. (H and I) C33A cells were transfected with 18E6/E7/E6/7 lentiviral vector, and HeLa cells were transfected with 18E6/E7/E6/7-specific siRNAs. The expression of Tim-3/galectin-9 was determined by western blot analysis. **P<0.01.

Article Snippet: The antibodies used were as follows: Rabbit polyclonal antibody against human Tim-3 (1:500 dilution, ab185703; Abcam), rabbit polyclonal antibody against human galectin-9 (1:100 dilution, ab123712; Abcam), mouse monoclonal antibody against human DNMT3A (1:200 dilution, sc-365769; Santa Cruz Biotechnology), rabbit monoclonal antibody against human EZH2 (1:1,000 dilution, 5246; Cell Signaling Technology), rabbit monoclonal antibody against human H3K27me3 (1:1,000 dilution, 9733; Cell Signaling Technology), mouse monoclonal antibody against HPV18 E6 (1:250 dilution, NB100-2729; Novus Biologicals) and mouse monoclonal antibody against HPV18 E7 (1:250 dilution, NB110-17215; Novus Biologicals), β-actin mouse monoclonal antibody (1:500 dilution, 60008-1-lg; Proteintech).

Techniques: Expressing, Knockdown, Transfection, Plasmid Preparation, Western Blot, Co-Immunoprecipitation Assay, Methylation

Journal: Oncology Reports

Article Title: Crosstalk between histone modification and DNA methylation orchestrates the epigenetic regulation of the costimulatory factors, Tim-3 and galectin-9, in cervical cancer

doi: 10.3892/or.2019.7388

Figure Lengend Snippet: HPV18 oncoprotein E6/E7 alters the expression EZH2 and H3K27me3 in cervical cancer cells. (A and B) C33A cells were transfected with 18E6/E7/E6/7 lentiviral vector and HeLa cells were transfected with 18E6/E7/E6/7 specific siRNAs and western blot analysis was then used to detect EZH2 and H3K27me3 expression. (C) EZH2 and H3K27me3 expression in HeLa and C33A cells detected by western blot analysis. (D) Western blot analysis of the HPV18 E6/E7 protein level in HeLa cells following transfection with siRNAs against EZH2. (E) Co-IP assay was used to analyzed the incorporation between EZH2 and HPV18 E6/E7. (F) Schematic representation of the 4 regions of the EZH2 promoter amplified in the chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) experiment. (G and H) Chromatin was cross-linked, fragmented and immunoprecipitated with either IgG (mock) or anti-E2F-1 and anti-FOXM1 ChIP-grade antibody and the purified DNA was used to amplify with respective primer pairs for indicated four regions in the EZH2 promoters in qPCR. The enrichment of E2F-1 and FOXM1 on EZH2 promoter relative to IgG in HeLa cells, H3 against RPL30 used as positive control. (I and J) Chromatin was cross-linked, fragmented and immunoprecipitated with either IgG (mock) or anti-E2F-1 and anti-FOXM1 ChIP-grade antibody and the purified DNA was used to amplify with respective primer pairs for the indicated 4 regions in the DNMT3A promoters in qPCR. The enrichment of E2F-1 and FOXM1 on DNMT3A promoter relative to IgG in HeLa cells, H3 against RPL30 used as a positive control. **P<0.01; ns, not significant.

Article Snippet: The antibodies used were as follows: Rabbit polyclonal antibody against human Tim-3 (1:500 dilution, ab185703; Abcam), rabbit polyclonal antibody against human galectin-9 (1:100 dilution, ab123712; Abcam), mouse monoclonal antibody against human DNMT3A (1:200 dilution, sc-365769; Santa Cruz Biotechnology), rabbit monoclonal antibody against human EZH2 (1:1,000 dilution, 5246; Cell Signaling Technology), rabbit monoclonal antibody against human H3K27me3 (1:1,000 dilution, 9733; Cell Signaling Technology), mouse monoclonal antibody against HPV18 E6 (1:250 dilution, NB100-2729; Novus Biologicals) and mouse monoclonal antibody against HPV18 E7 (1:250 dilution, NB110-17215; Novus Biologicals), β-actin mouse monoclonal antibody (1:500 dilution, 60008-1-lg; Proteintech).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Co-Immunoprecipitation Assay, Amplification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification, Positive Control

Journal: Oncology Reports

Article Title: Crosstalk between histone modification and DNA methylation orchestrates the epigenetic regulation of the costimulatory factors, Tim-3 and galectin-9, in cervical cancer

doi: 10.3892/or.2019.7388

Figure Lengend Snippet: The HPV18 pathway regulates Tim-3/galectin-9 through EZH2-H2K27me3-DNMT3A. HPV18 E6 and E7 regulates EZH2 through direct binding of FOXM1 and E2F-1 the EZH2 promoter in order to activate EZH2 expression; in turn, H3K27me3 is upregulated. EZH2 and H3K27me3 directly interact with the DNMT3A promoter to downregulate its expression. When DNMT3A is decreased, the HAVCR2/LGALS9 promoter region is also demethylated and Tim-3/galectin-9 expression is upregulated.

Article Snippet: The antibodies used were as follows: Rabbit polyclonal antibody against human Tim-3 (1:500 dilution, ab185703; Abcam), rabbit polyclonal antibody against human galectin-9 (1:100 dilution, ab123712; Abcam), mouse monoclonal antibody against human DNMT3A (1:200 dilution, sc-365769; Santa Cruz Biotechnology), rabbit monoclonal antibody against human EZH2 (1:1,000 dilution, 5246; Cell Signaling Technology), rabbit monoclonal antibody against human H3K27me3 (1:1,000 dilution, 9733; Cell Signaling Technology), mouse monoclonal antibody against HPV18 E6 (1:250 dilution, NB100-2729; Novus Biologicals) and mouse monoclonal antibody against HPV18 E7 (1:250 dilution, NB110-17215; Novus Biologicals), β-actin mouse monoclonal antibody (1:500 dilution, 60008-1-lg; Proteintech).

Techniques: Binding Assay, Expressing

Journal: Brazilian Journal of Microbiology

Article Title: A fluorometric hybridization assay for detecting and genotyping high-risk human papillomavirus 16 and 18 in archival tissues of cervical specimens

doi: 10.1007/s42770-019-00137-8

Figure Lengend Snippet: HPV types 16 and 18 prevalence in patients with different types of cervical dysplasia

Article Snippet: Source of plasmids DNA DNA plasmids containing the entire genomes of HPV16 (ATCC 45113D) and HPV18 (ATCC 45152D) were purchased from American Type Culture Collection (ATCC, Manassas, USA) and used as the control to develop efficient hybridization methods.

Techniques:

Journal: Brazilian Journal of Microbiology

Article Title: A fluorometric hybridization assay for detecting and genotyping high-risk human papillomavirus 16 and 18 in archival tissues of cervical specimens

doi: 10.1007/s42770-019-00137-8

Figure Lengend Snippet: HPV types 16 and 18 overall detection via three different methods

Article Snippet: Source of plasmids DNA DNA plasmids containing the entire genomes of HPV16 (ATCC 45113D) and HPV18 (ATCC 45152D) were purchased from American Type Culture Collection (ATCC, Manassas, USA) and used as the control to develop efficient hybridization methods.

Techniques:

Journal: Brazilian Journal of Microbiology

Article Title: A fluorometric hybridization assay for detecting and genotyping high-risk human papillomavirus 16 and 18 in archival tissues of cervical specimens

doi: 10.1007/s42770-019-00137-8

Figure Lengend Snippet: Comparison between the results obtained for the identification of HPV 16 and 18, with TSnPCR and with MCHA

Article Snippet: Source of plasmids DNA DNA plasmids containing the entire genomes of HPV16 (ATCC 45113D) and HPV18 (ATCC 45152D) were purchased from American Type Culture Collection (ATCC, Manassas, USA) and used as the control to develop efficient hybridization methods.

Techniques: Comparison

Journal: Brazilian Journal of Microbiology

Article Title: A fluorometric hybridization assay for detecting and genotyping high-risk human papillomavirus 16 and 18 in archival tissues of cervical specimens

doi: 10.1007/s42770-019-00137-8

Figure Lengend Snippet: Comparison of the results obtained for the identification of HPV 16 and 18, with TSnPCR and with MFHA

Article Snippet: Source of plasmids DNA DNA plasmids containing the entire genomes of HPV16 (ATCC 45113D) and HPV18 (ATCC 45152D) were purchased from American Type Culture Collection (ATCC, Manassas, USA) and used as the control to develop efficient hybridization methods.

Techniques: Comparison

Journal: Antiviral Research

Article Title: Alkyl-imino sugars inhibit the pro-oncogenic ion channel function of human papillomavirus (HPV) E5

doi: 10.1016/j.antiviral.2018.08.005

Figure Lengend Snippet: Viroporin inhibitors prevent activation of MAPK signalling by E5 proteins. (A) Hydrophobicity plot of HPV16 and HPV18 E5 using the Kyte Doolittle programme. (B) Molecular modelling of full-length HPV18 E5 hexamer. Models of the E5 channel complex were generated by using Maestro as described in materials and methods. (C) Merocyanine assay performed on C33A cells transfected with GFP or GFP-18E5. Cells were analysed on BD Fortessa cytometer and histograms were created using FlowJo software. C33A cells were transfected with GFP-18E5 (D), GFP-16E5 (E) or GFP-31E5 (F) in parallel with a GFP control. Cells were then incubated with Rimantadine (100 μM) and N N-DNJ (100 μM) for 36 h and lysates probed with antibodies against cyclin B1, phosphorylated and total forms of ERK1/2 and GFP. GAPDH served as a loading control. Representative blots are shown from at least three independent biological repeats. (G) Lysates from HeLa cells incubated with Rimantadine (100 μM) and N N-DNJ (100 μM) for 48 h were probed with antibodies against the total and phosphorylated forms of ERK1/2, cyclin B1, HPV E6 and E7 oncoproteins and the loading control GAPDH. Representative blots are shown from at least three independent biological repeats. (H) HPV18 E5 activates a cyclin B1 reporter plasmid. C33A cells were co-transfected with the CCNB1 reporter and GFP or GFP-18E5 then subsequently treated with EGF, rimantadine or N N-DNJ, before cells were lysed in Passive Lysis Buffer (Promega) and analysed using a dual luciferase system (Promega). Data are from 3 independent biological repeats and presented as fold change compared to untreated GFP. Error bars are ± Standard deviation. *p < 0.05, **p < 0.01.

Article Snippet: Total protein was extracted from keratinocytes in lysis buffer ( ) and resolved by SDS-PAGE (10–15% Tris-Glycine), transferred onto Hybond nitrocellulose membrane (Amersham biosciences) and probed with antibodies specific for cyclin B1 (H-433, Santa Cruz Biotechnology), HPV18 E6 (G-7, Santa Cruz Biotechnology), HPV18 E7 (8E2, Abcam (ab100953), phospho-ERK1/2 (43705, Cell Signalling Technology), GFP (sc-9996, Santa Cruz Biotechnology) and GAPDH (G-9, Santa Cruz Biotechnology).

Techniques: Activation Assay, Generated, Transfection, Cytometry, Software, Control, Incubation, Plasmid Preparation, Lysis, Luciferase, Standard Deviation

Journal: Antiviral Research

Article Title: Alkyl-imino sugars inhibit the pro-oncogenic ion channel function of human papillomavirus (HPV) E5

doi: 10.1016/j.antiviral.2018.08.005

Figure Lengend Snippet: Viroporin inhibitors prevent HPV18 E5 mediated mitogenic signalling in the context of productive HPV18 infection . Primary human keratinocytes containing wild type or E5KO HPV18 genomes were differentiated in high calcium media for 48 h in the presence of absence of (A) rimantadine (50 μM) and (B) N N-DNJ (50 μM) and cell lysates probed with antibodies against cyclin B1, the phosphorylated and total forms of ERK1/2, Filaggrin and the GAPDH loading control. Representative blots are shown from at least three independent biological repeats. Graphs represent densitometry analysis of cyclin B1 and phosphorylated ERK levels at the 48 h time point for untreated and drug-treated wild type and the E5KO cells. Graphs represent data from three independent repeats. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Total protein was extracted from keratinocytes in lysis buffer ( ) and resolved by SDS-PAGE (10–15% Tris-Glycine), transferred onto Hybond nitrocellulose membrane (Amersham biosciences) and probed with antibodies specific for cyclin B1 (H-433, Santa Cruz Biotechnology), HPV18 E6 (G-7, Santa Cruz Biotechnology), HPV18 E7 (8E2, Abcam (ab100953), phospho-ERK1/2 (43705, Cell Signalling Technology), GFP (sc-9996, Santa Cruz Biotechnology) and GAPDH (G-9, Santa Cruz Biotechnology).

Techniques: Infection, Control