Journal: PLoS ONE

Article Title: Fused toes homolog, a potential molecular regulator of human papillomavirus type 16 E6 and E7 oncoproteins in cervical cancer

doi: 10.1371/journal.pone.0266532

Figure Lengend Snippet: Expression of HPV16 E6/E7 and FTS in the cell lines (A). The expression levels of E6 and E7 in the control and FTS silenced group (B). The tumor suppressor proteins p53 and pRb were determined by western blot in the control and FTS silenced group (C). Actin was used as a loading control. The bar graphs adjacent to each blot represents mean ± standard error of the mean (SEM) of densitometric analyses performed on the blots obtained from three independent experiments. The data has been normalized to control. Differences between the groups were calculated by unpaired, parametric, two-tailed T-tests. p-values of statistically significant differences are shown. * p <0.05; ** p < 0.005; *** p < 0.001.

Article Snippet: This was followed by overnight incubation at 4°C with anti-FTS (cat. no. sc-134343, Santa Cruz) and either anti-E6 (cat. no. sc-1584, Santa Cruz) or anti-E7 (cat. no. bs-10446R, Bioss, USA) antibodies.

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: PLoS ONE

Article Title: Fused toes homolog, a potential molecular regulator of human papillomavirus type 16 E6 and E7 oncoproteins in cervical cancer

doi: 10.1371/journal.pone.0266532

Figure Lengend Snippet: The levels of tumor suppressor proteins p53, pRb, and cell survival marker Akt and pAKT were determined by western blot in the control and E6 (A) or E7 (B) silenced groups. Actin was used as a loading control. The bar graphs adjacent to each blot represents mean ± SEM of densitometric analyses performed on the blots obtained from three independent experiments. The data has been normalized to control. Differences between the groups were calculated by unpaired, parametric, two-tailed T-tests. p-values of statistically significant differences are shown. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p <0.0001.

Article Snippet: This was followed by overnight incubation at 4°C with anti-FTS (cat. no. sc-134343, Santa Cruz) and either anti-E6 (cat. no. sc-1584, Santa Cruz) or anti-E7 (cat. no. bs-10446R, Bioss, USA) antibodies.

Techniques: Marker, Western Blot, Two Tailed Test

Journal: PLoS ONE

Article Title: Fused toes homolog, a potential molecular regulator of human papillomavirus type 16 E6 and E7 oncoproteins in cervical cancer

doi: 10.1371/journal.pone.0266532

Figure Lengend Snippet: mRNA expression of HPV16 E6/E7 was evaluated in FTS intact/silenced group (A). Further, FTS mRNA levels were also verified in HPV16 E6/E7 intact/silenced group (B). GAPDH was used as positive control.

Article Snippet: This was followed by overnight incubation at 4°C with anti-FTS (cat. no. sc-134343, Santa Cruz) and either anti-E6 (cat. no. sc-1584, Santa Cruz) or anti-E7 (cat. no. bs-10446R, Bioss, USA) antibodies.

Techniques: Expressing, Positive Control

Journal: PLoS ONE

Article Title: Fused toes homolog, a potential molecular regulator of human papillomavirus type 16 E6 and E7 oncoproteins in cervical cancer

doi: 10.1371/journal.pone.0266532

Figure Lengend Snippet: Expression of FTS and E6 in CaSki cells (A), FTS and E7 in CaSki cells (B), FTS and E6 in SiHa cells (C), and FTS and E7 in SiHa cells (D). FTS was stained with Alexa Fluor 488 (green), while E6 or E7 was stained with Alexa Fluor 594 (red). The nuclei were counter-stained with DAPI. The Pearson’s coefficient for FTS co-localization with E6/E7 revealed that the co-localization ranged between 0.834–0.938.

Article Snippet: This was followed by overnight incubation at 4°C with anti-FTS (cat. no. sc-134343, Santa Cruz) and either anti-E6 (cat. no. sc-1584, Santa Cruz) or anti-E7 (cat. no. bs-10446R, Bioss, USA) antibodies.

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Fused toes homolog, a potential molecular regulator of human papillomavirus type 16 E6 and E7 oncoproteins in cervical cancer

doi: 10.1371/journal.pone.0266532

Figure Lengend Snippet: IP shows binding between FTS and E6/E7 in CaSki and SiHa cells (A), expression and co-localization of FTS (green color) and HPV16 E6 (brown color) in cervical cancer tissues (B), and expression and co-localization of FTS (green color) and HPV16 E7 (red color) in cervical cancer tissues (C). The co-localized area with maximum intensity is enclosed by a black box. The images of IHC shown are representative of 10 cervical cancer tissues. Black bar in (B) and (C) corresponds to 20 μm.

Article Snippet: This was followed by overnight incubation at 4°C with anti-FTS (cat. no. sc-134343, Santa Cruz) and either anti-E6 (cat. no. sc-1584, Santa Cruz) or anti-E7 (cat. no. bs-10446R, Bioss, USA) antibodies.

Techniques: Binding Assay, Expressing

Journal: PLoS ONE

Article Title: Fused toes homolog, a potential molecular regulator of human papillomavirus type 16 E6 and E7 oncoproteins in cervical cancer

doi: 10.1371/journal.pone.0266532

Figure Lengend Snippet: Docking conformation of the HPV16 E6/FTS (A) and E7/FTS (D), interacting residues of the HPV16 E6/FTS (B) and E7/FTS (E) protein-protein complex depicted in sticks representation, three intermolecular H-bonds present in the interface of the complex illustrated by yellow dotted lines, HPV16 E6/FTS (C), and E7/FTS (F). In all the cases, the HPV16 E6/E7 and FTS are represented in cyan and magenta colors, respectively.

Article Snippet: This was followed by overnight incubation at 4°C with anti-FTS (cat. no. sc-134343, Santa Cruz) and either anti-E6 (cat. no. sc-1584, Santa Cruz) or anti-E7 (cat. no. bs-10446R, Bioss, USA) antibodies.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel Affibody Molecules Targeting the HPV16 E6 Oncoprotein Inhibited the Proliferation of Cervical Cancer Cells

doi: 10.3389/fcell.2021.677867

Figure Lengend Snippet: Immunofluorescence and immunohistochemical staining analyses of the binding specificity of Z HPV16E6 affibodies to HPV16 E6. (A) Representative images showing TC-1, CaSki cells (HPV16 positive), HeLa229 (HPV18 positive), and C666-1 cells (HPV negative) stained with Z HPV16E6 1115, Z HPV16E6 1171, and Z HPV16E6 1235. The Z WT affibody was used as a negative control. The affibody molecule stain is shown in green, while the nuclear stain (PI) is shown in red; magnification at ×400. (B) Representative image of HPV16-positive cervical cancer sections and HPV-negative normal human sections by hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) staining with Z HPV16E6 affibodies. Sections from HPV16-positive cervical cancer sections (upper panel) and HPV-negative normal human sections (lower panel) were labeled with Z HPV16E6 affibodies. Polyclonal HPV16 E6 antibody was used as a positive control. Z WT and PBS were used as negative controls. Magnification at ×400.

Article Snippet: Human HPV-positive CaSki (HPV16), HeLa229 (HPV18 cell line, applied as HPV16 negative control) cells and nasopharyngeal carcinoma C666-1 cells (HPV negative control cell line) were bought from the ATCC (American Type Culture Collection) and cultured according to the supplier’s instructions.

Techniques: Immunofluorescence, Immunohistochemical staining, Staining, Binding Assay, Negative Control, Immunohistochemistry, Labeling, Positive Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel Affibody Molecules Targeting the HPV16 E6 Oncoprotein Inhibited the Proliferation of Cervical Cancer Cells

doi: 10.3389/fcell.2021.677867

Figure Lengend Snippet: In vivo testing of the target binding ability of Z HPV16E6 affibodies to tumor tissues. Tumor imaging in nude mice bearing TC-1 (A) or HeLa229 (C) xenografts (arrows) by using fluorescence-conjugated affibody molecules. NIR-based imaging was executed at various time points pi with DyLight 755-conjugated Z HPV16E6 affibodies and used DyLight 755-conjugated Z WT affibody as a negative control. Tumor-to-skin ratios were calculated at different time points pi of the indicated agents in nude mice bearing TC-1 (B) and HeLa229 (D) xenografts. Data are shown as the mean ± SD of triplicates.

Article Snippet: Human HPV-positive CaSki (HPV16), HeLa229 (HPV18 cell line, applied as HPV16 negative control) cells and nasopharyngeal carcinoma C666-1 cells (HPV negative control cell line) were bought from the ATCC (American Type Culture Collection) and cultured according to the supplier’s instructions.

Techniques: In Vivo, Binding Assay, Imaging, Fluorescence, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel Affibody Molecules Targeting the HPV16 E6 Oncoprotein Inhibited the Proliferation of Cervical Cancer Cells

doi: 10.3389/fcell.2021.677867

Figure Lengend Snippet: Z HPV16E6 1235 inhibits the proliferation of HPV16-positive cell lines by binding to and blocking the intracellular activity of the HPV16 E6 oncoprotein. (A) p53 was up-regulated in a dose-dependent manner by treatment with Z HPV16E6 1235 in CaSki cells. (B) The effect of Z HPV16E6 1235 on the intracellular binding between HPV16 E6 and p53 was assessed by immunoprecipitating the E6/E6AP/p53 trimeric complex using an anti-p53 antibody bound to Protein A/G agarose beads from CaSki cells treated for 24 h. A parallel negative control assay was run for each group by incubating cell lysates with control IgG. The bar graph represents the amount of E6 bound to the relative amount of immunoprecipitated p53 after the quantification of p53 and E6 protein bands with ImageJ software. Data are presented as the mean ± SD of three independent experiments. ** P < 0.01. (C) CaSki cells were treated with 10 μM Z HPV16E6 1235 for the indicated periods and the expression of p53 target genes, including PUMA, BAX and p21, was evaluated by Western blotting. Cells without any treatment (Mock) or treated with 10 μM Z WT for 48 h were used as negative controls. GAPDH served as an internal reference standard. (D) The effects of Z HPV16E6 1235 and Z HPV16E7 384 alone or in combination on the viability of HPV16-positive cancer cells (TC-1, CaSki), HPV18-positive cervical cancer cells (HeLa229) and HPV-negative cancer cells (C666-1) were assessed by CCK-8 assay after 48 h of treatment with the indicated concentrations; these cells were compared to Z WT -treated cells. Data are shown as the mean ± SD of three independent experiments. (E–F) Colony formation assays of HPV16-positive TC-1 and CaSki cells or HPV18-positive HeLa229 cells following treatment with 2.5 μM test affibody molecules for 14 days. The Z WT affibody and medium groups were set as controls. ** P < 0.01, *** P < 0.001 vs. the control group. # P < 0.05 vs. the Z HPV16E6 1235 or Z HPV16E7 384 alone treatment group.

Article Snippet: Human HPV-positive CaSki (HPV16), HeLa229 (HPV18 cell line, applied as HPV16 negative control) cells and nasopharyngeal carcinoma C666-1 cells (HPV negative control cell line) were bought from the ATCC (American Type Culture Collection) and cultured according to the supplier’s instructions.

Techniques: Binding Assay, Blocking Assay, Activity Assay, Negative Control, Control, Immunoprecipitation, Software, Expressing, Western Blot, CCK-8 Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel Affibody Molecules Targeting the HPV16 E6 Oncoprotein Inhibited the Proliferation of Cervical Cancer Cells

doi: 10.3389/fcell.2021.677867

Figure Lengend Snippet: Effect of Z HPV16E6 1235 in combination with Z HPV16E7 384 on the cell cycle, apoptosis, and cellular senescence. TC-1 and CaSki cells were treated with 10 μM of test affibody molecules for 24 h and analyzed by flow cytometry assay for the cell cycle (A,B) and apoptosis (C,D) with PI and Annexin V/PI. Data are presented as the mean ± SD in three independent experiments. (E,F) SA-β-gal staining of TC-1 and CaSki cells treated with 10 μM test affibody molecules for 24 h. Representative images taken using a bright-field inverted microscope (100 × magnification) are shown. In all panels, TC-1 and CaSki (HPV16-positive) cells treated with Z WT and HeLa229 (HPV18-positive) treated with selected affibodies were used as negative controls. Significance: * P < 0.05, ** P < 0.01 vs. the control group. # P < 0.05 vs. the Z HPV16E6 1235 or Z HPV16E7 384 alone treatment group.

Article Snippet: Human HPV-positive CaSki (HPV16), HeLa229 (HPV18 cell line, applied as HPV16 negative control) cells and nasopharyngeal carcinoma C666-1 cells (HPV negative control cell line) were bought from the ATCC (American Type Culture Collection) and cultured according to the supplier’s instructions.

Techniques: Flow Cytometry, Staining, Inverted Microscopy, Control

Journal: Oncotarget

Article Title: Generation of affibody molecules specific for HPV16 E7 recognition

doi: 10.18632/oncotarget.12174

Figure Lengend Snippet: Sensorgrams obtained after injection of the Z HPV16E7 127, Z HPV16E7 301, Z HPV16E7 384 and Z HPV16E7 745 affibody molecules with different concentrations over a sensor chip flow-cell surface containing HPV16 E7 protein. The wild Z wt molecule was set as control. All samples were run in duplicates, and the response obtained from an activated and deactivated reference surface has been subtracted from all curves.

Article Snippet: Human cervical cancer cells, including HPV16 positive SiHa cell (ATCC: HTB-35) and CaSki cell (ATCC: CRL-1550), HPV18 positive HeLa (ATCC: CCL-2) and HPV negative melanoma cell of A375 (ATCC: CRL-1619) were cultured on multi-well slides at 37°C.

Techniques: Injection, Control

Journal: Oncotarget

Article Title: Generation of affibody molecules specific for HPV16 E7 recognition

doi: 10.18632/oncotarget.12174

Figure Lengend Snippet: Combined with FITC-conjugated goat anti-mouse IgG, His-tag McAb which recognized His-tag of affibody molecules was used to detect the affibody molecules. The brightly dotted or crumby fluorescence signals were observed in both perinuclear area and nuclear membrane (200×). HPV-negative A375 cell and HPV18 positive HeLa cell were used as controls and did not show any fluorescence signal when the cells were stained with the 4 selected HPV16 E7-binding affibodies. Nuclei were counterstained with PI staining (red).

Article Snippet: Human cervical cancer cells, including HPV16 positive SiHa cell (ATCC: HTB-35) and CaSki cell (ATCC: CRL-1550), HPV18 positive HeLa (ATCC: CCL-2) and HPV negative melanoma cell of A375 (ATCC: CRL-1619) were cultured on multi-well slides at 37°C.

Techniques: Fluorescence, Membrane, Staining, Binding Assay

Journal: Oncotarget

Article Title: Generation of affibody molecules specific for HPV16 E7 recognition

doi: 10.18632/oncotarget.12174

Figure Lengend Snippet: A. Fluorescence imaging of tumor ex vivo at 24 h p.i. of Dylight755- conjugated Z HPV16E7 384 affibody injection. In each image: 1, tumor; 2, skin; 3, spleen; liver; 5, heart; 6, kidney; 7, brain; 8, lung; 9, muscle; 10, intestine. B. The relative fluorescence signal intensity of ex vivo imaging in the tumors and major organs at 24 h p.i. of Dylight755-labelling Z HPV16 E7 384 affibody injection. The value= fluorescence signal intensity in the tumors and other major organs / fluorescence signal intensity in the corresponding muscle tissue.

Article Snippet: Human cervical cancer cells, including HPV16 positive SiHa cell (ATCC: HTB-35) and CaSki cell (ATCC: CRL-1550), HPV18 positive HeLa (ATCC: CCL-2) and HPV negative melanoma cell of A375 (ATCC: CRL-1619) were cultured on multi-well slides at 37°C.

Techniques: Fluorescence, Imaging, Ex Vivo, Injection

Journal: Cancer letters

Article Title: A small molecule targeting the interaction between human papillomavirus E7 oncoprotein and cellular phosphatase PTPN14 exerts antitumoral activity in cervical cancer cells.

doi: 10.1016/j.canlet.2023.216331

Figure Lengend Snippet: Fig. 2. Effects of hit compounds on PTPN14/E7 binding. (A) Recombinant PTPN14-PTP domain was used to coat microtiter plates and increasing amounts of HPV16 E7, or another protein (HPV16 E6N) as a control, were added. Samples were then incubated with the appropriate primary and secondary HRP- conjugated antibody and bound proteins were detec ted by spectrophotometric measurements at 450 nm. (B) Increasing amounts of PTPN14-PTP were added together with a fixed amount of E7 to wells coated with PTPN14-PTP. Binding of E7 was detected as described above. The absorbance at 450 nm measured in the presence of the competitor is plotted. (C) Increasing amounts of E7 α1-peptide or PB11–15 pep tide as a negative control were added together with a fixed amount of E7 to wells coated with PTPN14-PTP. Binding of E7 was detected as described above. The percentage of the E7/PTPN14 binding measured in the presence of inhibitors with respect to that measured in the absence of inhibitors is plotted. (D) The ability of the 46 hit compounds to disrupt the E7/ PTPN14 interaction was assessed by the ELISA-based E7/PTPN14 interaction assay. The inhibitory con centration at half-maximal response (IC50) values are reported. The E7 α1-peptide was used as a positive control, while the anti-HPV E6 compound Cpd12, the anti-influenza compound 54 and PB11-15 peptide were used as negative controls. In all panels, data shown represent the mean ± SD of three independent experiments.

Article Snippet: Interactions were detected using either an anti-HPV16 E7 antibody (NM2, Santa Cruz) or an anti-HPV16 E6 antibody (N-17, Santa Cruz) followed by the incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody and the chromogenic 3,3′,5,5’ tetramethylbenzidine (TMB) substrate for spectrophotometric measurements.

Techniques: Binding Assay, Recombinant, Control, Incubation, Negative Control, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Cancer letters

Article Title: A small molecule targeting the interaction between human papillomavirus E7 oncoprotein and cellular phosphatase PTPN14 exerts antitumoral activity in cervical cancer cells.

doi: 10.1016/j.canlet.2023.216331

Figure Lengend Snippet: Fig. 5. Compound 20 is active against different HR-HPV genotypes. (A) Alignment of the C-terminal amino acids of high-risk HPV E7 protein sequences. Red arrows indicate conserved E7 residues crucial for the binding to PTPN14. Top numbering refers to HPV18 E7 sequence. The symbols indicate the residue conservation according to the color-coded structural similarity of the amino acids. ".", weak conservation; ":", strong conservation; "*", full conservation. Alignment generated with ClustalX. (B) The effects of compound 20 on the viability of HPV-positive MS751 (HPV45) and ME180 (HPV68) cells were tested by MTT assay after 48 h of treatment. Data on HeLa (HPV18), CaSki (HPV16), and C33A (HPV-negative) are also shown for comparison. IC50s represent the concentrations causing a decrease of 50% in cell viability. Data are the means ± SD derived from at least three independent experiments in duplicate. (C) Western blot analysis of PTPN14 levels in HPV-positive CaSki (HPV16), MS751 (HPV45), and ME180 (HPV68) cells upon treatment with different concentrations of compound 20. β-tubulin was used as a loading control. (D) Predicted binding mode of compound 20 with the CR3 domain of E7 from different HR-HPV genotypes: upper-left, docking pose of compound 20 on HPV18 E7 (PDB ID: 6IWD); upper-right, ligand-based homology model of compound 20 on HPV16 E7; lower-left, ligand-based homology model of compound 20 on HPV45 E7; lower-right, ligand-based homology model of compound 20 on HPV68 E7. Hydrogen bonds are depicted as dashed lines. Protein electrostatic properties are represented in either red (negatively charged) or blue (posi tively charged).

Article Snippet: Interactions were detected using either an anti-HPV16 E7 antibody (NM2, Santa Cruz) or an anti-HPV16 E6 antibody (N-17, Santa Cruz) followed by the incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody and the chromogenic 3,3′,5,5’ tetramethylbenzidine (TMB) substrate for spectrophotometric measurements.

Techniques: Binding Assay, Sequencing, Residue, Generated, MTT Assay, Comparison, Derivative Assay, Western Blot, Control

Journal: Inflammopharmacology

Article Title: Plant-based chimeric HPV-virus-like particles bearing amyloid-β epitopes elicit antibodies able to recognize amyloid plaques in APP-tg mouse and Alzheimer's disease brains.

doi: 10.1007/s10787-017-0408-2

Figure Lengend Snippet: Fig. 4 Immune sera obtained from chimeric VLP-immunized C57Bl/6 mice were tested by Western blot (a). From left to right, the Ab1–42, pyroGluAb3–42 and pyroGluAb11–42 peptides were loaded in triplets in a 4–12% gradient gel. The gel was transferred onto PVDF membrane which was cut in triplets. Each triplet was incubated with sera obtained from VLP-immunized mice or BAM90.1 monoclonal antibody (recognizing the Ab12–28 epitope) as positive control. IgG isotypes found in immune sera obtained from VLP–L1a- and VLP–L1b-immunized mice were determined by ELISA (b). IgG1 and IgG2b antibodies, indicative of anti-inflammatory response, were found, while IgG2c, indicative of pro- inflammatory Th1 response, was not detected. Mice inoculated with PBS and immunized with VLP–L1 wt did not produce anti-Ab Abs. Each bar represents the mean OD values of three different experiments

Article Snippet: Membranes were incubated with mouse anti-L1 antibodies (Santa Cruz Biotechnology, IncTM Catalog Number sc-53324) at 1:2000 dilution.

Techniques: Western Blot, Membrane, Incubation, Positive Control, Enzyme-linked Immunosorbent Assay