hpv 16 e6 e7  (ATCC)

Journal: bioRxiv

Article Title: CKII-Phosphorylated HPV-16 E7 Disrupts Planar Cell Polarity by Recruiting Vangl1 Phospho-E7 Hijacks Vangl1 Trafficking

doi: 10.64898/2026.02.25.707969

Figure Lengend Snippet: (a) Schematic of the HPV-16 E7 CR2 region showing the Casein Kinase II (CKII) phosphorylation site at serines 31 and 32/33. (b) Sequences of biotin-tagged peptides corresponding to the HPV-16 E7 CR2 region, synthesized in phosphorylated and non-phosphorylated forms and used as bait in peptide pulldown assays. (c) Mass spectrometry analysis of HaCaT lysates pulled down with phosphorylated or non-phosphorylated E7 peptides. The phosphorylated peptide enriched known E7 interactor pRB and selectively precipitated Vangl1. (d) GST pulldown assays showing strong binding of endogenous Vangl1 to phosphorylated HPV-16 E7, with weak or undetectable binding to HPV-11 E7, non-phosphorylated HPV-16 or HPV-18 E7, phosphorylated HPV-18 E7, or HPV-5 E7. ‘P’ indicates phosphorylated forms of the respective E7s. (e) Co-immunoprecipitation of FLAG-Vangl1 with FLAG/HA-tagged HPV-16 E7 expressed in HEK293 cells confirms the in vivo interaction. Beta galactosidase (β-gal) protein expression is used as a loading control in the western blotting analyses. (f) TCGA analysis shows that Vangl1 expression is associated with increased clinical risk in multiple tumour types, including cervical and endocervical cancer (CESC), lower-grade glioma (LGG), lung adenocarcinoma (LUAD), and pancreatic adenocarcinoma (PAAD). The statistical significance is represented as z-score and p -value.

Article Snippet: CaSki, HeLa, C-33A and HaCaT cells were transfected with siRNAs targeting Vangl1 (Dharmacon SMARTpool), AP1M1 (Dharmacon SMARTpool), HPV-16 E6/E7 (Eurofins), or Scramble control (Dharmacon siSTABLE) as previously described ( ).

Techniques: Phospho-proteomics, Synthesized, Mass Spectrometry, Binding Assay, Immunoprecipitation, In Vivo, Expressing, Control, Western Blot

Journal: bioRxiv

Article Title: CKII-Phosphorylated HPV-16 E7 Disrupts Planar Cell Polarity by Recruiting Vangl1 Phospho-E7 Hijacks Vangl1 Trafficking

doi: 10.64898/2026.02.25.707969

Figure Lengend Snippet: (a) Co-immunoprecipitation of FLAG-tagged Vangl1 or Vangl2 with FLAG/HA-tagged HPV-16 E7 expressed in HEK293 cells. HPV-16 E7 binds strongly to Vangl1 but only minimally to Vangl2. (b) Peptide pulldown assays using phosphorylated and non-phosphorylated HPV-16 E7 N29S CR2 peptides. Vangl1 binds strongly to the phosphorylated N29S peptide but not to its non-phosphorylated form. (c) Pulldown assays using phosphomimic HPV-16 E7 CR2 peptides in which CKII-targeted serines were substituted with aspartic acid. The phosphomimic fails to bind Vangl1. ‘ p’ indicates phosphorylation. (d) Co-immunoprecipitation of FLAG-Vangl1 with FLAG/HA-tagged HPV-16 E7 in HEK293 cells treated with the CKII inhibitor CX-4945. CKII inhibition markedly reduces Vangl1–E7 association. Beta galactosidase (β-gal) protein expression is used as a loading control in the western blotting analyses.

Article Snippet: CaSki, HeLa, C-33A and HaCaT cells were transfected with siRNAs targeting Vangl1 (Dharmacon SMARTpool), AP1M1 (Dharmacon SMARTpool), HPV-16 E6/E7 (Eurofins), or Scramble control (Dharmacon siSTABLE) as previously described ( ).

Techniques: Immunoprecipitation, Phospho-proteomics, Inhibition, Expressing, Control, Western Blot

Journal: bioRxiv

Article Title: CKII-Phosphorylated HPV-16 E7 Disrupts Planar Cell Polarity by Recruiting Vangl1 Phospho-E7 Hijacks Vangl1 Trafficking

doi: 10.64898/2026.02.25.707969

Figure Lengend Snippet: Immunoblot analysis of Vangl1 and E7 protein levels following siRNA-mediated knockdown of E6/E7 or Vangl1. (a, b) In HPV-16 E7-positive CaSki cells and HPV-18 E7-positive HeLa cells, E6/E7 depletion increases total Vangl1 abundance. (c, d) In HPV-negative C33A cells and non-transformed HaCaT keratinocytes, Vangl1 remains unchanged following E6/E7 knockdown. GAPDH expression is shown as loading control. Vangl1 band density was normalized to GAPDH band density, and the data was used to plot the densitometry graphs for all cell lines. Data is shown as the fold-changes of normalized Vangl1 levels relative to the control (siScramble) ± standard deviation. p values of 3 independent experiments were calculated by Student’s t test. ( p values of 3 independent experiments were calculated by Student’s t test. (*: p -value < 0.05; **: p -value <0.01; ***: p -value <0.001).

Article Snippet: CaSki, HeLa, C-33A and HaCaT cells were transfected with siRNAs targeting Vangl1 (Dharmacon SMARTpool), AP1M1 (Dharmacon SMARTpool), HPV-16 E6/E7 (Eurofins), or Scramble control (Dharmacon siSTABLE) as previously described ( ).

Techniques: Western Blot, Knockdown, Transformation Assay, Expressing, Control, Standard Deviation

Journal: bioRxiv

Article Title: CKII-Phosphorylated HPV-16 E7 Disrupts Planar Cell Polarity by Recruiting Vangl1 Phospho-E7 Hijacks Vangl1 Trafficking

doi: 10.64898/2026.02.25.707969

Figure Lengend Snippet: (a) Immunoblot analysis of Vangl1 in CaSki cells transfected with control, Vangl1 or E6/E7 siRNAs and treated with the proteasome inhibitor CBZ, the lysosomal inhibitor chloroquine (CQ), or DMSO. E6/E7 knockdown alone increases Vangl1 levels more strongly than either inhibitor. (b) Cycloheximide chase assays in CaSki cells shows that Vangl1 half-life in control cells is longer than in E7-depleted cells. GAPDH expression is shown as loading control. Vangl1 band density was normalized to GAPDH band density, and the data was used to plot densitometry graphs for the cell lines. Data is shown as the fold changes of normalized Vangl1 levels relative to the control (siScramble) ± standard deviation. (c) Long exposure inset of the blot in (b) shows siScramble and siE6/E7 at similar start-point intensities, highlighting biphasic degradation of unphosphorylated and phosphorylated Vangl1 forms in siScramble cells. (d) Cycloheximide chase assays in HEK293 cells overexpressing Vangl1 with or without HPV-16 E7. Vangl1 half-life increases when co-expressed with E7. (e) Densitometry showing reciprocal stabilization of E7 by Vangl1. E7 half-life increases when co-expressed with Vangl1. Dotted lines on time-kinetic graphs indicate specific half-life. β-gal expression is shown as loading control. Vangl1 band density was normalized to the β-gal band density, and the data was used to plot a densitometry graph. Data is shown as the fold changes of normalized Vangl1 levels relative to the control (Vangl1 only) ± standard deviation. p values of 3 independent experiments were calculated by Student’s t test. ( p values of 3 independent experiments were calculated by Student’s t test. (*: p -value < 0.05; **: p -value <0.01; ***: p -value <0.001).

Article Snippet: CaSki, HeLa, C-33A and HaCaT cells were transfected with siRNAs targeting Vangl1 (Dharmacon SMARTpool), AP1M1 (Dharmacon SMARTpool), HPV-16 E6/E7 (Eurofins), or Scramble control (Dharmacon siSTABLE) as previously described ( ).

Techniques: Western Blot, Transfection, Control, Knockdown, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: CKII-Phosphorylated HPV-16 E7 Disrupts Planar Cell Polarity by Recruiting Vangl1 Phospho-E7 Hijacks Vangl1 Trafficking

doi: 10.64898/2026.02.25.707969

Figure Lengend Snippet: (a) Subcellular fractionation of CaSki cells transfected with control or E6/E7 siRNAs. Vangl1 is absent from the cytoskeletal fraction in control cells, but becomes detectable upon E6/E7 knockdown. (b) GST pulldown assays showing that AP1M1 selectively binds phosphorylated HPV-16 E7. (c–d) Immunoblot analysis of CaSki and HeLa cells shows that Vangl1 abundance increases upon E6/E7 depletion and is further increased by AP1M1 knockdown. (e) Immunoblot analysis of HaCaT keratinocytes shows that neither E6/E7 knockdown nor AP1M1 knockdown results in increased Vangl1 levels. (f) Subcellular fractionation of CaSki cells after AP1M1 knockdown shows Vangl1 accumulation across cytoplasmic, membrane, and cytoskeletal compartments mirroring E6/E7 knockdown. GAPDH expression is shown as loading control. Vangl1 band density was normalized to GAPDH band density, and the data was used to plot densitometry graphs for the cell lines. Data is shown as the fold changes of normalized Vangl1 levels relative to the control (siScramble) ± standard deviation. p values of 3 independent experiments were calculated by Student’s t test. ( p values of 3 independent experiments were calculated by Student’s t test. (*: p -value < 0.05; **: p -value <0.01; ***: p -value <0.001).

Article Snippet: CaSki, HeLa, C-33A and HaCaT cells were transfected with siRNAs targeting Vangl1 (Dharmacon SMARTpool), AP1M1 (Dharmacon SMARTpool), HPV-16 E6/E7 (Eurofins), or Scramble control (Dharmacon siSTABLE) as previously described ( ).

Techniques: Fractionation, Transfection, Control, Knockdown, Western Blot, Membrane, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: CKII-Phosphorylated HPV-16 E7 Disrupts Planar Cell Polarity by Recruiting Vangl1 Phospho-E7 Hijacks Vangl1 Trafficking

doi: 10.64898/2026.02.25.707969

Figure Lengend Snippet: (a) Confocal microscopy of HEK293 cells expressing Vangl1 alone, HPV-16 E7 alone, or both proteins together, show diffuse, cytoplasmic Vangl1 with reduced cortical levels in cells expressing E7. Merged and zoomed images show co-localization of Vangl1 and E7 within intracellular compartments. (b) Brightfield images of CaSki spheroids following siRNA-mediated knockdown of E6/E7 or Vangl1 showing spheroids with pronounced structural disintegration, irregular edges, and reduced cohesion. (c) 3D surface plots of spheroids highlight architectural defects. Control spheroids show uniform signal intensity and well-defined boundaries, while E6/E7 and Vangl1 knockdown spheroids display diffuse signal, fragmented contours, and disrupted core organization. Micrographs were prepared using the Quickfigures plugin of ImageJ. The brightness of the panels was uniformly enhanced post image processing.

Article Snippet: CaSki, HeLa, C-33A and HaCaT cells were transfected with siRNAs targeting Vangl1 (Dharmacon SMARTpool), AP1M1 (Dharmacon SMARTpool), HPV-16 E6/E7 (Eurofins), or Scramble control (Dharmacon siSTABLE) as previously described ( ).

Techniques: Confocal Microscopy, Expressing, Knockdown, Control

Journal: bioRxiv

Article Title: CKII-Phosphorylated HPV-16 E7 Disrupts Planar Cell Polarity by Recruiting Vangl1 Phospho-E7 Hijacks Vangl1 Trafficking

doi: 10.64898/2026.02.25.707969

Figure Lengend Snippet: (a) Brightfield representative images from 2D Matrigel invasion assays in CaSki cells show dense invasion in control cells, whereas Vangl1-depleted cells show markedly reduced invasion. (b) Quantification of invading cells using the ImageJ Cell counter tool confirms a significant decrease in invasion upon Vangl1 knockdown. (c) Brightfield images of spheroid invasion across increasing collagen content (Matrigel:Collagen 1:1 and 1:3) show collective invasion in control spheroids; (d) E6/E7-depleted spheroids fail to generate organized invasive protrusions in either matrix condition; (e) Vangl1-depleted spheroids similarly lack robust invasive structures and display matrix-dependent architectural defects. Corresponding 3D surface plots below the micrographs reflect spheroidal architectural integrity. Invasiveness of the spheroids was monitored for 7 days and imaged at 20x magnification. (f) Brightfield representative images from 2D Matrigel invasion assays following AP1M1 knockdown reveal a pronounced reduction in invasion. Invading cells in the 2D assays were stained with crystal violet and imaged using a brightfield microscope at 10x magnification. p values of 3 independent experiments were calculated by Student’s t test. (*: p -value < 0.05; **: p -value <0.01; ***: p -value <0.001; ****: p -value <0.0001). Micrographs were prepared using the Quickfigures plugin of ImageJ. The brightness of the panels was uniformly enhanced post-image processing.

Article Snippet: CaSki, HeLa, C-33A and HaCaT cells were transfected with siRNAs targeting Vangl1 (Dharmacon SMARTpool), AP1M1 (Dharmacon SMARTpool), HPV-16 E6/E7 (Eurofins), or Scramble control (Dharmacon siSTABLE) as previously described ( ).

Techniques: Control, Knockdown, Staining, Microscopy

Journal: bioRxiv

Article Title: CKII-Phosphorylated HPV-16 E7 Disrupts Planar Cell Polarity by Recruiting Vangl1 Phospho-E7 Hijacks Vangl1 Trafficking

doi: 10.64898/2026.02.25.707969

Figure Lengend Snippet: (a) Brightfield images of CaSki spheroids following siRNA transfection and 48 h treatment with DMSO. Control spheroids remain compact and cohesive, whereas E6/E7- and Vangl1-depleted spheroids exhibit architectural changes; (b) Palbociclib treatment induces mild disintegration in siScramble spheroids, but causes pronounced disintegration in E6/E7- and Vangl1-depleted spheroids; (c–d) Cisplatin and Etoposide treatments show moderate disruption in siScramble spheroids, whereas E6/E7- and Vangl1-depleted spheroids undergo rapid dissociation and display debris-rich halos. The 3D surface plots of the spheroids are presented on the right panels. Brightfield images were acquired at 20x magnification. Micrographs and 3D surface plots were prepared using ImageJ. The brightness of the panels was uniformly enhanced post-image processing.

Article Snippet: CaSki, HeLa, C-33A and HaCaT cells were transfected with siRNAs targeting Vangl1 (Dharmacon SMARTpool), AP1M1 (Dharmacon SMARTpool), HPV-16 E6/E7 (Eurofins), or Scramble control (Dharmacon siSTABLE) as previously described ( ).

Techniques: Transfection, Control, Disruption

Journal: NPJ Vaccines

Article Title: Plasmablast, memory B cell and T follicular helper cell responses after human papillomavirus vaccination: effect of dose number and age

doi: 10.1038/s41541-026-01408-w

Figure Lengend Snippet: HPV 16/18-specific IgG and IgM plasmablasts were enumerated from freshly isolated peripheral blood mononuclear cells at baseline (pre-vaccination), day 7 post-dose 1, and pre- and day 7 post-dose 2 or 3 of Gardasil 9 vaccination ( n = 20, 19, and 16 for the 4-8-, 9-14-, and 15-26-year-old groups, respectively). A Representative FluoroSpot readouts are shown for one participant in the 15–26-year-old group. Box and whisker plots show HPV 16-specific ( B ) IgG and ( C ) IgM secreting cells, with the lower, central, and upper lines denoting the minimum, median, and maximum values, respectively. Age groups are colour-coded, with each dot representing an individual. Statistical analyses evaluating the effects of dose number and age compared plasmablast numbers between baseline and post-dose 1, 2, or 3 timepoints. Paired one-way ANOVA with Dunnett’s adjustment and paired two-way ANOVA with Tukey’s adjustment were performed for pooled and age-stratified analyses, respectively. Statistically significant p-values ( p < 0.05) are highlighted in blue. Line graphs show the kinetics of HPV 16-specific ( D ) IgG and ( E ) IgM secreting cells across the four evaluated timepoints. yrs—years.

Article Snippet: Ninety-six-well plates were coated with HPV 16/18 VLPs (Merck Sharp and Dohme Corp., USA) at 4 °C overnight, washed and incubated with pre-stimulated PBMC for 20 h. During incubation, HPV 16/18-specific IgG antibodies secreted by B cells were captured by the HPV VLPs and the cells were removed by washing in PBS.

Techniques: Isolation, Whisker Assay

Journal: NPJ Vaccines

Article Title: Plasmablast, memory B cell and T follicular helper cell responses after human papillomavirus vaccination: effect of dose number and age

doi: 10.1038/s41541-026-01408-w

Figure Lengend Snippet: HPV 16/18-specific IgG Bmem were enumerated from freshly isolated peripheral blood mononuclear cells at baseline (pre-vaccination), day 14 post-dose 1, and pre- and day 14 post-dose 2 or 3 of Gardasil 9 vaccination ( n = 18, 19 and 17 for 4-8-, 9-14- and 15–26-year-olds, respectively). A Representative ELISpot readouts are shown for one participant in the 4–8-year-olds group. Box and whisker plots show ( B ) HPV 16- and ( C ) HPV 18-specific IgG Bmem responses, with the lower, central and upper lines denoting minimum, median and maximum values, respectively. Age groups are colour-coded with each dot representing an individual. Statistical tests to evaluate the effect of dose number and age compared Bmem numbers between baseline and post-dose 1, 2 or 3 timepoints. Paired One-way ANOVA with Dunnett’s adjustment and paired two-way ANOVA with Tukey adjustment were performed for pooled and age-stratified analyses, respectively. Statistically significant p values ( p < 0.05) are highlighted in blue. Line graphs show the kinetics of ( D ) HPV 16- and ( E ) HPV 18-specific IgG secreting cells across the four evaluated timepoints. yrs – years.

Article Snippet: Ninety-six-well plates were coated with HPV 16/18 VLPs (Merck Sharp and Dohme Corp., USA) at 4 °C overnight, washed and incubated with pre-stimulated PBMC for 20 h. During incubation, HPV 16/18-specific IgG antibodies secreted by B cells were captured by the HPV VLPs and the cells were removed by washing in PBS.

Techniques: Isolation, Enzyme-linked Immunospot, Whisker Assay

Journal: NPJ Vaccines

Article Title: Plasmablast, memory B cell and T follicular helper cell responses after human papillomavirus vaccination: effect of dose number and age

doi: 10.1038/s41541-026-01408-w

Figure Lengend Snippet: Frozen PBMCs were thawed and pre-stimulated in culture for 18 h with HPV 16 or HPV 18 VLPs, a mitogen (SEB) positive control, or cultured in medium only without stimulation (Medium). Stimulated cells were stained with the AIM antibody panel, and data were acquired on BD LSRFortessa III flow cytometer with gating performed in FlowJo. A Lymphocytes were first gated from all cells, followed by exclusion of dead cells and CD14+ monocytes. Total Tfh cells were then identified as FoxP3-CD4 + CD45RO + CXCR5+. B Activated cells were identified using AIM markers as OX40 + CD25 + , OX40 + PD-L1+ and PD-L1 + CD25+ across all stimulation conditions. Shown is a representative gating strategy from one subject in the 15–26-year-old group. VLPs - virus-like particles; SEB staphylococcal enterotoxin B; AIM activation-induced marker.

Article Snippet: Ninety-six-well plates were coated with HPV 16/18 VLPs (Merck Sharp and Dohme Corp., USA) at 4 °C overnight, washed and incubated with pre-stimulated PBMC for 20 h. During incubation, HPV 16/18-specific IgG antibodies secreted by B cells were captured by the HPV VLPs and the cells were removed by washing in PBS.

Techniques: Positive Control, Cell Culture, Staining, Flow Cytometry, Virus, Activation Assay, Marker

Journal: NPJ Vaccines

Article Title: Plasmablast, memory B cell and T follicular helper cell responses after human papillomavirus vaccination: effect of dose number and age

doi: 10.1038/s41541-026-01408-w

Figure Lengend Snippet: Activated HPV 16/18-specific Tfh cells were identified using an AIM flow cytometry assay with the phenotypes OX40 + CD25+, OX40 + PD-L1+ and PD-L1 + CD25+ within the total circulating Tfh cell pool. Box and whisker plots show activated HPV 16-specific Tfh cells based on A OX40 + CD25+, B OX40 + PD-L1+ and C PD-L1 + CD25+, and HPV 18-specific Tfh cells based on D OX40 + CD25+, E OX40 + PD-L1+ and F PD-L1 + CD25+. The lower, central, and upper lines indicate the minimum, median, and maximum values, respectively. Age groups are colour-coded, with each dot representing an individual. Statistical evaluation of the effects of dose number and age compared activated Tfh cell frequencies between baseline and post-dose 1, 2, or 3 timepoints. Unpaired one-way ANOVA with Dunnett’s adjustment and unpaired two-way ANOVA with Tukey adjustment were performed for pooled and age-stratified analyses, respectively. Statistically significant p values ( p < 0.05) are highlighted in blue. VLPs virus-like particles, SEB staphylococcal enterotoxin B, AIM activation-induced marker.

Article Snippet: Ninety-six-well plates were coated with HPV 16/18 VLPs (Merck Sharp and Dohme Corp., USA) at 4 °C overnight, washed and incubated with pre-stimulated PBMC for 20 h. During incubation, HPV 16/18-specific IgG antibodies secreted by B cells were captured by the HPV VLPs and the cells were removed by washing in PBS.

Techniques: Flow Cytometry, Whisker Assay, Virus, Activation Assay, Marker