hpv18 genome  (ATCC)

Journal: bioRxiv

Article Title: Human Papillomavirus Infection Blunts STING Signaling and Alters Downstream Response

doi: 10.1101/2025.11.06.686957

Figure Lengend Snippet: Both oncogenes are needed to impact cGAMP levels. (A) western blot analysis for functional markers p53 and PTPN14, N= 3 using TTL system (B) cGAMP ELISA following DNA stimulation, N=4. (C) p-values for cGAMP ELISA of each cell line relative to HPV18 at specified time points. (D) Western blot showing function expression of E6 and/or E7 in overexpression cell lines. N=3 (E) cGAMP ELISA following DNA transfection, N=3 Statistics for (C and E) were calculated with two-way ANOVA, followed by Dunnett’s multiple comparison test. * p < 0.05; **p < 0.01; *** p < 0.001. Error bars indicate SEM.

Article Snippet: Primary human foreskin keratinocytes (HFKs) with or without HPV18 genomes were treated with 25 μg/mL 2′3′-cGAMP (InvivoGen tlrl-nacga23) to directly activate STING.

Techniques: Western Blot, Functional Assay, Enzyme-linked Immunosorbent Assay, Expressing, Over Expression, Transfection, Comparison

Journal: bioRxiv

Article Title: Human Papillomavirus Infection Blunts STING Signaling and Alters Downstream Response

doi: 10.1101/2025.11.06.686957

Figure Lengend Snippet: HPV18(+) cells produce more cGAMP than parental control cells. Quantification of cGAMP by ELISA following DNA stimulation of HPV(-) cells (black) and HPV18(+) cells (red). N=3 donors, 3 reps in each donor, average of the 9 replicates, (A) replicates separated by donor (B). Statistics for (A) were calculated using a linear mixed-effects model with “HPV” and “Time” as fixed effects and donor as a random effect to account for inter-donor variability. Statistics for (B) were calculated with a two-way ANOVA followed by a Dunnett’s post hoc test. * p<0.05; ** p<0.01; *** p<0.001Error bars for represent SEM, calculated by first averaging technical replicates (n=3 per donor) with propagated error, and then computing SEM across biological replicates (n=3 donors).

Article Snippet: Primary human foreskin keratinocytes (HFKs) with or without HPV18 genomes were treated with 25 μg/mL 2′3′-cGAMP (InvivoGen tlrl-nacga23) to directly activate STING.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Human Papillomavirus Infection Blunts STING Signaling and Alters Downstream Response

doi: 10.1101/2025.11.06.686957

Figure Lengend Snippet: HPV decreases STING and IRF3 activation. (A) Western blot showing DNA-treated HPV18(+) and parental cells. Quantification of (B) pSTING, average of the 9 replicates (C) pSTING, separated by donor (D) pIRF3 western blots, average of the 9 replicates, (E) pIRF3, separated by donor. N=3 donors, 3 replicates in each. Statistics for the average of 9 (B and D) were calculated using a linear mixed-effects model with “HPV” and “Time” as fixed effects and donor as a random effect to account for inter-donor variability. Statistics for each cell line (C and E) were calculated with a two-way ANOVA followed by a Dunnett’s post hoc test. * p < 0.05; ** p<0.01; *** p<0.001. Error bars represent SEM, calculated by first averaging technical replicates (n=3 per donor) with propagated error, and then computing SEM across biological replicates (n=3 donors). Slope was calculated by fitting linear regressions within each time interval, and the values were subsequently analyzed using a linear mixed-effects model.

Article Snippet: Primary human foreskin keratinocytes (HFKs) with or without HPV18 genomes were treated with 25 μg/mL 2′3′-cGAMP (InvivoGen tlrl-nacga23) to directly activate STING.

Techniques: Activation Assay, Western Blot

Journal: bioRxiv

Article Title: Human Papillomavirus Infection Blunts STING Signaling and Alters Downstream Response

doi: 10.1101/2025.11.06.686957

Figure Lengend Snippet: Both oncogenes are needed to impact STING activation. (A) Western blot for total STING, pSTING, and αTubulin for the TTL cell lines, N=3. (B) Densitometry of pSTING normalized to αTubulin and a loading control lysate run on all blots. (C) p-values for pSTING densitometry of each cell line relative to HPV18 cells at specified time points. (D) Western blot for total STING, pSTING, and αTubulin for the over expression cell lines, N=3. (E) Densitometry of pSTING normalized to αTubulin and a loading control lysate run on all blots. (F) p-values for pSTING densitometry of each cell line relative to E6/E7 expressing cells at specified time points. Statistics for (C and F) were calculated with two-way ANOVA, followed by Dunnett’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001. Error bars indicate SEM.

Article Snippet: Primary human foreskin keratinocytes (HFKs) with or without HPV18 genomes were treated with 25 μg/mL 2′3′-cGAMP (InvivoGen tlrl-nacga23) to directly activate STING.

Techniques: Activation Assay, Western Blot, Control, Over Expression, Expressing, Comparison

Journal: Current protocols in microbiology

Article Title: Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes.

doi: 10.1002/cpmc.101

Figure Lengend Snippet: Figure 4 Production of HPV18 minicircles. (A) Map of pMC.BESPX-HPV18. (B) Agarose gel showing pMC.BESPX-HPV18 DNA before and after induction. E. coli ZYCY10P3S2T transformed with pMC.BESPX- HPV18 were induced to generate minicircles.Plasmid DNA isolated before and after induction was digested with BglII. DNAs were separated on a 0.8% TAE agarose gel and imaged on a UV lightbox. Lane 1: Pre-induction pMC.BESPX-HPV18, undigested; lane 2: pre-induction pMC.BESPX-HPV18, digested; lane 3: post-induction pMC.BESPX-HPV18, undigested;lane 4:post-induction pMC.BESPX-HPV18, digested.CCC, covalently closed circular DNA.

Article Snippet: Materials 293TT cells (available from NCI’s Developmental Therapeutics Program) 293TT medium (see recipe) with and without antibiotics Opti-MEM serum-free medium (Thermo Fisher #31985070) Lipofectamine 2000 (Invitrogen #11668019) HPV genomes prepared using Support Protocol 1 or Support Protocol 2 using either of these plasmids: Plasmid pMC.BESPX-HPV18 minicircle HPV18 (Support Protocol 1; Henno et al., 2017) Recircularized HPV18 genomes (Support Protocol 2; Boshart et al., 1984; Cole & Danos, 1987) derived from cloned HPV genomes; HPV genomes can be requested from the HPV Reference Center p16SheLL (Addgene #37320) or p18SheLL (Addgene #37321) 0.25% trypsin/EDTA (Thermo Fisher #25200056) Dulbecco’s phosphate-buffered saline (DPBS with calcium and magnesium; Invitrogen #14040-141) MgCl2 (Quality Biological #351-033-721) Triton X-100 (Sigma #T8787) 1 M ammonium sulfate, pH 9 (Sigma #204501) Plasmid Safe (Lucigen #E3110K) Benzonase (Millipore #70664-3) 5 M NaCl (Sigma #59222C) DPBS/NaCl (see recipe) 46% OptiPrep solution (see recipe) 75-cm2 (T-75) tissue culture flasks (Cellstar #658175) 5% and 10% CO2 incubators, e.g., Heracell VIOS 160i (Thermo Fisher # 51030400) Serological pipettes 5-ml polypropylene round-bottom tubes (Corning #352063) 50-ml conical tubes (Corning #352070) Centrifuge such as Sorvall Legend XTR Centrifuge (Thermo Fisher #75004521) Refrigerated tabletop centrifuge such as Centrifuge 5415R (Eppendorf #5401000137) Siliconized conical microcentrifuge tubes with screw caps (Bio Plas #4202SLS) Heat block such as ThermoMixer C (Eppendorf #5382000023) 5-ml open-top thinwall polypropylene ultracentrifuge tubes (Beckman #326819) Porter and McBride 3 of 32 Current Protocols in Microbiology 3-ml syringe (VWR #53548-017) 150-mm stainless steel needle (Kemtech #S389150) Ultracentrifuge, such as OPTIMA XPN 90 (Beckman # A94468) Clamp stand 22-G needles (Monoject #15141-136) Part 1: Transfection of 293TT cells 1.

Techniques: Agarose Gel Electrophoresis, Transformation Assay, Plasmid Preparation, Isolation

Journal: Current protocols in microbiology

Article Title: Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes.

doi: 10.1002/cpmc.101

Figure Lengend Snippet: Figure 5 Traditional HPV genome recircularization. (A) Map of pUC-HPV18 showing the NcoI sites used to remove the vector and recircularize the viral genome. (B) Agarose gel showing a comparison of uncut, digested, and religated viral DNA. Lane 1: uncut pBR-HPV18; Lane 2, pBR-HPV18 restriction digested to free the HPV genome from plasmid backbone; Lane 3: pBR-HPV18 fragments ligated under dilute conditions to promote intramolecular recombination. Samples were separated on a 0.8% agarose TAE gel containing 0.5 μg/ml EtBr and imaged on a UV imager.

Article Snippet: Materials 293TT cells (available from NCI’s Developmental Therapeutics Program) 293TT medium (see recipe) with and without antibiotics Opti-MEM serum-free medium (Thermo Fisher #31985070) Lipofectamine 2000 (Invitrogen #11668019) HPV genomes prepared using Support Protocol 1 or Support Protocol 2 using either of these plasmids: Plasmid pMC.BESPX-HPV18 minicircle HPV18 (Support Protocol 1; Henno et al., 2017) Recircularized HPV18 genomes (Support Protocol 2; Boshart et al., 1984; Cole & Danos, 1987) derived from cloned HPV genomes; HPV genomes can be requested from the HPV Reference Center p16SheLL (Addgene #37320) or p18SheLL (Addgene #37321) 0.25% trypsin/EDTA (Thermo Fisher #25200056) Dulbecco’s phosphate-buffered saline (DPBS with calcium and magnesium; Invitrogen #14040-141) MgCl2 (Quality Biological #351-033-721) Triton X-100 (Sigma #T8787) 1 M ammonium sulfate, pH 9 (Sigma #204501) Plasmid Safe (Lucigen #E3110K) Benzonase (Millipore #70664-3) 5 M NaCl (Sigma #59222C) DPBS/NaCl (see recipe) 46% OptiPrep solution (see recipe) 75-cm2 (T-75) tissue culture flasks (Cellstar #658175) 5% and 10% CO2 incubators, e.g., Heracell VIOS 160i (Thermo Fisher # 51030400) Serological pipettes 5-ml polypropylene round-bottom tubes (Corning #352063) 50-ml conical tubes (Corning #352070) Centrifuge such as Sorvall Legend XTR Centrifuge (Thermo Fisher #75004521) Refrigerated tabletop centrifuge such as Centrifuge 5415R (Eppendorf #5401000137) Siliconized conical microcentrifuge tubes with screw caps (Bio Plas #4202SLS) Heat block such as ThermoMixer C (Eppendorf #5382000023) 5-ml open-top thinwall polypropylene ultracentrifuge tubes (Beckman #326819) Porter and McBride 3 of 32 Current Protocols in Microbiology 3-ml syringe (VWR #53548-017) 150-mm stainless steel needle (Kemtech #S389150) Ultracentrifuge, such as OPTIMA XPN 90 (Beckman # A94468) Clamp stand 22-G needles (Monoject #15141-136) Part 1: Transfection of 293TT cells 1.

Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Comparison

Journal: Current protocols in microbiology

Article Title: Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes.

doi: 10.1002/cpmc.101

Figure Lengend Snippet: Figure 6 Screening of quasivirus fractions. (A) qPCR was performed to measure HPV18 DNA in fractions prepared by standard (Basic Protocol 1) and Ripcord (Alternate Protocol 2). (B) Total proteins in each quasivirus fractions prepared by normal (Basic Protocol 1) and Ripcord (Alternate Protocol 2) methods were analyzed by SDS-PAGE and SYPRO Ruby detection.

Article Snippet: Materials 293TT cells (available from NCI’s Developmental Therapeutics Program) 293TT medium (see recipe) with and without antibiotics Opti-MEM serum-free medium (Thermo Fisher #31985070) Lipofectamine 2000 (Invitrogen #11668019) HPV genomes prepared using Support Protocol 1 or Support Protocol 2 using either of these plasmids: Plasmid pMC.BESPX-HPV18 minicircle HPV18 (Support Protocol 1; Henno et al., 2017) Recircularized HPV18 genomes (Support Protocol 2; Boshart et al., 1984; Cole & Danos, 1987) derived from cloned HPV genomes; HPV genomes can be requested from the HPV Reference Center p16SheLL (Addgene #37320) or p18SheLL (Addgene #37321) 0.25% trypsin/EDTA (Thermo Fisher #25200056) Dulbecco’s phosphate-buffered saline (DPBS with calcium and magnesium; Invitrogen #14040-141) MgCl2 (Quality Biological #351-033-721) Triton X-100 (Sigma #T8787) 1 M ammonium sulfate, pH 9 (Sigma #204501) Plasmid Safe (Lucigen #E3110K) Benzonase (Millipore #70664-3) 5 M NaCl (Sigma #59222C) DPBS/NaCl (see recipe) 46% OptiPrep solution (see recipe) 75-cm2 (T-75) tissue culture flasks (Cellstar #658175) 5% and 10% CO2 incubators, e.g., Heracell VIOS 160i (Thermo Fisher # 51030400) Serological pipettes 5-ml polypropylene round-bottom tubes (Corning #352063) 50-ml conical tubes (Corning #352070) Centrifuge such as Sorvall Legend XTR Centrifuge (Thermo Fisher #75004521) Refrigerated tabletop centrifuge such as Centrifuge 5415R (Eppendorf #5401000137) Siliconized conical microcentrifuge tubes with screw caps (Bio Plas #4202SLS) Heat block such as ThermoMixer C (Eppendorf #5382000023) 5-ml open-top thinwall polypropylene ultracentrifuge tubes (Beckman #326819) Porter and McBride 3 of 32 Current Protocols in Microbiology 3-ml syringe (VWR #53548-017) 150-mm stainless steel needle (Kemtech #S389150) Ultracentrifuge, such as OPTIMA XPN 90 (Beckman # A94468) Clamp stand 22-G needles (Monoject #15141-136) Part 1: Transfection of 293TT cells 1.

Techniques: SDS Page