Isolation and Characterization of HPV-Positive Keratinocytes

Healthy human foreskin keratinocytes (HFKs) were isolated from deidentified neonatal specimens. HFK cells were transfected with HPV16 or HPV31 genomes to generate HFK-16 and HFK-31 cell lines that contain episomal HPV DNA as previously described (18 (link)). CIN 612 cells were isolated from a cervical cancer biopsy specimen and contain HPV31 episomes (19 (link)). All keratinocytes were cocultured in E-media with growth-arrested NIH 3T3 fibroblasts as described previously (3 (link)). Antibodies to phosphorylated CHK1 (pCHK1) (catalog no. 12302; Cell Signaling Technology [CST]), phosphorylated CHK2 (pCHK2) (catalog no. 2661; CST), FANCD2 (catalog no. 100182; Novus), phosphorylated H2Ax (γH2AX) (catalog no. 05636; Millipore), phosphorylated SMC1 (pSMC1) (catalog no. 4805S; CST), BRCA1 (catalog no. OP92; Millipore), RAD51 (catalog no. NB100148; Novus), and TOP2β (catalog no.A300-950A; Bethyl Laboratories) were used.

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Kaminski P., Hong S., Kono T., Hoover P, & Laimins L. (2021). Topoisomerase 2β Induces DNA Breaks To Regulate Human Papillomavirus Replication. mBio, 12(1), e00005-21.

Publication 2021

FANCD2 phosphorylated H2Ax (γH2AX) BRCA1 RAD51

Antibodies Biopsy Brca1 Cell lines Cells Cervical cancer Episomes Fancd2 Fibroblasts Foreskin Genomes Hpv16 Hpv31 Human Keratinocytes Neonatal Nih 3t3 Novus

Corresponding Organization : Northwestern University

Other organizations : Chongqing Medical University

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Variable analysis

independent variables

Transfection of HFK cells with HPV16 or HPV31 genomes to generate HFK-16 and HFK-31 cell lines

dependent variables

Levels of phosphorylated CHK1 (pCHK1)
Levels of phosphorylated CHK2 (pCHK2)
Levels of FANCD2
Levels of phosphorylated H2Ax (γH2AX)
Levels of phosphorylated SMC1 (pSMC1)
Levels of BRCA1
Levels of RAD51
Levels of TOP2β

control variables

Healthy human foreskin keratinocytes (HFKs) without HPV transfection
CIN 612 cells containing HPV31 episomes
Co-culture of all keratinocytes with growth-arrested NIH 3T3 fibroblasts

positive controls

CIN 612 cells containing HPV31 episomes

negative controls

Healthy human foreskin keratinocytes (HFKs) without HPV transfection

Annotations

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