Data associated with: "HaCaT Keratinocytes: A Differentiation-Competent Platform for Episomal Replication of HPV"
Description
Current studies of HPV (and PyV) intranuclear processes, such as viral genome replication and transcription often rely on episome transfection of transformed or tumor-derived cell lines (e.g., C33A, HEK293/HEK293T, CIN612); however, these cell lines lack keratinocyte differentiation, and their transformed status and expression of oncogenes may complicate interpretation of some studies. While primary keratinocytes can undergo differentiation, they are not readily transfected and have a limited lifespan. Cell lines have been isolated that have naturally occurring HPV episomes, and can be induced to differentiate, however, as the episomes are already present and in multiple copies, creating mutations in the HPV genome/proteins for analysis of their function is very difficult. Hence, there is still a need for a non-transformed immortalized cell line that undergoes keratinocyte differentiation and can be readily transfected for molecular studies of intra-nuclear viral functions. To address this gap, we have investigated the differentiation and transfection characteristics of HaCaT human keratinocytes, a spontaneously immortalized but non-transformed cell line capable of reversible differentiation. We show that using the appropriate growth and transfection conditions, HaCaT cells can serve as a tractable epithelial platform for studying early intranuclear events associated with episomal viral DNA replication. Our study makes three contributions. First, we optimize extracellular Ca²⁺ conditions to reproducibly switch HaCaT cells between basal-like and differentiated states, allowing controlled experimental access to differentiation-linked epithelial biology. Second, we refine transfection parameters to achieve substantial consistent DNA delivery in HaCaT cells, suitable for quantitative assays. Third, using a luciferase-based origin replicon readout, we demonstrate robust, origin-dependent replication of HPV11 episomal plasmids in HaCaT cells in both basal-like and differentiated states—providing, to our knowledge, the first demonstration that HaCaT supports transient HPV origin-driven episomal replication in a differentiation-competent keratinocyte context. Here, we upload: 1) Making of Figures and Supplementary Figures in the paper- raw and assembled 2) Supplementary Table 1-3
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HaCaT keratinocytes were cultured in high-glucose DMEM (Gibco, ThermoFisher) supplemented with 10 % fetal bovine serum (FBS; Thermo Fisher) and 4 mM L-glutamine (Gibco) (standard “full” medium, containing ~1.8 mM Ca²⁺) or in calcium-free DMEM (high-glucose, no glutamine, no Ca²⁺; Gibco) supplemented with 10 % calcium-depleted FBS and CaCl₂ to a final Ca²⁺ concentration of either ~0.06 mM (low Ca²⁺ medium) or 2.8 mM (high Ca²⁺ medium). Calcium was removed from FBS by batch incubation with Chelex-100 resin (Bio-Rad) for 1 h at 4 °C with gentle rocking, followed by sterile filtration through a 0.22 μm filter unit. For the in vitro differentiation model, proliferating HaCaT cells were adapted to low Ca²⁺ medium [0.06 mM] for ≥3 passages to maintain a basal, undifferentiated phenotype. Cells were then shifted to high Ca²⁺ medium (1.8–2.8 mM) for up to 120 h (5 days) to in-duce differentiation. Differentiation was monitored by (a) phase contrast microscopy, b) Western Blotting. Transfection of HaCaT and HEK293T Cells: Three transfection reagents were evaluated in HaCaT cells: (a) Lipofectamine 3000 (Invitrogen; lipid based), (b) TransIT X2 (Mirus), and (c) FuGENE 6 (Promega; non liposomal polymeric reagent). At 72 h post transfection, live cells were stained with Hoechst 33342 (0.001 µg/mL, added directly to warm medium) and imaged without washing. Fluorescence images were acquired in overlay mode. DNA replication assays: Transient viral DNA replication was quantified using a dual luciferase plasmid replication assay. Two assay systems were used: SV40 (polyomavirus) and HPV11 (papillomavirus). For both, cells were co-transfected with a Firefly ori⁺ reporter, viral replication protein expression plasmid(s), and a control ori⁻ Renilla plasmid. For SV40, the reporter was pFLORI 40 (Firefly linked to the SV40 ORI) and replication was driven by SV40 large T antigen (LT). For HPV11, the reporter was pFLORI 11 (Firefly linked to the HPV11 ORI), with E1 and E2 supplied in trans from separate expression vectors. DNA amounts were empirically optimized, and very low ori plasmid input was used so that increases in Firefly signal emphasized replicated DNA over initial plasmid uptake . Dual luciferase assays were performed 72 h after transfection of DNA into HaCaT cells to assess DNA replication efficiency. Replication efficiency was expressed as a percentage of the positive control (e.g., C33A, AD293, or HaCaT cells transfected with 10 ng LT or E1, defined as 100% for SV40 or HPV11 assays, respectively).
Institutions
- University at Buffalo - The State University of New York
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Funders
- National Institutes of HealthUnited States Department of Health and Human ServicesUnited StatesGrant ID: AI188176