Data associated with: "HaCaT Keratinocytes: A Differentiation-Competent Platform for Episomal Replication of HPV"

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