FIGURE 1.

Generation of human cervical keratinocyte cell lines conditionally expressing the defined viral and cellular oncogenes and potential lineage‐determining factors. A, Schematic illustration of a single polycistronic lentiviral vector, which induces the expression of E6 and E7 of human papillomavirus (HPV)16 or HPV18, MYC^T58A and KRAS^G12V (EMR) under the control of tetracycline (tet)‐responsible promoter. Ψ, packaging signal; 2A, 2A peptide sequences; CMVmini, CMV minimal promoter; LTR, long terminal repeat; TRE, tet‐responsive elements. B, Expression profiles of FOXA2, POU5F1, and HNF4α in cervical squamous carcinoma and adenocarcinoma in The Cancer Genome Atlas. P value was computed using the Wilcoxon rank‐sum test. **P ≤ .01, ***P ≤ .001. C, D, Western blot analyses to verify the tet‐responsible expression of the transgenes. HCK1T‐tre16EMR or HCK1T‐tre18EMR were further transduced with a lentivirus expressing FOXA2, a microRNA targeting SMAD4 (SMAD4mi) or POU5F and HNF4α (PH) from tet‐responsible promoter. The indicated cells were cultured with or without doxycycline (DOX; 1000 ng/mL) for 5 d, and the whole‐cell lysates were collected. The expression of the transgenes was detected using a specific Ab as indicated on the left of the panels. Vinculin was also detected as a loading control. E, Western blot analysis for endogenous FOXA2 in HCK1T cells expressing the indicated genes. HCK1T lines expressing E6 or E6 and E7 through retrovirus (LXSN)‐mediated transduction were described elsewhere. ¹⁰ , ¹⁴ Levels of FOXA2 in the indicated cells relative to that in HCK1T‐tetOFF cells are indicated at the bottom. Average values of three independent experiments are shown