Fig 1: Oncogenicity assays (p53/Rb and anchorage-independent growth). (a) Interaction of the proteins translated from WT E6, mutant E6-E7-S2, WT E6-E7-S2 with p53 in the C33A cell line. Ten picomoles of the indicated mRNA-LNPs were transfected into C33A cells. Cells were collected 72 h after transfection, and the whole cell extracts (50 μg protein/well) were analyzed by Western blot for detection of the target antigen. β-actin was used as a loading control. Results are representative of three independent experiments. (b–d) Interaction of the proteins translated from WT E6-E7, mutant E7 and mutant E6-E7 fused to S2 with Rb in primary keratinocytes. (b) Representative FACS plots showing percentage of RFP+ cells in lentivirus-transduced vs. untransduced primary human keratinocytes. (c) Quantification of the percentage of RFP+ cells in lentivirus-transduced vs. untransduced primary human keratinocytes. (d) Mean fluorescence intensity (MFI) of underphosphorylated Rb expression in lentivirus-transduced keratinocytes. Underphosphorylated Rb expression was determined by intracellular staining of both un-transduced and transduced RFP+ keratinocytes. (e) Transforming activity measured with Cell Transformation Assay Kit (Colorimetric) (Abcam, ab235698). Equal numbers of transduced primary keratinocytes (1 × 104 cells) were seeded into plates. After 7 days, cell numbers were quantified by spectrophotometry absorbance at 450 nm. Statistical analysis using two-tailed unpaired Student’s t-test for (c,e). Statistical analysis using nonparametric Mann–Whitney test for (d). ** p < 0.01, *** p < 0.001; ns, not significant.