Fig 2. KLF4 is required for HPV DNA amplification and late gene expression.

2A. KLF4 was transiently silenced in CIN-612 cells using lentiviral shRNAs. Differentiation was induced by suspending cells in methylcellulose [7,40]. (i) The reductions in KLF4 protein levels were observed by western analysis in both undifferentiated and differentiated conditions of shKLF4 cells compared to mock and shGFP controls. (ii) Silencing KLF4 with shRNAs impaired the ability of the cells to amplify episomal DNA upon differentiation as shown by Southern blot analysis. (iii) Silencing KLF4 also resulted in the reduction in levels of late mRNA transcripts upon differentiation when compared to the controls as shown by northern blot analysis. 2B. KLF4 was stably silenced in HFK-31gen cells using lentiviral shRNAs. Following infection with shRNA lentiviruses, cells were expanded and selected with puromycin. (i) KLF4 protein levels were reduced in cells stably transduced with shKLF4 lentivirus compared to both mock and shGFP lentivirus controls as shown in the western blot. (ii) Stable silencing of KLF4 reduced the amount of viral DNA amplification upon differentiation compared to the controls as shown by Southern blot analysis.