Figure 2. HPV-18 E2 expression extends replicative lifespan of HFK and modulates expression of ncmtRNAs. (A) A triplicate determination of proliferation rate was plotted as time (days) versus cell doubling or passage number (p). Cells were arrested at p26. (B) At p26 the expression of HPV-18 E2 was suppressed as compared to 18Nco cells, transduced with the complete genome of HPV-18 (Bars = 50 µm). (C) E2 protein expression was progressively reduced in HFK-E2, from p8 to p26. Calnexin (CANX) was used as loading control. (D) A triplicate analysis of the results in (C) show a quantification of the gradual decrease of E2 from p8 to p26 (**p=0.001; ***p=0.0001). (E) Determination of the relative expression of ncmtRNAs by RT-PCR using 18S rRNA as loading control. A triplicate analysis shows downregulation of ASncmtRNA-1 at p3 (**p<0.01), which is sustained until p26 (F). In contrast, ASncmtRNA-2 is also downregulated at p3 but is later progressively upregulated from p15 to senescence at p26 (**p=0.001) (G). (H) SncmtRNA is downregulated at p26 (**p=0.001).