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. 2018 Dec 17;10(12):521. doi: 10.3390/cancers10120521

Figure 5.

Figure 5

E6 regulates the expression levels of AKNA and CD40 in the HPV positive cell line HeLa. HeLa cells were seeded in a 60 mm dishes and transfected with siRNAs directed against E6, siE6/E7 and Luciferase (siLuc) as an unspecific control. After 72 h cells were either fixed or collected and total protein extracted using sample buffer. (A) Levels of AKNA and CD40 were evaluated by western blot using specific antibodies, α-actinin was used as a loading control, and p53 was evaluated to assess the silencing efficiency of E6. Cells showed a clear increase in p53 levels indicating the efficient ablation of E6 expression and, in cells were p53 was restored showed also an increase in AKNA and CD40 levels. Densitometric analysis expressing data as the ratio of relative units between AKNA/α-actinin (B), and CD40/α-actinin (C). Data are shown as the mean ± SD. Tukey’s test * p < 0.05, ** p < 0.001 and *** p < 0.0001 vs. pCA control. n = 3. (D,F) Fixed HeLa cells were stained for immunofluorescence analysis using anti-CD40, AKNA anti-serum and anti-p53 antibody followed by secondary Alexafluor antibodies as specified, finally cells were mounted using Prolong DAPI media (Molecular Probes) and observed in a confocal NIKON Eclipse Ti microscope. E6 knocked down cells exhibited p53 positive signal due to E6 inactivation, p53 positive cells showed a clear accumulation of AKNA in the cytoplasm and in the nucleus as well as a co-localization with p53. CD40 showed an increase in the siE6 and siE6/E7 cells mainly in the cytoplasm. All images were acquired under the 40× objective using a NIKON camera. (E,G) AKNA and CD40 fluorescence quantification using the corrected total cell fluorescence (CTCF) method. Data are shown as the mean ± SD. Student’s t-test ** p < 0.01 and *** p < 0.0001 vs. siLuc control. n = 3.