Figure 9
HPV E1 protein decreases IFNβ1 and IFNλ1 expression in the presence of the Poly I:C agonist. HaCaT cells were transfected using 0.01, 0.1, or 1 μg of Poly I:C and pCA vector to a final amount of 4.5 μg. After 24 h post transfection total RNA was extracted and the IFNβ1 expression was evaluated by pPCR. (A) The Poly I:C increases the IFNβ1 and IFNλ1 expression in HaCaT cells in all concentrations tested. The 0.1 μg concentration was selected for further analyses. Bars represent the mean ± SD. ***p < 0.001 vs control (pCA). (B) IFNβ1 and (C) IFNλ1 expression is repressed in the presence of HPV16, 18 and 11 E1 proteins. After 24 h of transfection with 0.1 μg of Poly I:C and the indicated HPV E1 plasmid, total RNA was extracted, cDNA synthesized, and qPCR performed to evaluate the IFNβ1 and IFNλ1 expression. In presence of the different HPV E1 proteins there is a decrease of IFNβ1 and IFNλ1 expression. Values are expressed as the difference in double delta-Ct compared with cells transfected with the pCA vector. The expression of the housekeeping gene 18S was used for normalizing RNA expression. Bars represent the mean ± SD. ***p < 0.001 vs pCA control. (D) IkBα and p52-NFκB protein levels. HaCaT cells transfected with pCA vector and HPV E1 expressing plasmids were collected and total protein extracted 24 h after transfection. Levels of IkBα and p52-NFκB were ascertained by western blot. Proteins were separated in a 10% SDS-PAGE and membrane was probed with anti-phospho-IκBα (Ser32/36)(5A5) (9246; Cell signaling Technology Europe, B.V) or anti-p52 (NFκB) (Santa Cruz Biotechnology, California) observing decreased levels of IkBα and an increase of p52-NFκB levels in the presence of HPV16, 18 and 11 E1 proteins. GAPDH was used as loading protein control (GAPDH, 37 KDa).
