Skip to main content
. Author manuscript; available in PMC: 2014 May 24.
Published in final edited form as: Oncogene. 2012 Dec 3;32(43):5176–5185. doi: 10.1038/onc.2012.533

Fig. 4. NF90 regulates HPV E6 oncogene expression.

Fig. 4

(a) Schematic showing HPV-mediated p53 degradation via E6 and E6AP. (b) Knockdown of NF90 suppresses the expression of HPV18 E6 RNA in HeLa cells. Left panel: RT-PCR indicating the levels of viral E6 and cellular E6AP RNA in siRNA-transfected cells. Right panel: immunoblots showing the expression of proteins. (c) Diagrams of HPV18 genome organization (linear form) and open reading frames (top) (105) and of pGL3-HPV18URR-Firefly luciferase reporter construct (bottom). URR, upstream regulatory region; P105, early promoter; P811, late promoter; AE, early polyadenylation site; AL, late polyadenylation site; E1, E2, E4, E5, E6 and E7: early genes; L1 and L2, late genes. (d) NF90/NF45 depletion does not destabilize E6 RNA. Top panel: northern blot showing E6/E7 RNA in siRNA-transfected HeLa cells treated with actinomycin D (0.5 μg/ml) compared to untreated cells (0 h time point). Lower panel: 18S and 28S ribosomal RNA stained with ethidium bromide. (e) RNA half-life estimation. Quantitation of E6/E7 transcripts from two independent northern blotting experiments as in panel (d). (f) Depletion of NF90/NF45 inhibits the HPV18 early promoter. Expression of Firefly luciferase from pGL3-HPV18URR-Firefly luciferase plasmid normalized to Renilla luciferase expression in HeLa cells transfected with C, D3 and D5 siRNA (n=4 independent experiments). Standard deviations and P values are relative to respective controls: **, < 0.01; ***, < 0.001; ****, < 0.0001. Similar results were obtained with 600 ng reporter plasmid. Immunoblot analysis for a representative experiment is shown.