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. 2017 Jul 10;45(16):9654–9678. doi: 10.1093/nar/gkx606

Figure 1.

Figure 1.

(A) Schematic representation of the HPV16 genome. Rectangles represent open reading frames, promoters p97 and p670 are indicated as arrows, filled and open triangles represent 5′- and 3′-splices sites respectively, HPV16 early and late polyA signals pAE and pAL are indicated. Below the HPV16 genome, a schematic representation of the pBELsLuc reporter plasmid stably integrated in the genome of the C33A2 cells. Transcription of the HPV16 sequences in the pBELsLuc plasmid is driven by the human cytomegalovirus promoter (CMV). The sLuc gene inserted into the L1 region is indicated and is preceded by the poliovirus 2A internal ribosome entry site (IRES). HPV16 E2 and E4 mRNAs mRNAs produced by C33A2 cells are indicated in light grey and HPV16 late mRNAs encoding sLuc that can be induced in this reporter cell line are indicated in black. (B and C) Fold induction of secreted luciferase enzyme activity (sLuc) in the cell culture medium of reporter cell line C33A2 treated with various concentrations of the indicated inhibitors over DMSO treated cells. sLuc activity was monitored at the indicated time points. sLuc activity is displayed as fold over DMSO-treated C33A2 cells at the various time points. (B) Results show the effect on C33A2 cells of inhibitors to the cellular kinases Akt (AZD5363), ERK1/2 (PDO325901) and Src (PP2). (C) Induction of sLuc activity by the Akt kinase inhibitor GDC0068. (D). Western blot of the Akt kinase, various phosphorylated versions of the Akt kinase and phosphorylated GSK3b in C33A2 cells in the absence or presence of Akt kinase inhibitor GDC0068 (100 uM) at different time points. (E) Western blot of the same kinases as in (D) in various cell lines treated with Akt kinase inhibitor GDC0068. Note that Akt kinase inhibitor GDC0068 locks the Akt kinase in an inactive, but phosphorylated state appearing as a band detected with antibodies to phosphorylated forms of Akt. (F) RT-PCR on total RNA extracted from C33A2 cells of the Akt1 mRNA in C33A2 cells transfected with siRNAs to the Akt1 kinase (si-Akt), compared to cells transfected with scrambled siRNAs (scr). (G) Relative sLuc activity induced by transfection of C33A2 cells with siRNAs to the Akt1 kinase (si-Akt), compared to cells transfected with scrambled siRNAs (scr).