Fig. 2

OCT1 Down-regulates Replication of HPV18 and HPV5 Genomes (A) OCT1 was overexpressed in U2OS cells. WCEs were analysed using immunoblotting and anti-OCT1 antibody 2 days post-transfection. GAPDH is shown as a loading control. (B) U2OS cells were cotransfected with either HPV18-Nluc or HPV5-Nluc genomes and the indicated amounts of the OCT1 expression construct. Nluc activity was measured in triplicate using the Live Cells Assay and normalized to viability values (MTS assay) 3 days post-transfection. Normalized Nluc activity in the control samples (cotransfected with an empty vector) was set as 1, and data from the OCT1-treated samples are expressed relative to the control. (C) The OCT1 expression vector at the amounts indicated was electroporated together with either HPV5 or HPV18 genomes per million U2OS cells, which were split into separate wells for acquiring multiple timepoints (shown by days). Total DNA was treated with DpnI and BglI or SacI restriction endonucleases to linearize the HPV18 and HPV5 genomes, respectively. The replication rate of HPV genomes was analysed using SB (left panel), and the obtained signals were quantified (right panels). (D) U2OS cells were transfected with the HPV18 genome together with carrier DNA, an empty vector or various quantities of the OCT1 expression vector. Cell cycle profiles were analysed using propidium iodide by flow cytometry 3 days post-transfection. (E) U2OS cells were transfected with the HPV18-E1^- genome and OCT1 expression construct. Total RNA was isolated 3 days post-transfection. Viral gene mRNA expression levels were analysed in triplicate using qPCR, normalized to GAPDH expression levels, and set as 1 in the samples cotransfected with an empty vector. Data from the OCT1-treated samples are presented relative to 1. All panels: data are presented as average means ± SDs of 3 independent experiments (* - p < 0.05; ** - p < 0.01; *** - p < 0.001)