Fig. 3

POU-HD TFs Inhibit Replication of HPV18, HPV5, and HPV11 Genomes (A) U2OS cells were transfected with the appropriate expression vector and incubated for 2 days, and WCEs were prepared. Equal amounts of the WCEs were incubated with 32-P-labelled double-strand oligonucleotides for 30 min and resolved on 4% PAAG. The relative mobility of different protein-DNA complexes was deduced from published scientific literature. Right panel: the image has been cropped for clarity of the presentation. (B) U2OS cells were cotransfected with viral genomes and either an empty vector or the indicated expression constructs using 500 ng of the plasmids per 10⁶ cells. Cells were split after transfection to acquire different time points and incubated for 2, 3, and 4 days. Total DNA was purified, treated with the restriction enzymes DpnI and BglI, SacI or HindIII to linearize the HPV18, HPV5, and HPV11 genomes, respectively, and analysed using SB. (C) HPEKs were transfected with the HPV18-Nluc genome and the indicated expression constructs or an empty vector. The days after transfection, HPEKs were treated with 1.5 mM CaCl₂ to induce differentiation. Nluc activity was measured 3 days post-transfection, normalized to alkaline phosphatase values, and set as 100% in the control samples transfected with an empty vector. Data from other samples are presented as a percentage ± SD of the control (n = 3, * - p < 0.05, *** - p < 0.001)