Primers used for the PCR.

<p>^aAccession nr. AJ293656</p><p>Primers used for the PCR.</p

Localisation of the ZFN binding domains (set 1–3) in the sequence of PERV provirus.

<p>In addition, sequences of siRNA (gag2, pol1, pol2) successfully used for inhibition of PERV expression were indicated.</p

Expression of ZFN proteins in PK-15 cells.

<p><b>A.</b> Kinetic of expression of ZFN protein in PK-15 cells after nucleofection of different amounts (0.1 to 7.5 μg) of plasmids corresponding to ZFN set 1 (ZFN1, ZFN2, ZFN1/2). Both expressed proteins carry a 3xFLAG tag and for detection an antibody against the 3xFLAG tag was used. Cell lysate from PK-15 cells expressing an unrelated protein with a 3xFLAF tag and incubated for 2 days after nucleofection was used as positive control (Pos. ctrl). The arrows indicate the marker proteins. <b>B.</b> Detection of ZFN proteins in cytoplasmic and nuclear lysates. PK-15 cells were nucleofected with ZFN1 and ZFN2 plasmids together or separately and about 2 x 10⁶ cells per sample were fractionated. Each lane was loaded with nuclear or cytoplasmic extract from about 5 x 10⁵ cells. Whole cell lysate (WCL) from PK-15 cells transfected with pCMV, a vector with a FLAG tag was used as positive control (1 x 10⁵ cells). Anti-Flag antibodies were used for detection of the proteins. The purity of the cytoplasmic and nuclear fractions was analysed using antibodies against β-actin and DDX3, respectively.</p

Results of the surveyor nuclease assay.

<p><b>A.</b> PCR using genomic DNA as template for four different PCR (PCR1 = 645bp, PCR3 = 517 bp, PCR4 = 718 bp). After running on agarose gel, DNA concentration was estimated using ImageJ software. Lane 1–5, PK-15 cells transfected with 100 ng, 500 ng, 1 μg, 2 μg ZFN1/2 and 4 μg pLVTHM; lanes 8 and 12, PK-15 transfected with 2 μg ZFN1/2 each, lanes 9 and 13, PK-15 cells transfected with 4 μg pLVTHM. Lanes 6, 10 and 14 PERV-infected 293 cells transfected with 2 μg ZFN1/2 each and lanes 7, 11 and 15 with 4 μg pLVTHM. <b>B.</b> Agarose gel analysis of the rehybridisation of PCR amplicons shown in A. <b>C.</b> PAGE analysis of dehybridised and hybridised samples after incubation with 1 μl nuclease, 1 μl MgCl₂ and 1 μl enhancer for 20 minutes. The sample numbers correspond to the samples described in A. <b>D.</b> Analysis of the G/C control of the surveyor nuclease assay. Three different amounts (200, 400, 600 ng) of DNA were treated with nuclease as described by the manufacturer and analyzed on an agarose gel.</p

Interaction of ZFN1-CFP and ZFN2-YFP as shown by FRET confocal imaging.

<p><b>A.</b> FRET images. 293 cells were transfected with the vector pECFP-EYFP expressing CFP-YFP (row 1), pcDNA-CFP/pCMV-YFP expressing CFP and YFP (row 2) or pZFN1-CFP/pZFN2-YFP expressing ZFN1-CFP and ZFN2-YFP (row 3), fixed 24 h after transfection and then imaged in the following channels: Donor CFP (first column), FRET (second column) and acceptor YFP (third column). The last column displays a corrected and normalized FRET image. NFRET was calculated from the first 3 channels as described in the methods section. Scale bars, 20 μm. NFRET color lookup bar values range from black (0) to red (1). <b>B.</b> NFRET intensities of 9–16 cells were measured and the mean NFRET values ±SD are represented. <b>C.</b> Expression and localization of the separately transfected ZFN1 fused to CFP or ZFN2 fused to YFP, respectively.</p

Expression of the ZFN as detected by PLA.

<p>PK-15 cells were transfected with the pZFN1 or pZFN2 vectors expressing the 3xFLAG tagged fluorescent proteins ZFN1-CFP or ZFN2-YFP, the nucleus was stained by DAPI and a PLA against the FLAG epitope was performed demonstrating the specific localization of the ZFN in the nucleus. Scale bars, 5 μm.</p

Design of zinc finger nucleases targeting the PERV genes.

<p>^a Accession nr. AJ293656</p><p>^b Compared with positive control</p><p>Design of zinc finger nucleases targeting the PERV genes.</p

Cell viability after nucleofection with ZFN plasmids.

<p>PK-15, PERV-infected and uninfected 293 cells were nucleofected and at days 1, 3 and (in two experiments also 5) post nucleofection the cell number in the cultures was determined. <b>A.</b> PK-15 cells were nucleofected with both ZFN, ZFN1 and ZFN2, or with the control plasmid pLVTHM-GFP encoding GFP, or were left untreated. <b>B.</b> PERV-infected 293 cells were nucleofected with both ZFN, ZFN1 and ZFN2, or with GFP or were left untreated. <b>C.</b> PK-15 cells were left untreated or were nucleofected with different amounts of plasmids expressing ZFN1 and ZFN2 at day 0 and the cell number increased in dependence on the amount of plasmids. <b>D.</b> PK-15 cells were left untreated or were nucleofected with pLVTHM-GFP expressing GFP, with ZFN1 alone or ZFN2 alone and both ZFNs together. A double amount of DNA was used in case of transfection with a single plasmid. <b>E.</b> Uninfected 293 cells were nucleofected with pLVTHM-GFP (4μg) or ZFN1/ZFN2 (2 μg each) or were left untreated.</p

Donor-dependence of IL-10 and MMP-1 release.

<p>a, PBMCs from three donors were incubated with the same batch of the isu peptide polymer or medium and IL-10 release and MMP-1 mRNA expression were measured simultaneously. The donor-dependence of IL-10 release was shown using PBMCs from more than 50 donors some are shown in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s002" target="_blank">Figure S2</a>. b, Kinetic date of IL-10 release from PBMCs from a high responder donor A (column 1, dark grey) and low responder donor B (column 3, dark grey), both treated with the isu peptide homopopymer. Untreated medium control cells from both donors (Colum 2 and 4, light grey) did not release IL-10. The p values were calculated using the Student’s t-test, n = 3, p = 0.00015 in the case of a high responder and p = 0.03 in the case of a low responder.</p

Summary of the changes in cytokine expression.

<p>Cytokine release was measured 24 hrs or 3 days after incubation of normal PBMCs with isu-peptide homopolymers alone or in the presence of a mitogen, respectively. The full name of the abbreviated molecules is given in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s008" target="_blank">Table S4</a>.</p