Targeting DNA-PKcs by siRNA.

<p>293 cells are transfected with siRNA (100 pmole) targeting DNA-PKcs mRNA and non-specifically control siRNA using Lipofectamine™ 2000. Two days after transfection, cells were harvested. (A) RT-PCR for the detection of DNA-PKcs mRNA. Amplified cDNA fragments were separated on a 1% agarose gel. (B) Western blot for the detection of DNA-PKcs protein using beta-actin protein as an internal control.</p

rAAV replication in MO59K cells and 293 cell treated with wortmannin.

<p>Hirt DNA was purified two days after viral infection and subjected to Southern blot analysis. All samples were triplicated and hybridized with ³²P labeled CMV probe. Replicated forms of rAAV include double-stranded monomer (about 3.4 kb), or dimer (about 6.8 kb), and concatamers DNA (high molecular weight). Notice that treatment of wortmannin reduced rAAV replication in a dose-dependent manner in both MO59K cells (A) and in 293 cells (B).</p

Targeting DNA-PKcs reduced rAAV replication.

<p>293 cells were transfected with DNA-PKcs specific siRNA (DNA-PKcs siRNA) or control siRNA. Two days after transfection, cells were infected with rAAV-UF5 and rHSV, or transfected with pUF5 and pDG. rAAV replication was evaluated by Southern blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015073#pone-0015073-g001" target="_blank">Figure 1</a>. (A) Effect of DNA-PKcs siRNA on rAAV replication by Southern blot after vector infection. Concentration of DNA-PKcs siRNA and control siRNA was 100 pmole. (B) Densitometry analysis of data from Figure A. **, <i>P<0.001</i> for monomer; *, <i>P<0.05</i> for dimer and concatamers when compared with control siRNA group. (C) Dose dependent effect of DNA-PKcs siRNA on rAAV DNA replication. (D) Effect of DNA-PKcs siRNA on rAAV after vector and helper plasmid transfection. Left panel, without DpnI digestion; Right panel after. Note: DpnI digestion removes transfected plasmid DNA and shows all <i>de novo</i> replicated rAAV forms (monomer, dimer and concatamers).</p

AAT-ITR interacts with Ku proteins.

<p> (A) construction of AAV-ITR on a magnetic particle. (B) ITR on magnetic bead interacts with Rep78 and Ku proteins. AAV-ITR was bound to purified proteins or HeLa nuclear extract (NE) at room temperature or 37°C and then subjected to western blot analysis using antibodies toDNA-PKcs, Ku80 and Ku70. Rep78 are used as a positive control. (C) T-shaped closed ITR interact with Ku proteins in dose-dependent manner. AAV-ITRs on the bead were treated with Exonuclease III and incubated with different amount of HeLa NE (65µg/µl). The ITR binding proteins were subjected to western blot analysis for DNA-PKcs, Ku80 and Ku70. (D) Competition assay (right panel). When AAV-ITR on bead was incubated with HeLa nuclear extract, free AAV-ITR was added (2.5 fold) as a competitor. Streptavidin-coated magnetic beads and the beads with AAT-ITR (left panel) served as a control and showed addition of AAV-ITR increased pull down of Ku proteins.</p

Structure analysis of replicated DNA.

<p>(A) Map of the rAAV-UF5 vector; X (XbaI), S (SacI), and N (NotI), Two bold lines indicate the position of CMV and Neo^R probes. (B) All possible genome sizes generated from different AAV junctions. I. F., internal fragment; H-H, head-to-head; T-T, tail-to-tail; H-T, head-to-tail. (C and D) Southern hybridization probed with CMV (C), and Neo^R (D) after restriction enzyme digestion of Hirt DNA.</p

Human AAT inhibited pDC maturation and cytokine secretion.

<p>BM pDCs from B6 mice were differentiated with or without hAAT using Flt3L for 8 days then stimulated with 10μg/ml CpG for an additional 24 hr prior to FACS analysis. (A) CD80, (B) CD40, and (C) CCR9 expression. (D) TNF-α and (E) IL-6 levels in pDC culture media. P values of One-Way-ANOVA using Tukey’s post-hoc test are indicated as * P<0.05; ** P<0.01; *** P<0.001, n = 3.</p

Detection of endogenous mouse AAT, exogenous human AAT, and anti-human AAT neutralizing antibody in MRL/lpr mice (N = 10) by ELISA.

<p>(A) Mouse AAT levels (relative unit to C57BL/6) in PBS-treated MRL mice. (B) hAAT levels detected in hAAT injected group. Note: at week 2 and 8, animals were bled at 2 days after hAAT injection; at week 4, animals were bled at 1 day after the injection; at week 11, animals were bled 3 days after the injection. Dashed line is the lower limit of quantification (LLOQ). The serum concentration of human AAT from the PBS-treated group was below LLOQ. (C) Relative levels of anti-human AAT neutralizing antibody in MRL/lpr mice following multiple-dose human AAT administrations in MRL/lpr mice.</p

Human AAT inhibited cDC maturation and cytokine secretion.

<p>BM cDCs from B6 mice were generated in vitro in the presence of GM-CSF and IL-4 for 4 days with or without hAAT and then stimulated with 0.5 μg/ml LPS or 10μg/ml CpG for an additional 24 hr prior to FACS analysis. (A) CD80, CD86 and I-A^b expression (measured as mean fluorescence intensity, MFI) in B6 DCs stimulated with LPS. (B) TNF-α, IFNI, and IL-1β secretions in supernatants of B6 DCs stimulated with LPS. (C) CD80, CD86 and I-Ab expression in B6 DCs stimulated with CpG. (D) Secretion of TNF-α, IFN-I, IL-1β, and IL-6 by B6 DCs stimulated with CpG. P values of One-Way-ANOVA using Tukey’s post-hoc test are indicated as * P<0.05; ** P<0.01; *** P<0.001, n = 3.</p

<i>In vivo</i> hAAT treatment attenuated BMDCs differentiation and maturation in MRL/lpr mice.

<p>BMDCs from MRL/lpr mice treated with hAAT or PBS for 11 weeks were stimulated with LPS for 24 hrs. (A) Percentages of CD11c⁺ and (B) CD11b⁺CD11c⁺. (C-D) Percentages of CD80⁺ (C) and I-A (D) expressing BMDCs. P values of Student’s <i>t</i>-test are indicated as * P<0.05; ** P<0.01; *** P<0.001.</p

Human AAT inhibited autoantibodies production in MRL/lpr mice.

<p>Disease development was evaluated in MRL/lpr mice after 11 weeks of treatment with hAAT or PBS (n = 10 per group). (A) Interscapular lesion surface (cm²). (B) Lymph node weight (cervical, brachial, and inguinal). (C) Terminal serum IFN-I, (D) BAFF and (E) TNF-α. (F) Terminal serum anti-dsDNA IgG. (G) Serum ANA staining. Representative images are shown on the left, and FITC relative intensities are graphed on the right. * P<0.05, and*** P<0.001 by Student’s <i>t</i>-test.</p