Multiple compounds improve aged MDSPC proliferation (self-renewal).

<p>(A) Aged MDSPCs were cultured in medium containing aspirin, nordihydroguaiaretic acid (NDGA), or rapamycin, and proliferation was monitored by live cell imaging for up to 60 hours, or (B) cells were treated in fusion medium and assessed for myogenic differentiation after 5 days (*p≤0.05 Aged WT vs Treated; ANOVA followed by Tukey post-hoc analysis).</p

NF-κB inhibition decreases senescence in BM-MSCs.

<p>BM-MSCs were treated for 48 hours with IKK inhibitor VII and then stained for senescence-associated beta-galactosidase (SA β-gal) expression, which demonstrated a decrease in cell senescence (*p≤0.05, T-test).</p

NF-κB inhibition improves the oxidative stress resistance of MDSPCs isolated from old mice.

<p>MDSPCs were exposed to 250 μM H₂O₂ and monitored by live cell imaging for up to 24 hours. IKKi treated aged WT MDSPCs and aged <i>p65</i>^+/- MDSPCs demonstrated improved survival compared to vehicle treated aged WT MDSPCs (*p≤0.05 Aged WT vs +IKKi; +p≤0.05 Aged WT vs Aged <i>p65</i>^+/-; ANOVA followed by Tukey post-hoc analysis).</p

NF-κB inhibition improves the differentiation of MDSPCs isolated from old mice.

<p>(A) Brightfield images demonstrate a dose dependent increase in myotube formation following IKKi administration (10x images). (B) Immunofluorescent staining for MyHC demonstrated significantly improved differentiation following treatment at the 5μM dose (20x magnification), quantified in (C). (D) The total number of nuclei per field of view for experiment in B, C (*p≤0.05; ANOVA followed by Tukey post-hoc analysis). (E) IKKi treatment of young WT MDSPCs did not significantly improve differentiation (*p≤0.05, T-test).</p

Correction of <i>XPF</i> mutant cell NER defect by microinjection of XPF-ERCC1.

<p>Primary fibroblasts from XFE progeroid patient XP51RO were fused to create homopolykaryons by treatment with inactivated Sendai virus then plated on glass coverslips. Only homopolykaryons were injected with recombinant XPF-ERCC1 protein complex (A) wild-type (B) XPF^R799W-ERCC1 (C) XPF^R153P-ERCC1. The cultures were irradiated with 10 J/m² UV-C and ³H-thymidine was added to the culture. UV-induced unscheduled DNA synthesis was detected by autoradiography. Homopolykaryons are indicated with arrows. (D) Histogram indicating the average number of radiographic grains in nuclei injected with each of the recombinant protein complexes and uninjected cells in the same sample. Error bars indicate the standard deviation. N indicates the number of nuclei analyzed in each population. P values for the comparison between injected and uninjected cells were calculated using an unpaired two-tailed Student's t-test.</p

Characteristics of <i>XPF</i> mutant cell lines.

<p>UDS unscheduled DNA synthesis</p><p>*The patient had normal levels of <i>XPF</i> transcript, suggesting one allele encodes a full-length mRNA.</p>♦<p>Mutation could not be confirmed on genomic DNA.</p><p>° Siblings.</p><p>n.d.  =  not determined</p

Characterization of XPF-YFP and XPF¹⁵³-YFP in CHO cells.

<p>(A) Western blot analysis of XPF-YFP expressed in <i>Xpf</i> mutant cells. XPF-deficient hamster cell line, UV41, was transiently transfected with wild type <i>XPF-YFP</i> or <i>XPF¹⁵³-YFP</i> and the fusion proteins were detected using an antibody against XPF or GFP. C5RO was used as positive control for the XPF blot and as a negative control for the GFP blot. UV41 cells transfected with YFP alone was used as a negative control for XPF blot and as a positive control for GFP blot. (B) Clonogenic survival of wild-type (wt), XPF-deficient CHO cell line UV41, and UV41 transfected with wild type XPF-YFP and XPF¹⁵³-YFP after UV and MMC treatment. Colonies were counted 7–10 days after treatment and results are plotted as mean 3 independent experiments. (C) Subcellular localization of wild type XPF-YFP and XPF¹⁵³-YFP after transient transfection in XPF-deficient the CHO cell line UV41 detected by fluorescence microscopy.</p

Biochemical characterization of XPF^R153P-ERCC1 and XPF^R799W-ERCC1 mutants.

<p>(A) Gel filtration profiles from the purification of recombinant XPF-ERCC1, XPF^R153P-ERCC1 and XPF^R799W-ERCC1 from baculovirus-infected Sf9 insect cells using a His₆ tag on ERCC1. Aggregated proteins elute at ∼45 ml in the void volume of the column; heterodimeric XPF-ERCC1 elutes at ∼65 ml, corresponding to ∼200 kD, and monomeric ERCC1 elutes at ∼78 ml (∼50kD). (B) SDS PAGE analysis of purified protein complexes. Lane 1, 3 and 5 (D): XPF-ERCC1, XPF^R153P-ERCC1 and XPF^R799W-ERCC1, respectively, after purification over NTA-agarose, gel filtration and heparin columns. Lanes 2 and 4 (A) show the proteins present in the fractions eluting at 45 ml in the gel filtration column step of XPF^R153P-ERCC1 and XPF^R799W-ERCC1, respectively. (C) Immunodetection of XPF in normal (C5RO) and <i>XPF</i> mutant cells. The star indicates the migration of a cross-reactive band demonstrating equal loading <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000871#pgen.1000871-Niedernhofer2" target="_blank">[32]</a>. (D) Incision activities of XPF-ERCC1, XPF^R153P-ERCC1 and XPF^R799W-ERCC1 (200 fmol) on a 5′-³²P-labeled stem-loop DNA substrate (100 fmol) in the presence of either 0.4 mM MnCl₂ (lanes 2, 4 and 6) or 2 mM MgCl₂ (lanes 3, 5 and 7). Reactions were analyzed on a 15% denaturing polyacrylamide gel. The 46-mer substrate and 9–10-mer products are indicated.</p

Development of novel NEMO-binding domain mimetics for inhibiting IKK/NF-κB activation

<div><p>Nuclear factor κB (NF-κB) is a transcription factor important for regulating innate and adaptive immunity, cellular proliferation, apoptosis, and senescence. Dysregulation of NF-κB and its upstream regulator IκB kinase (IKK) contributes to the pathogenesis of multiple inflammatory and degenerative diseases as well as cancer. An 11–amino acid peptide containing the NF-κB essential modulator (NEMO)-binding domain (NBD) derived from the C-terminus of β subunit of IKK, functions as a highly selective inhibitor of the IKK complex by disrupting the association of IKKβ and the IKKγ subunit NEMO. A structure-based pharmacophore model was developed to identify NBD mimetics by in silico screening. Two optimized lead NBD mimetics, SR12343 and SR12460, inhibited tumor necrosis factor α (TNF-α)- and lipopolysaccharide (LPS)-induced NF-κB activation by blocking the interaction between IKKβ and NEMO and suppressed LPS-induced acute pulmonary inflammation in mice. Chronic treatment of a mouse model of Duchenne muscular dystrophy (DMD) with SR12343 and SR12460 attenuated inflammatory infiltration, necrosis and muscle degeneration, demonstrating that these small-molecule NBD mimetics are potential therapeutics for inflammatory and degenerative diseases.</p></div

The IC₅₀ of NBD mimetics.

<p>Inhibitory effects of NBD mimetics on NF-κB activation were measured by luciferase assays at multiple concentrations: 0, 25, 50, 100, and 150 μM. IC₅₀ of NBD mimetics was determined based on the dose-dependent curve by using GraphPad.</p