Sustained Egr1 expression and binding upstream of target genes in response to NGF versus EGF.

<p><b>(a)</b> PC12 cell cultures were treated with 50 ng/ml NGF or 25 ng/ml EGF for 0–4 h. Total cell lysates were harvested and subjected to SDS-PAGE and Western blot for Egr1. Egr1 levels were quantified by densitometry and converted to % maximum levels for ten independent experiments and plotted (average ± S.E.). <b>(b)</b> PC12 cultures were treated with or without 25 ng/ml EGF for 1 h and subjected to ChIP assay using antibodies against Egr1 or an IgG control. Real-time PCR was then conducted on the immunoprecipitated DNA using primers within 250 bp of predicted Egr binding sites (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170076#pone.0170076.g001" target="_blank">Fig 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170076#pone.0170076.s003" target="_blank">S2 Table</a> for primer locations and sequences). Primers to amplify a region approximately 100 bp upstream of the <i>Myog</i> gene were used as a negative control for Egr1 binding. Data are plotted as % input and are averages from three to six independent experiments ± S.E. *, One-tailed Student’s <i>t</i> tests were conducted comparing the % input value for <i>Myog</i> after Egr1 IP to the % input values for each predicted Egr target after Egr1 IP, which yielded <i>p</i> values ≤ 0.05. <b>(c)</b> PC12 cell cultures were treated with 50 ng/ml NGF or 25 ng/ml EGF for 0–4 h and subjected to ChIP with anti-Egr1 antibody. Immunoprecipitated DNA was then subjected to real-time PCR with primers to detect Egr1 binding to a subset of its target genes, which was quantified as % input. Percent input values were then converted into % maximum values, which were plotted. *, One-tailed Student’s <i>t</i> tests were conducted comparing the % input values at each time point after NGF versus EGF treatment, which yielded <i>p</i> values ≤ 0.05 at the 3-hour time point for <i>Arc</i>, <i>Kctd11</i>, <i>Trib1</i>, <i>Tph1</i>, and <i>Vgf</i>.</p

Predicted Egr binding sites and ChIP primer locations upstream of genes preferentially expressed during sustained ERK signaling in response to NGF.

<p>Of the 69 genes that Mullenbrock et al. (2011) determined are preferentially expressed during sustained ERK signaling in response to NGF, 21 contained putative Egr binding sites within 5 kb upstream of their transcription start site (TSS). The locations of each Egr binding site are denoted by the vertical blue lines. The red horizontal lines denote the relative locations of primer sets used for real time PCR to detect Egr binding to nearby Egr binding site(s). For genes with multiple dispersed Egr binding sites, multiple primers sets were designed (denoted P1, P2, etc.) to detect Egr binding to the nearest predicted Egr binding site.</p

AP-1, CREB, and Egr1 cooperatively regulate 28 genes during their preferential expression in response to NGF and sustained ERK signaling.

<p>The Venn diagram summarizes genes bound by AP-1, CREB, and/or Egr1 during their preferentially expression in response to NGF and sustained ERK signaling, as detected by Mullenbrock et al. (2011) and the present study.</p

Combined pharmacokinetic monitoring of MSCs and MSC-secreted IL-6.

<p>(A) Bioluminescent images of C57Bl/6 mice over a period of three days after IV cell administration of one million luciferase-engineered human MSCs. (B) Photon flux of bioluminescent signal over time after IV cell administration. Durable BLI signals were detected up to 24 hours in mice that were injected IV with MSCs. (C) Serum ELISA measurements of human IL-6 released by IV cell transplants over time. Time points for serum and imaging analyses were 0.5, 8, 24, and 72 hours after cell injection. Pooled mouse serum was serially analyzed as batches of N = 5.</p

Enhanced delivery of IL-6 by MSC transplants compared to MSC conditioned medium.

<p>(A) Serum profiles of human IL-6 after IV administration of concentrated conditioned medium into C57Bl/6 mice. The plot was normalized to the dose of conditioned medium that was contributed by 1×10⁶ cells. Pharmacokinetic parameters (B) Cmax, (C) AUC, (D) Tmax, (E) Half-life, and (F) Elimination constant were calculated for IL-6 exposure by cell transplants compared to CM administration. Significant differences between cell transplants compared to CM whereby higher levels of IL-6 and longer artificial duration was observed in plasma after cell transplantation. Time points for serum analyses were 0.5, 8, and 24 hours after cell or media injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P>0.01.</p

Golgi-dependent secretion mechanism of MSC-derived IL-6 in vivo.

<p>Brefeldin A pre-treatment of MSCs was used to evaluate blockade of IL-6 release in vitro and in vivo. (A) MTT assay of MSCs treated at different concentrations of brefeldin. A non-toxic dose of 5 ug/ml was used for functional studies. (B) Human IL-6 levels in vitro after brefeldin pre-treatment. Significant reduction in 24 hour release of IL-6 was observed across all doses. (C) Alteration in serum IL-6 delivery by MSCs pretreated with a Golgi-apparatus inhibitor, Brefeldin A. MSCs were incubated with 5 µg/ml of BFA for one day and then injected into C57Bl/6 mice and compared to untreated MSCs in terms of serum IL-6 delivery. Brefeldin treatment of MSCs led to diminished release of human IL-6 in vitro and in vivo. (D) Area-under-curve analysis of human IL-6 after MSC pre-treatment with brefeldin A and transplantation. Exposure to IL-6 was significantly reduced by inhibition of the Golgi apparatus. Time points for serum analyses were 0.5, 8, and 24 hours after cell injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P>0.01.</p

Blood Monitoring of Engineered Human MSCs with the Secreted Gaussia Luciferase Reporter.

<p>A lentivirus vector expressing GLuc and GFP was transduced into human MSCs at a confluence of 70% and multiplicity of infection of 4∶1 in complex with 8 ug/ml of polybrene. (A) GFP micrograph and (B) flow cytometry showing high expression level and therefore transduction efficiency of construct. (C) The activity of GLuc was successfully measured in MSCs conditioned medium using a luminometer. (D) Five different engineered cell lines were infused into NOD-SCID mice and serum was individually collected at 0.5, 8, 24, 72, and 168 hours after cell injection in batches of N = 5 per study. MSCs constitutively expressing GLuc were detected in many cases over a week in duration.</p