VOLUME 291 (2016) PAGES 24607–24617
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There were several errors in the original figure legend to Fig. 4. The figure legend should be replaced with the following.
FIGURE 4. MSC-derived EVs activate NF-κB and Notch signaling to promote HSPC differentiation. A, diagrammatic representation of the lentiviral vector design used to generate the NF-κB enhancer-luciferase reporter cell line. Stably transduced and sorted SIM-A9 microglial cells were used for EV exposure followed by luciferase assay. The SIM-A9-NF-κB-Luc cells were exposed with MSC EVs and incubated for 48 h followed by luciferase assay. Non-treated SIM-A9-NF-κB-Luc and LPS-treated SIM-A9-NF-κB-Luc cells were used as negative and positive controls, respectively. Luciferase activity was measured as relative fluorescence units (RFU). Data represent n = 4 independent experiments; error bars depict S.D. All p values have been calculated using two-tailed Student's t test. B, quantitative mRNA expression of NF-κB and Notch-1 signaling and downstream targets involved in cell proliferation in MSC EV-exposed HSPCs. Data represent n = 3 independent experiments; error bars are S.D. C, we next tested a candidate panel of canonical TLR4 responsive cytokines for both transcriptional activation and secretion, either after EV or S100 exposure. Transcriptional analysis of EV-exposed HSPCs reveals an up-regulation of several known TLR4-responsive cytokine genes (IL6, TNFa, Stat1, and EGFR) in WT but not MyD88−/− HSPCs. Data represent n = 3 independent experiments; error bars are S.D. D, TLR4 signaling was specifically inhibited in WT MSC-EV-exposed HSPCs using TAK-242. Transcriptional analysis of EV-exposed HSPCs with and without TLR4 inhibitor showed down-regulation of the same TLR-responsive genes described above. Data represent n = 2 independent experiments; error bars represent S.D. E, cytokines released by HSPCs after 48 h co-culture, with MSC EVs compared with S100, were measured using a Luminex assay, whereby error bars are S.D. and p values are an indication of the variance across three technical replicates in this screening panel.