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Figure 4

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IRF-1 is required for IFN-γ – mediated quiescence

(A) Whole cell lysates obtained from O-2A/OPCs treated for indicated times were probed for phospho-Rb, quantified, and normalized against total Rb expression. Total Rb protein was quantified and normalized to actin intensity. (B) Western blot analysis was performed for cyclin E and p27 and normalized to β-tubulin. (C, D) O-2A/OPCs were treated for the indicated times with control or IFN-γ and RNA was isolated for RT-qPCR of p27, IRF-1, and GAPDH. Error bars represent mean +/− SEM. p27 expression increased by 1 hour but was only significant at 3 days (p<0.001, t-test vs control-treated cells). IRF-1 gene expression was significantly greater at each time point tested (p<0.05, p<0.001, t-test vs control-treated cells). (E) O-2A/OPCs grown at clonal density were infected with lentivirus expressing shRNA constructs against p27, IRF-1 and a scrambled non-targeting control prior to control or IFN-γ treatment. IFN-γ significantly decreased self-renewal in scrambled shRNA infected cells (*p<0.05, t-test). p27 shRNA lentivirus infection had a significant effect on self-renewal in control treated cells(**p<0.001, t-test versus control treated scrambled shRNA infected cells). IFN-γ treatment reduced self-renewal in p27 shRNA-treated cells to control levels. IRF-1 shRNA abrogated IFN-γ – mediated quiescence. (F) Representative images of clones under each condition. Scale bar, 25 μm.

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