Fig. 7. Up-regulation of NAC1 by proinflammatory cytokines breaks immune tolerance via down-regulation of FoxP3.
(A) NAC1 and FoxP3 in Tregs from the FIR reporter mice by immunoblots. (B) NAC1 and FoxP3 mRNA of WT Tregs generated in vitro and with ectopic expression of NAC1 by reverse transcription polymerase chain reaction. ***P < 0.001. (C) FoxP3 protein of the sorted Tregs generated in vitro and with ectopic expression of NAC1 by immunoblots. (D) Immunofluorescence staining of 4′,6-diamidino-2-phenylindole, NAC1, and FoxP3 in Tregs generated in vitro. (E) Expressions of FoxP3 and NAC1 in Tregs from WT and NAC1−/− mice by immunoblots. (F) FoxP3 was immunoprecipitated and examined for its acetylation of lysine. (G) WT Tregs were cultured in the presence of various cytokines for 12 hours, and then the expressions of FoxP3 and NAC1 were analyzed by immunoblots. (H) FoxP3 from WT Tregs treated in (G) was immunoprecipitated and examined for its acetylation. (I) WT or NAC1−/− Tregs were treated with cycloheximide (150 μg/ml) and chased for the indicated hours. FoxP3 protein level was analyzed by immunoblotting. (J) HDAC expression. (K) HDAC9 and FoxP3 of the sorted WT or NAC1−/− Tregs generated in vitro and with ectopic expression of either the control Mig vector or human HDAC9 vector were analyzed by immunoblots. All the data shown are the representative of three identical experiments. (L) Proposed model of regulation of FoxP3 by NAC1 in immunity.