Journal: Nature Communications

Article Title: Inflammation-induced Id2 promotes plasticity in regulatory T cells

doi: 10.1038/s41467-018-07254-2

Figure Lengend Snippet: Id2 is induced by IL-1β and IL-6 mediated signaling and downstream transcription factors STAT3, IRF4 and BATF in T reg cells. a Strategy for sorting T Naive , iT reg , and ex-Foxp3 T H 17 cells from Foxp3 Thy1.1 reporter mice (left). Flow cytometry analysis of IL-1r1 and IL-6rα among the T Naive , iT reg , and ex-Foxp3 T H 17 cells (right). b , c FACS-sorted iT reg cells were re-stimulated with cytokines as indicated and analyzed for Id2 and Foxp3 mRNA expression by RT-qPCR ( b ) as well as for Id2 protein levels by flow cytometry ( c ). d Cartoon depicting highly putative STAT3, IRF4, and BATF binding motifs upstream of Id2 transcription start site (TSS). e Cartoon depicting Id2 promoter constructs used for luciferase reporter assay. f Id2 promoter-luciferase construct (−582/+36) were co-transfected with the combination of STAT3, IRF4 and BATF expressing vectors in HEK-293 T cells. Lysates were prepared 30 h after transfection, and luciferase activities were measured with the reporter activities normalized to renilla luciferase activity. *P < 0.05, **P < 0.005, ***P < 0.001 (Student’s t -test). All data are representative three independent experiments with similar results (error bars, s.d.)

Article Snippet: 0.5 μg of the Id2 promoter-luciferase constructs or pGL4.17 empty vector was co-transfected with STAT3 (MR227265; Origene), IRF4 (MR226642; Origene) and BATF (MR222114; Origene) expression plasmids (HEK-293 T) and 1 μg of the Foxp3 promoter-luciferase construct was co-transfected with E2A (MG209745; Origene) and Id2 (MR200792; Origene) expression plasmids (Jurkat).

Techniques: Flow Cytometry, Expressing, Quantitative RT-PCR, Binding Assay, Construct, Luciferase, Reporter Assay, Transfection, Activity Assay