Figure S6 . " width="100%" height="100%">
Journal: Immunity
Article Title: Indoleamine 2,3-dioxygenase 1 activation in mature cDC1 promotes tolerogenic education of inflammatory cDC2 via metabolic communication
doi: 10.1016/j.immuni.2022.05.013
Figure Lengend Snippet: AhR cooperates with RelB to induce IDO1 in isolated cDC2 treated with l -kynurenine (A) Heatmap of tryptophan metabolic enzymes gene expression in cDC2 from AhR f/f mice versus Ahr f/f Vav1 iCre mice treated as depicted. Bright blue (lowest) to bright red (highest). (B) IDO1 immunoblot in cDC2 treated as shown for 48 h (n = 4). (C) IL-6 analysis in supernatants of cDC2 treated as in (A) for 48 h (n = 4). (D) AhR enrichment at the κB sequences of Il6 promoter by ChIP in sorted cDC2 untreated or treated overnight with LPS and conditioned for 2 h with l -kynurenine (n = 2). (E) IDO1 immunoblot in purified cDC2 were transfected and treated as depicted for 48 h (n = 4). (F) l -kynurenine uptake in purified cDC2. MFI is shown (n = 4). (G) IDO1 expression by IS in cDC2 treated as (A) in the presence or absence of BCH for 48 h (n = 5). (H) Predicted canonical AhR binding sites in the murine Ido1 promoter. (I) MFI of GFP expression in pre-gated as Thy1.1 + cDC2 transduced with RV vector containing regions as described in (H). Gray histograms show empty reporter (n = 4). (J) Quantification of the +1,340 bp and +1,601 bp Ido1 enhancer activity in WT and Ahr − / − cDC2s using retroviral reporters as in (I) (n = 3). (K) ChIP-PCR analysis of AhR binding on +1,340 bp and +1,601 bp Ido1 enhancer elements in cDC2 treated as in (C) (n = 3). (L) IDO1 expression by IS in gated CCR7 + cDC2 derived from Rosa26 Cas9−GFP/+ c-Kit hi progenitors infected with RV expressing sgRNAs targeting +1,340 bp and +1,601 bp Ido 1 enhancer elements treated as shown for 48 h (n = 2). (M) Schematic representation of BM chimera model. (N) IDO1 expression by IS in cDC1 and cDC2 from BMDC derived as in (M) and cultured as shown (n = 3). (O) ChIP-PCR analysis of RelB binding on +1,340 bp and +1,601 bp Ido1 enhancer elements in cDC2 treated as in (C) (n = 3). (P) AhR and RelB Immunoblot in purified cDC2 treated as in (K) where AhR was immunoprecipitated (n = 3). (Q) AhR and RelB interaction in purified cDC2 treated as indicated by PLA. Red spots show a single AhR/RelB interaction. Scale bars, 10 μm (n = 3). Data are shown as means ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, one-way (L) or two-way (C, D, G, J, K, N, and O) ANOVA followed by Bonferroni multiple comparison test. Dots represent a biological replicate (G and N). β-actin used as loading control (B, E, and P). Data are normalized to IgG control (D, K, and O). Please also see Figure S6 .
Article Snippet: Rabbit monoclonal anti-mouse RelB (clone C1E4) , Cell Signaling , Cat#: 4922; RRID: AB_2179173.
Techniques: Isolation, Gene Expression, Western Blot, Purification, Transfection, Expressing, Binding Assay, Transduction, Plasmid Preparation, Activity Assay, Retroviral, Derivative Assay, Infection, Cell Culture, Immunoprecipitation, Comparison, Control
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