Figure 3: WASH promotes AHR expression in NKp46⁺ ILC3s.

(a) Expression levels of the indicated genes were examined in the indicated cells sorted from the indicated mice by RT-PCR analysis. (b) AHR expression levels were examined in the indicated ILC3 subsets from the indicated mice. For a and b, n=5. (c) Sorted NKp46⁺ ILC3 cells were subjected to ChIP assay with antibody against WASH, followed by detection of Ahr promoter (right panel) with different primers shown as in the left panel. (d) The indicated ILC3 subsets were subjected to ChIP assay with anti-WASH antibody, followed by detection of Ahr promoter through PCR. (e) The indicated ILC3 subsets were subjected to nuclear run-on assay, followed by RT-PCR analysis of Ahr. (f) Vector or WASH overexpressing ILC3 subsets were subjected to nuclear run-on assay, followed by RT-PCR analysis of Ahr. For c–f, n=5. (g) pGL3-AHR promoter expressing NK92 cells were transfected with the indicated WASH variants with or without cytochalasin D, followed by luciferase assays (upper panel). Endogenous expression levels of AHR were examined by immunoblotting with anti-AHR antibody (lower panel). (h) In situ hybridization of Ahr promoter and WASH in sorted NKp46⁺ ILC3 cells (left panel). White arrow head denotes Ahr promoters colocalized with WASH. Percentages of cells with WASH colocalized with Ahr promoter were calculated (right panel). At least 200 NKp46⁺ ILC3 cells were counted. Scale bar, 5 μm. (i,j) ChIP analysis of Ahr promoter in NKp46⁺ ILC3 cells sorted from the indicated mice with antibodies against H3K9K14ac (i) or H3K4me3 (j). (k) NKp46⁺ ILC3 nuclei of the indicated mice were treated with indicated units of DNase I. DNA was extracted and examined by PCR (lower panel). Intensities of PCR products were calculated (upper panel). (l) ChIP analysis of Ahr promoter in NKp46⁺ ILC3 cells sorted from the indicated mice with anti-H3K27me3 antibody. (m) NKp46⁺ ILC3 cells sorted from the indicated mice were subjected to nuclear run-on assay, followed by RT-PCR analysis of Ahr. For (i–m), n=9. Data are shown as means±s.d. *P<0.05; **P<0.01; ***P<0.001. Data are representative of at least three independent experiments.