Fig. 3: CircKcnt2 suppresses Batf transcription.
From: An inducible circular RNA circKcnt2 inhibits ILC3 activation to facilitate colitis resolution
a Heatmap analysis according to differentially expressed TFs in circKcnt2+/+ and circKcnt2−/− ILC3s isolated from DSS-treated mice. b ILC3s were isolated from circKcnt2+/+ and circKcnt2−/− mice after DSS treatment, followed by RNA extraction and qRT-PCR analysis for top ten upregulated TFs. n = 3 independent samples. c Batf expression was tested by western blotting in circKcnt2+/+ and circKcnt2−/− ILC3s isolated from DSS-treated mice. d ILC3s from DSS-treated mice were lyzed and subjected to CHIRP assay using biotin-labeled probes against circKcnt2. Precipitated DNA fragments were extracted and enrichment of circKcnt2 on Batf promoter was analyzed by PCR. n = 3 independent samples. e CircKcnt2 linearized RNA was immobilized on NC membranes, followed by probing with indicated biotin-labeled DNA probes. f Luciferase reporter assay using Batf truncated promoters with overexpression of circKcnt2 or vector control. n = 3 independent samples. g CircKcnt2 linearized RNAs were immobilized onto membrane, followed by probing with biotin-labeled single-strand DNA. HR hairpin region. h A base pairing between Batf promoter (−1800 to −1600) and circKcnt2 HR4 was predicted. WT or mutant circKcnt2 HR4 RNAs were immobilized onto membrane, followed by probing with WT or mutant DNA probes. i, j ChIP assay was performed using anti-H3K27ac or anti-H3K27me3 with circKcnt2+/+ and circKcnt2−/− ILC3 cell lysates after DSS treatment (***P = 0.0003; ***P = 0.0006). n = 3 independent samples. k DNaseI accessibility assay was conducted using circKcnt2+/+ and circKcnt2−/− ILC3s from DSS-treated mice (*P = 0.0102). n = 3 independent samples. l CircKcnt2+/+ and circKcnt2−/− ILC3s were subjected to nuclear run-on assay, followed by detection of Batf transcription by RT-PCR analysis (***P = 0.0001). n = 3 independent samples. m CircKcnt2 enrichment on Batf promoter was tested by CHIRP assay using circKcnt2WT and circKcnt2Mut ILC3 lysates after DSS treatment (***P = 0.0005). n = 3 independent samples. n Batf expression was measured using western blotting in circKcnt2WT and circKcnt2Mut ILC3s after DSS treatment. o H&E staining of colon tissues from circKcnt2WTRag1−/− and circKcnt2MutRag1−/− mice treated with DSS. Scale bar, 100 μm. p Colitis scores of colon tissues in o (**P = 0.0053). n = 5 independent samples. q CircKcnt2WTRag1−/− and circKcnt2MutRag1−/− mice were treated with DSS and weight changes were monitored (***P < 0.0001). n = 5 independent samples. r ILC3s were isolated from DSS-treated circKcnt2WTRag1−/− and circKcnt2MutRag1−/− mice, followed by ELISA assay (**P = 0.0011). Veh vehicle. n = 3 independent samples. s ILC3s isolated from circKcnt2WT and circKcnt2Mut mice were transplanted into Rag1−/−Il2rg−/− mice. Two weeks later, recipient mice were administrated with DSS. Colons were isolated and analyzed by H&E staining. Scale bar, 100 μm. t Colitis scores of indicated mice treated as in s were analyzed (***P < 0.0001). n = 5 for each group. *P < 0.05, **P < 0.01, and ***P < 0.001. Data were analyzed by an unpaired Student’s t-test and shown as means ± SD. Data are representative of at least three independent experiments. Source data are provided as a Source Data file.
