Fig 2: Model of ADAM‐17/EGFR signalling axis activation induced by deoxycholic acid in intestinal carcinogenesis. DCA stimulates ADAM‐17 activation and AREG release, which is required for EGF receptor activation, EGFR/Akt signalling pathway activation and apoptosis resistance in intestinal tumour cells. ADAM‐17, a disintegrin and metalloprotease domain‐containing protein 17; AREG, amphiregulin; DCA, deoxycholic acid; EGFR, epidermal growth factor receptor [Colour figure can be viewed at http://wileyonlinelibrary.com]

Fig 3: GPR174 regulates neovascularization by inhibiting AREG expression in Tregs.a Heatmap of RNA-seq analysis of the ischemic muscles of WT and Gpr174-/Y mice 7 days after HLI (n = 3) (q-value < 0.05; |log2Fc| > 1). b Serum AREG, IL-10, and VEGF protein content in Rag1-/- mice receiving Tregs 7 days after adoptive transplantation experiments (n = 6 for PBS ? Rag1-/- mice; n = 7 for wild-type Tregs?Rag1-/- mice; n = 7 for GPR174-deficient Tregs?Rag1-/- mice). c Relative mRNA levels of Areg, Il-10, and Vegf in Tregs sorted from the gastrocnemius tissues of WT and Gpr174-/Y mice 7 days after HLI (n = 4). d Representative immunofluorescent images of AREG (red) and DAPI (blue) staining in Tregs isolated from ischemic muscle of WT and Gpr174-/Y mice 7 days after HLI. Scale bar, 10 µm. e, f Representative images and quantification of vascular sprouting of vessel segments cocultured with AREG neutralizing antibody and Tregs isolated from WT and Gpr174-/Y mice for 5 days (n = 5). Scale bar, 200 µm. g, h Representative Laser Doppler images and quantification of hindlimb blood perfusion in WT and Gpr174-/Y mice injected with AAV9-shAreg at indicated times after HLI (n = 7 for WT mice + AAV9-shN and Gpr174-/Y mice + AAV9-shN; n = 8 for WT mice + AAV9-shAreg and Gpr174-/Y mice + AAV9-shAreg). i, j Representative immunofluorescent images of CD31 (i) staining and quantification of CD31 (j) in muscle cross sections (n = 6). Scale bar, 50 µm. k, l Representative immunofluorescent images of aSMA (k) staining and quantification of lumen perimeter (l) in muscle cross sections (n = 6). Scale bar, 50 µm. For all statistical plots, the data are presented as mean ± SD. One-way ANOVA with Bonferroni multiple comparisons test in (b). Two-way ANOVA with Bonferroni multiple comparisons test in (c, f, j, l). Two-way repeated measures ANOVA with Sidak’s multiple comparisons test in (h). Source data are provided as a Source Data file.

Fig 4: GPR174 downregulates AREG expression by inhibiting EGR1 nuclear localization.a Top transcription factors of AREG. b Egr1 mRNA expression in Tregs isolated from the muscle tissues of WT and Gpr174-/Y mice 7 days after HLI (n = 5). c Representative immunofluorescent images of EGR1 (red), and DAPI (blue) staining in Tregs isolated from the ischemic muscles of WT and Gpr174-/Y mice 7 days after HLI. Scale bar, 10 µm. d Quantification of nuclear localization of EGR1 (n = 5). e Areg mRNA expression in WT or GPR174-deficient Tregs treated with shEgr1 and non-ischemic or ischemic muscle lysates (n = 4). For all statistical plots, the data are presented as mean ± SD. Two-tailed unpaired t-tests in (d). Two-way ANOVA with Bonferroni multiple comparisons test in (b, e). Source data are provided as a Source Data file.

Fig 5: GPR174 deficiency mitigates the inflammation in ischemic muscles through AREG.a Representative flow cytometric dot plots of leukocyte populations in gastrocnemius tissues of WT and Gpr174-/Y mice 7 days after HLI. b Quantification of all leukocytes, neutrophils, total macrophages, Ly6Clow macrophages, and Ly6Chigh macrophages in WT and GPR174-deficient gastrocnemius 7 days post HLI (n = 7 for WT mice; n = 8 for Gpr174-/Y mice). c Scheme of Tregs-macrophages co-culture. d Relative mRNA levels of proinflammatory (upper panel) and anti-inflammatory (lower panel) genes in macrophages co-cultured with Tregs and AREG neutralizing antibodies for 24 h (n = 4). e Quantification of bioactive TGF-ß and VEGF in macrophages stimulated with recombinant AREG in the presence or absence of inhibitors for the EGFR (Gefitinib) for 24 h (n = 8). For all statistical plots, the data are presented as mean ± SD. Two-tailed unpaired t-tests in (b). Two-way ANOVA with Bonferroni multiple comparisons test in (d, e). Source data are provided as a Source Data file.