Journal: Nature Communications

Article Title: GPR174 knockdown enhances blood flow recovery in hindlimb ischemia mice model by upregulating AREG expression

doi: 10.1038/s41467-022-35159-8

Figure Lengend Snippet: 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; |log 2 Fc| > 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-sh Areg at indicated times after HLI ( n = 7 for WT mice + AAV9-sh N and Gpr174 −/Y mice + AAV9-sh N ; n = 8 for WT mice + AAV9-sh Areg and Gpr174 −/Y mice + AAV9-sh Areg ). 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 αSMA ( 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.

Article Snippet: The levels of AREG and IL-10 in serum of recipient mice 10 days after HLI were determined using Mouse amphiregulin ELISA kit (BOSTER, EK0591) and Mouse IL-10 ELISA kit (ABclonal, RK00016).

Techniques: RNA Sequencing, Muscles, Transplantation Assay, Staining, Isolation, Injection

Journal: Nature Communications

Article Title: GPR174 knockdown enhances blood flow recovery in hindlimb ischemia mice model by upregulating AREG expression

doi: 10.1038/s41467-022-35159-8

Figure Lengend Snippet: a Representative immunofluorescent image of CD31 (red), Ki67 (green) and DAPI (blue) and quantification of CD31 + Ki67 + cells in muscle cross sections of 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). Scale bar, 50 μm. b Representative immunofluorescent images of CD31 (red), TUNEL (green), and DAPI (blue) staining and quantification of CD31 + TUNEL + cells in muscle cross sections of 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). Scale bar, 50 μm. c Representative immunofluorescent images of CD31 (red), Ki67 (green) and DAPI (blue) and quantification of CD31 + Ki67 + cells in muscle cross sections of WT and Gpr174 −/Y mice injection with AAV9-sh Areg 14 days after HLI ( n = 6 for WT mice + AAV9-sh N and WT mice + AAV9-sh Areg ; n = 7 for Gpr174 −/Y mice + AAV9-sh N and Gpr174 −/Y mice + AAV9-sh Areg ). Scale bar, 50 μm. d Representative immunofluorescent images of CD31 (red), TUNEL (green), and DAPI (blue) staining and quantification of CD31 + TUNEL + cells in muscle cross sections of WT and Gpr174 −/Y mice injection with AAV9-sh Areg 14 days after HLI ( n = 6 for WT mice + AAV9-sh N and WT mice + AAV9-sh Areg ; n = 7 for Gpr174 −/Y mice + AAV9-sh N and Gpr174 −/Y mice + AAV9-sh Areg ). Scale bar, 50 μm. e Representative images of capillary-like structures and quantification of branch points and total tube length in mouse aortic endothelial cells stimulated with recombinant AREG for 16 h ( n = 6). Scale bar, 200 μm. For all statistical plots, the data are presented as mean ± SD. One-way ANOVA with Bonferroni multiple comparisons test in ( a , b ). Two-way ANOVA with Bonferroni multiple comparisons test in ( c , d ). Two-tailed unpaired t-tests in ( e ). Source data are provided as a Source Data file.

Article Snippet: The levels of AREG and IL-10 in serum of recipient mice 10 days after HLI were determined using Mouse amphiregulin ELISA kit (BOSTER, EK0591) and Mouse IL-10 ELISA kit (ABclonal, RK00016).

Techniques: Transplantation Assay, TUNEL Assay, Staining, Injection, Recombinant, Two Tailed Test

Journal: Nature Communications

Article Title: GPR174 knockdown enhances blood flow recovery in hindlimb ischemia mice model by upregulating AREG expression

doi: 10.1038/s41467-022-35159-8

Figure Lengend Snippet: 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, Ly6C low macrophages, and Ly6C high 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-β 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.

Article Snippet: The levels of AREG and IL-10 in serum of recipient mice 10 days after HLI were determined using Mouse amphiregulin ELISA kit (BOSTER, EK0591) and Mouse IL-10 ELISA kit (ABclonal, RK00016).

Techniques: Co-Culture Assay, Cell Culture, Recombinant, Two Tailed Test

Journal: Nature Communications

Article Title: GPR174 knockdown enhances blood flow recovery in hindlimb ischemia mice model by upregulating AREG expression

doi: 10.1038/s41467-022-35159-8

Figure Lengend Snippet: 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 sh Egr1 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.

Article Snippet: The levels of AREG and IL-10 in serum of recipient mice 10 days after HLI were determined using Mouse amphiregulin ELISA kit (BOSTER, EK0591) and Mouse IL-10 ELISA kit (ABclonal, RK00016).

Techniques: Expressing, Isolation, Staining, Muscles, Two Tailed Test

Journal: Acta Biochimica et Biophysica Sinica

Article Title: HDAC11 in ovarian granulosa cells coordinates LH in the maturation of oocytes in Tan sheep

doi: 10.3724/abbs.2025036

Figure Lengend Snippet: Expressions and secretion of AREG, EREG and BTC in cultured granulosa cells of Tan sheep by HDAC11 inhibition (A) Expressions of AREG, EREG, BTC were verified by western blot analysis. (B) The quantification of the protein levels of panel (A). (C) Protein quantification of AREG, EREG and BTC at 24 h by ELISA. (D–F) The expression levels of AREG (D), EREG (E) and BTC (F) in cultured granulosa cells after HDAC11 inhibition. Scale bar = 200 μm. (G) The quantification of the fluorescence intensity of panel (D–F). All experiments were performed in triplicate and the data were presented as the mean ± SEM. ns, no significance, *P < 0.05, **P < 0.01.

Article Snippet: Subsequently, the membranes were incubated for 1 h at room temperature with 5% BSA, followed by an overnight incubation with the primary antibodies against AREG (PU595967S, 1:1000; ABmart, Shanghai, China), EREG (A16372, 1:1000; Abclonal, Wuhan, China), BTC (DF7038, 1:1000; Affinity), PCNA (10205-2-AP, 1:1000; Proteintech, Wuhan, China), H3K9ac (ab32129, 1:500; Abcam, Cambridge, UK), H3K27ac (ab52946, 1:2000; Abcam), H4K12ac (ab177793, 1:10,000; Abcam), H4K5ac (ab51997, 1:10,000, Abcam), GAPDH (10494-1-AP, 1:1000; Proteintech), acetylated-Lysine (9814, 1:1000; Cell Signaling, Danvers, USA), YAP1 (ab52771, 1:1000; Abcam), P53 (AF0879, 1:1000; Affinity), or histone H3 (AF863, 1:1000; Affinity) at 4°C.

Techniques: Cell Culture, Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence