Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Mas Signaling Potentiates Neutropil Extracellular Traps Formation Induced by Endothelial Cells Derived S1P in Mice with Acute Liver Failure.

doi: 10.1002/advs.202411428

Figure Lengend Snippet: Figure 1. Systemic Mas1 deficiency protects mice from L/G challenge. A) Representative immunohistochemical staining (left) with the quantification (right) of Mas in HC and ALF patients (n = 6 per group, two-sided Student’s t-test, p = 3.68 × 10−4). Scale bars are shown as indicated. B) Representative mIHC staining of Mas on hepatocytes (HNF4a+), endothelial cells (ECs, CD31+), macrophages (F4/80+) and neutrophils (Ly6G+) in liver sections from WT-solvent and WT-L/G groups with the quantification (left) of Mas+ neutrophils and ECs (two-sided Student’s t-test, p = 3.19 × 10−4 and p = 2.85 ×

Article Snippet: Mas1−/− mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques: Immunohistochemical staining, Staining, Solvent

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Mas Signaling Potentiates Neutropil Extracellular Traps Formation Induced by Endothelial Cells Derived S1P in Mice with Acute Liver Failure.

doi: 10.1002/advs.202411428

Figure Lengend Snippet: Figure 2. Systemic Mas1 deficiency reduces NETs formation in mice exposed to L/G. A) GSEA of KEGG enrichment plot in livers from WT-L/G and Mas1−/−-L/G mice (both n = 3, bulk RNA-seq). B) Plasma levels of cell-free DNA in WT-solvent, WT-L/G, and Mas1−/−-L/G groups (n = 6 per group, One- way ANOVA with Tukey’s test, two-sided Student’s t-test, p = 5.12 × 10−4 and p = 2.67 × 10−4). C) Representative mIHC staining with the quantification (n = 6 per group) of intrahepatic H3Cit+ and total neutrophils (One-way ANOVA with Tukey’s test, two-sided Student’s t-test, p = 5.69 × 10−4, p = 3.12 × 10−4, p = 4.07 × 10−4 and p = 7.21 × 10−4 from left to right, from top to bottom). Scale bar: 20 μm. D) Representative immunoblots (Raf-MEK-ERK pathway and markers for NETs formation: H3Cit, NE) of liver tissues with the quantification (below). WT-L/G mice were prophylactically treated with GSK484 (the NETs inhibitor) or solvent control (n = 6 per group, E–G). E) Representative liver photographs and immunohistochemical staining with the quantification (below) of H&E, TUNEL, and Ly6G (two-sided Student’s t-test, p = 4.65 × 10−4, p = 5.30 × 10−4 and p = 6.02 × 10−3 from left to right). Scale bars are shown as indicated. F) Serum levels of ALT and TBA (two-sided Student’s t-test, p = 5.30 × 10−4 and p = 3.27 × 10−4 from left to right). G) Representative immunoblots of liver tissues with the quantification (below). WT-L/G mice were pre-treated with Ang-(1-7) or solvent control (n = 6 per group, H,I). H) Plasma levels of cell-free DNA (two-sided Student’s t-test, p = 6.70 × 10−3). I) Representative immunoblots of liver tissues with the quantification (below). Data are presented as mean ± SD (**p < 0.01, ***p < 0.001).

Article Snippet: Mas1−/− mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques: RNA Sequencing, Clinical Proteomics, Solvent, Staining, Western Blot, Control, Immunohistochemical staining, TUNEL Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Mas Signaling Potentiates Neutropil Extracellular Traps Formation Induced by Endothelial Cells Derived S1P in Mice with Acute Liver Failure.

doi: 10.1002/advs.202411428

Figure Lengend Snippet: Figure 3. Single-cell annotation of Cldn1+CD177+ neutrophils in Mas1−/−-L/G mice. Mouse livers were analyzed by single-cell RNA-seq (scRNA-seq, n = 1 per group). A) The uniform manifold approximation and projection (UMAP) plot of main cell types. B) Percentage of neutrophils in total immune cells. C) The DEGs (red, upregulated; green, downregulated) based on cell types in Mas1−/−-L/G versus WT-L/G group. D) KEGG enrichment of DEGs in neutrophils. Top 20 downregulated are shown in Mas1−/−-L/G versus WT-L/G. E) Histogram illustrating the distribution of neutrophil cluster proportions

Article Snippet: Mas1−/− mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques: RNA Sequencing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Mas Signaling Potentiates Neutropil Extracellular Traps Formation Induced by Endothelial Cells Derived S1P in Mice with Acute Liver Failure.

doi: 10.1002/advs.202411428

Figure Lengend Snippet: Figure 4. Col4a1+ ECs trigger NETs formation in Cldn1+CD177+ neutrophils via Col4a1-CD44 interaction. A,B) Heatmap shows the number of in- teractions between neutrophil clusters and other cell types in the WT-LG (A) and Mas1−/−-L/G (B) groups. C) Bubble plots display the likelihood of ligand-receptor pairs for ECs signaling to Neu2 in both WT-LG and Mas1−/−-L/G mice. D) KEGG enrichment of DEGs in ECs. Top 20 downregulated are shown in Mas1−/−-L/G versus WT-L/G. E) Violin plots illustrate the score of sphingolipid signaling pathway for EC clusters. F) Dot plots show scaled

Article Snippet: Mas1−/− mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Mas Signaling Potentiates Neutropil Extracellular Traps Formation Induced by Endothelial Cells Derived S1P in Mice with Acute Liver Failure.

doi: 10.1002/advs.202411428

Figure Lengend Snippet: Figure 5. Systemic Mas1 deficiency inhibits S1P-induced NETs formation. A) GSEA of GO enrichment plot in livers from WT-L/G and Mas1−/−-L/G mice (both n = 3, bulk RNA-seq). B) PCA of liver metabolomics (n = 6 per group, left) and the relative abundance of intrahepatic S1P (right) (two-sided Mann–Whitney U test, p = 2.58 × 10−2). C) Serum S1P levels (all, n = 6, One-way ANOVA with Tukey’s test, two-sided Student’s t-test, p = 4.28 × 10−4

Article Snippet: Mas1−/− mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques: RNA Sequencing, MANN-WHITNEY

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Mas Signaling Potentiates Neutropil Extracellular Traps Formation Induced by Endothelial Cells Derived S1P in Mice with Acute Liver Failure.

doi: 10.1002/advs.202411428

Figure Lengend Snippet: Figure 6. Systemic Mas modulates gut microbiota and DCA production in mice after L/G challenge. A) Gram’s staining of liver sections (red arrows indicate positive staining). Scale bar: 200 μm. B,C) The mRNA expression (B) (both n = 6, two-sided Student’s t-test, p = 2.12 × 10−2, p = 3.10 × 10−2, p = 2.68 × 10−2 and p = 6.52 × 10−3 from left to right) and representative immunoblots (C, the quantification, below) of markers for mucosal barrier integrity in the ileum. Mas1−/−-L/G mice were pre-treated with oral antibiotics (ABx) or solvent control (n = 6 per group, D–F). D) The timeline for ABx treatment.

Article Snippet: Mas1−/− mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques: Staining, Expressing, Western Blot, Solvent, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Mas Signaling Potentiates Neutropil Extracellular Traps Formation Induced by Endothelial Cells Derived S1P in Mice with Acute Liver Failure.

doi: 10.1002/advs.202411428

Figure Lengend Snippet: Figure 7. FXR serves as the pivotal upstream regulator of S1P in L/G challenge. A) Serum levels of DCA (all, n = 6, One-way ANOVA with Tukey’s test, two-sided Student’s t-test, p = 7.29 × 10−4, p = 4.74 × 10−3 and p = 5.69 × 10−3 from left to right). B) Representative immunoblots of liver tissues with the quantification (below). C) The intrahepatic mRNA expression of FXR and its target genes in WT, WT-L/G and Mas1−/−-L/G mice (all, n = 6, One-way ANOVA with Tukey’s test, two-sided Student’s t-test, p = 6.58 × 10−4, p = 3.81 × 10−4, p = 2.39 × 10−4, p = 4.01 × 10−4, p = 4.96 × 10−3, p = 8.23 ×

Article Snippet: Mas1−/− mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques: Western Blot, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Mas Signaling Potentiates Neutropil Extracellular Traps Formation Induced by Endothelial Cells Derived S1P in Mice with Acute Liver Failure.

doi: 10.1002/advs.202411428

Figure Lengend Snippet: Figure 8. Intrahepatic SHP2 functions downstream of Mas to regulate the FXR-S1P-NETs axis during L/G challenge. A) Intrahepatic mRNA expression of Ptpn11 in WT-Solvent, WT-L/G and Mas1−/−-L/G groups (all, n = 6; One-way ANOVA with Tukey’s test, two-sided Student’s t-test, p = 6.01 × 10−4

Article Snippet: Mas1−/− mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques: Expressing, Solvent

Journal: Advanced Science

Article Title: Myeloid‐Mas Signaling Modulates Pathogenic Crosstalk among MYC + CD63 + Endothelial Cells, MMP12 + Macrophages, and Monocytes in Acetaminophen‐Induced Liver Injury

doi: 10.1002/advs.202306066

Figure Lengend Snippet: EC‐sourced lactate polarizes Mψ to the mixed M1/M2‐like phenotype by inducing glycolysis. A) The metabolic pathway activity analysis of EC clusters. The circle size and color darkness both represent the scaled metabolic score. B) mIHC of CD31 + MYC + ECs and key glycolytic enzymes (PKM and PFKFB3). Scale bar: 20 µm. C) The UMAP plot shows the subpopulation of Mψ between groups. D) Histogram showing the proportion of each cluster between groups. E) The MIA map of Mψ clusters and ST‐defined regions. F) mIHC of F4/80 + MMP12 + Mψ. Scale bar: 50 µm. The white arrows represent the F4/80 + MMP12 + Mψ, and the quantification of F4/80 + MMP12 + Mψ is shown ( n = 4 mice per group; One‐way ANOVA with Tukey's test, p = 8.41 × 10 −4 , 1.59 × 10 −3 and 1.72 × 10 −3 from left to right). G) mIHC of CD31 + MYC + ECs and F4/80 + MMP12 + Mψ. Scale bar: 20 µm. H) Representative intravital images of Mψ polarization by DAOSLIMIT. The white arrows represent the CD80 + CD163 + Mψ. Scale bar: 50 µm. I) Pathway enrichment analysis of Mψ clusters. GSVA was used to perform the pathway enrichment analysis (Methods). Only selected pathways were shown. J) Multiplex fluorescence of F4/80 + MMP12 + kupffer cells and key glycolytic enzymes (PKM and PFKFB3). Scale bar: 50 µm. The mouse primary kupffer cells were treated with or without LSEC‐CM for 24 h. K) Quantification of F4/80 + MMP12 + PKM + PFKFB3 + kupffer cells for multiplex fluorescence as shown in J ( n = 4 samples per group; two‐sided Student's t‐test, p = 3.63 × 10 −3 ). L) Representative H&E staining and mIHC of CD31 + MYC + ECs and F4/80 + MMP12 + Mψ. Scale bar: 50 µm. Pkm f/f and Cdh5 cre Pkm f/+ mice were administrated with APAP for 24 h ( n = 4 mice per group). M) Representative H&E staining and mIHC of CD31 + MYC + ECs, F4/80 + MMP12 + Mψ, and CCR2 + monocytes. Scale bar: 50 µm. Mas1 −/− mice were administrated with or without 3PO under APAP challenge ( n = 4 mice per group). In all graphs, data are presented as mean ± SD, ** p < 0.01.

Article Snippet: Mas1 −/− , Mas1 f/f , Pkm f/f , Alb ‐cre, LysM ‐cre, Lrat ‐cre and Cd4 ‐cre mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques: Activity Assay, Multiplex Assay, Fluorescence, Staining

Journal: Advanced Science

Article Title: Myeloid‐Mas Signaling Modulates Pathogenic Crosstalk among MYC + CD63 + Endothelial Cells, MMP12 + Macrophages, and Monocytes in Acetaminophen‐Induced Liver Injury

doi: 10.1002/advs.202306066

Figure Lengend Snippet: Monocyte‐derived TNF‐α acts on MYC + CD63 + EC through TNFR1. A) Spatial feature plots showing the cell types in EC7 expansion units for ST. B) CellPhoneDB analysis showing the cell–cell interactions for scRNA‐seq. C) Mas1 −/− mice were pre‐administrated with clodronate liposomes or control liposomes for 24 h before APAP challenge ( n = 4 per group). Representative stainings of H&E and mIHC are shown. Scale bar: 50 µm. D) WT and Mas1 −/− mice were pre‐administrated with cenicriviroc for 2 h before APAP challenge ( n = 4 per group). Representative stainings of H&E and mIHC are shown. Scale bar: 50 µm. E) CellPhoneDB analysis showing the major interaction pairs between Ly6c hi monocyte‐Mψ5 or Ly6c hi monocyte‐EC7 for scRNA‐seq. In F‐G, Mas1 −/− mice were pre‐administrated with cediranib or control for 2 h before APAP challenge ( n = 4 per group). F) Representative staining of H&E and mIHC are shown. Scale bar: 50 µm. G) Quantification of necrotic area for H&E as shown in F ( n = 4 mice per group; two‐sided Student's t‐test; p = 1.55 × 10 −4 ) and serum ALT ( n = 4 mice per group; two‐sided Student's t‐test; p = 2.64 × 10 −8 ). H) Multiplex fluorescence of CD31 + MYC + CD63 + LSECs and key glycolytic enzymes (PKM and PFKFB3). Scale bar: 100 µm. The mouse primary LSECs were treated with or without BMDM‐CM for 24 h. I) Quantification of CD31 + MYC + CD63 + PFKFB3 + PKM + LSECs for multiplex fluorescence as shown in H ( n = 4 samples per group; two‐sided Mann‐Whitney U test, p = 2.86 × 10 −2 ). J) Glycolytic rate assay showing glycolytic function of LSECs treated with or without BMDM‐CM for 24 h. ( n = 5 or 6 samples per group; two‐sided Student's t‐test, p = 7.50 × 10 −3 and 3.77 × 10 −3 from left to right). K) Multiplex fluorescence of CD31 + MYC + CD63 + LSECs and key glycolytic enzymes (PKM and PFKFB3). Scale bar: 100 µm. The mouse primary LSECs were treated with or without TNF‐α (20 ng ml −1 ) for 24 h. L) Quantification of CD31 + MYC + CD63 + PFKFB3 + PKM + LSECs for multiplex fluorescence as shown in K ( n = 4 samples per group; two‐sided Student's t‐test, p = 2.00 × 10 −2 ). M) Glycolytic rate assay showing glycolytic function of LSECs treated with or without TNF‐α (20 ng ml −1 ) for 24 h. ( n = 5 or 6 samples per group; two‐sided Student's t‐test, p = 8.00 × 10 −6 and 1.30 × 10 −5 from top to bottom). N) mIHC of CD31 + MYC + ECs, F4/80 + MMP12 + Mψ and CCR2 + monocytes are shown. Scale bar: 200 µm, 50 µm and 20 µm. Spatial localizations of CD31 + MYC + ECs, CCR2 + monocytes, and F4/80 + MMP12 + Mψ within injured region are shown. In all graphs data are presented as mean ± SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Mas1 −/− , Mas1 f/f , Pkm f/f , Alb ‐cre, LysM ‐cre, Lrat ‐cre and Cd4 ‐cre mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques: Derivative Assay, Liposomes, Control, Staining, Multiplex Assay, Fluorescence, MANN-WHITNEY

Journal: Advanced Science

Article Title: Myeloid‐Mas Signaling Modulates Pathogenic Crosstalk among MYC + CD63 + Endothelial Cells, MMP12 + Macrophages, and Monocytes in Acetaminophen‐Induced Liver Injury

doi: 10.1002/advs.202306066

Figure Lengend Snippet: Myeloid Mas modulates AILI through the highly pro‐inflammatory and glycolytic microenvironment. A) Mas1 f/f , LysM cre Mas1 f/f , Alb cre Mas1 f/f , Cdh5 cre Mas1 f/f , Lrat cre Mas1 f/f and Cd4 cre Mas1 f/f mice were administrated with APAP for 24 h ( n = 6 mice per group). Representative stainings of H&E and mIHC are shown. Scale bar: 50 µm. B) mIHC of CD31 + MYC + ECs, F4/80 + MMP12 + Mψ, and CCR2 + monocytes are shown. Scale bar: 50 µm and 20 µm. C) The UMAP plot showing the subpopulation of Mψ. D) mIHC of CD31 + MYC + ECs, F4/80 + MMP12 + Mψ, and key glycolytic enzymes (PKM and PFKFB3). Scale bar: 20 µm. E) LysM cre Mas1 f/f mice were pre‐administrated with or without 3PO before APAP challenge ( n = 4 per group). Representative stainings of H&E and mIHC are shown. Scale bar: 50 µm. F) Quantification of necrotic area for H&E as shown in E ( n = 4 mice per group; two‐sided Student's t‐test, p = 9.53 × 10 −7 ), serum ALT ( n = 4 mice per group; two‐sided Student's t‐test, p = 3.77 × 10 −7 ) and quantification of CD31 + MYC + ECs for mIHC as shown in E ( n = 4 mice per group; two‐sided Student's t‐test, p = 2.70 × 10 −5 ). G) Timelapse data of neutrophils (Ly6G) and monocytes (Ly6C) in the vessels (WGA) of living mouse livers were captured by DAOSLIMIT. Representative intravital images and the temporal traces of their number are shown. LysM cre Mas1 f/f mice were pre‐administrated with or without 3PO before APAP challenge. Scale bar: 50 µm. In all graphs, data are presented as mean ± SD, *** p < 0.001.

Article Snippet: Mas1 −/− , Mas1 f/f , Pkm f/f , Alb ‐cre, LysM ‐cre, Lrat ‐cre and Cd4 ‐cre mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques:

Journal: Advanced Science

Article Title: Myeloid‐Mas Signaling Modulates Pathogenic Crosstalk among MYC + CD63 + Endothelial Cells, MMP12 + Macrophages, and Monocytes in Acetaminophen‐Induced Liver Injury

doi: 10.1002/advs.202306066

Figure Lengend Snippet: Myeloid Mas modulates AILI microenvironment via the NF‐κB/TNF‐α pathway. A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs in LysM cre Mas1 f/f ‐APAP versus Mas1 f/f ‐APAP groups for monocytes. The top twenty pathways are presented. B) Violin plots showing the key genes of TNF signaling pathway and NF‐kappa B signaling pathway for monocytes between groups. In C‐D, BMDMs from WT and Mas1 −/‐ mice were stimulated with LPS (100 ng ml −1 ) for 12 h to drive pro‐inflammatory activation. Activated BMDMs were collected for bulk RNA‐seq ( n = 5 or 6 samples per group). C) KEGG pathway enrichment analysis of DEGs in Mas1 −/− versus WT BMDMs. The top twenty pathways are presented. D) Volcano plot showing the DEGs in Mas1 −/− versus WT BMDMs for TNF signaling pathway and NF‐kappa B signaling pathway. E) BMDMs from WT and Mas1 −/‐ mice were stimulated with LPS (100 ng ml −1 ) for 12 h to drive pro‐inflammatory activation. Representative IF images and the statistical results of nuclear translocation of p65 ( n = 4 samples per group; two‐sided Student's t‐test, p = 3.44 × 10 −3 ) and TNF‐α ( n = 4 samples per group; two‐sided Student's t‐test, p = 4.07 × 10 −3 ) are shown. Scale bar: 10 µm. F) BMDMs from WT and Mas1 −/− mice were stimulated with LPS (100 ng ml −1 ) for 12 h to drive proinflammatory activation. The TNF‐α of the CM were tested ( n = 8 samples per group; two‐sided Student's t‐test, p = 4.14 × 10 −3 ). G) LysM cre Mas1 f/f mice were pre‐administrated with or without (Rac)‐Benpyrine (30 mg kg −1 , ig) before APAP challenge ( n = 4 mice per group). Representative stainings of H&E and mIHC are shown. Scale bar: 50 µm and 20 µm. H) LysM cre Mas1 f/f mice were pre‐administrated with or without R‐7050 (10 mg kg −1 , ip) for 2 h before APAP challenge ( n = 4 mice per group). Representative stainings of H&E and mIHC are shown. Scale bar: 50 µm and 20 µm. In all graphs, data are presented as mean ± SD, ** p < 0.01.

Article Snippet: Mas1 −/− , Mas1 f/f , Pkm f/f , Alb ‐cre, LysM ‐cre, Lrat ‐cre and Cd4 ‐cre mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques: Activation Assay, RNA Sequencing, Translocation Assay

Journal: Advanced Science

Article Title: Myeloid‐Mas Signaling Modulates Pathogenic Crosstalk among MYC + CD63 + Endothelial Cells, MMP12 + Macrophages, and Monocytes in Acetaminophen‐Induced Liver Injury

doi: 10.1002/advs.202306066

Figure Lengend Snippet: The characteristic AILI microenvironment visualized by DAOSLIMIT in living mice correlates well with human DILI progression. A‐B) Timelapse data captured by DAOSLIMIT showing the interactions between monocytes and CD63 + ECs in LysM cre Mas1 f/f ‐APAP mouse (A) and LysM cre Rosa26(R26)‐tdTomato ‐APAP mouse (B). The curves of cell distances are shown on the right. Scale bar: 50 µm. C) mIHC of CD31 + MYC + ECs, CD68 + CD14 − MMP12 + Mψ and CD14 + monocytes in the livers of healthy individuals and patients with DILI ( n = 14 healthy and 47 with DILI). Scale bar: 50 µm and 20 µm. D) Spatial localization of CD31 + MYC + ECs, CD68 + CD14 − MMP12 + Mψ and CD14 + monocytes within injured region ( n = 14 healthy and 47 with DILI). Scale bar: 200 µm. E) mIHC of CD31 + MYC + ECs, CD68 + CD14 − MMP12 + Mψ and CD14 + monocytes in the livers of patients with chronic hepatitis B (CHB), alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), Non‐AILI and AILI ( n = 10 with CHB, 5 with ALD, 5 with NAFLD, 5 with AIH, 5 with PBC, 32 with Non‐AILI and 15 with AILI). Scale bar: 100 µm and 20 µm. F) Quantification of CD31 + MYC + ECs, CD68 + CD14 − MMP12 + Mψ and CD14 + monocytes for mIHC as shown in E ( n = 10 with CHB, 5 with ALD, 5 with NAFLD, 5 with AIH, 5 with PBC, 32 with Non‐AILI and 15 with AILI; two‐sided Student's t‐test and two‐sided Mann‐Whitney U test; p = 5.00 × 10 −6 , 5.30 × 10 −4 , 5.30 × 10 −4 , 6.24 × 10 −4 , 6.24 × 10 −4 , 1.00 × 10 −5 , 5.00 × 10 −6 , 8.00 × 10 −6 , 1.20 × 10 −5 , 1.30 × 10 −5 , 5.84 × 10 −1 from left to right for quantification of CD31 + MYC + ECs; p = 9.00 × 10 −6 , 1.01 × 10 −3 , 1.01 × 10 −3 , 8.62 × 10 −4 , 1.18 × 10 −3 , 8.20 × 10 −5 , 2.80 × 10 −5 , 1.70 × 10 −5 , 1.00 × 10 −5 , 3.70 × 10 −5 , 9.13 × 10 −2 from left to right for quantification of CD68 + CD14 − MMP12 + Mψ; p = 6.79 × 10 −1 , 8.24 × 10 −1 , 6.72 × 10 −3 , 4.77 × 10 −1 , 1.10 × 10 −1 , 2.22 × 10 −1 , 1.90 × 10 −1 , 5.20 × 10 −3 , 8.87 × 10 −2 , 4.46 × 10 −2 , 2.18 × 10 −1 from left to right for quantification of CD14 + monocytes). G) Correlation analysis between CD31 + MYC + ECs, CD68 + CD14 − MMP12 + Mψ, CD14 + monocytes and DILI pathology parameters ( n = 47 patients; Pearson correlation; color and circle size indicate correlation). In all graphs data are presented as mean ± SD, * p < 0.05; ** p < 0.01; *** p < 0.001; NS, non‐significant.

Article Snippet: Mas1 −/− , Mas1 f/f , Pkm f/f , Alb ‐cre, LysM ‐cre, Lrat ‐cre and Cd4 ‐cre mice were purchased from Cyagen Biosciences Inc (Suzhou, China).

Techniques: MANN-WHITNEY

Journal: Journal of Biological Chemistry

Article Title: Control of Adipogenesis by the Autocrine Interplays between Angiotensin 1–7/Mas Receptor and Angiotensin II/AT1 Receptor Signaling Pathways

doi: 10.1074/jbc.m113.459792

Figure Lengend Snippet: FIGURE 3. Ang(1–7) stimulates adipogenesis and neutralizes the antiadipogenic effect of AngII-AT1 receptor activation in 3T3-L1 preadipocytes. The cells were transfected with control siRNA or AT1 receptor siRNA or AT2 receptor siRNA and cultured until confluence. This was followed by induction of differentiation for 7–8 days, without () (control) or with () 1 M Ang(1–7) and/or 1 M AngII. A, representative images of Oil Red O-stained 3T3-L1 adipocytes differentiated under different conditions (day 8). B–G, Western blot analyses of the expression of PPAR, C/EBP, FAS, ACC, aP2, and actin in 3T3-L1 adipocytes (day 7–8). Representative immunoblots are shown in B. The statistics (mean S.E., n 5–6) of the optical densities of the blots normalized to that of actin are showninC–G.H,intracellulartriglyceridecontent(meanS.E.,n4–5)in3T3-L1adipocytes(day7–8).Student’sttestwasused:*,p0.05;**,p0.01versus control; #, p 0.05; ##, p 0.01 between indicated pairs.

Article Snippet: All other chemicals and reagents were purchased from Sigma-Aldrich. siRNA Silencing—Gene silencing of Mas receptor (Mas1), ACE2 (Ace2), AT1 (Agtr1), AT2 (Agtr2), ERK1 (Mapk3), ERK2 (Mapk1), or Akt (Akt1 and Akt2) in 3T3-L1 cells was achieved using a mouse MAS1 siRNA (sc62601, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), a mouse ACE2 siRNA (sc41401, Santa Cruz Biotechnology, Inc.), a mouse AT1 siRNA (sc29751, Santa Cruz Biotechnology, Inc.), a mouse AT2 siRNA (sc29753, Santa Cruz Biotechnology, Inc.), mouse ERK1 and ERK2 siRNAs (sc-29308 and sc-35336, Santa Cruz Biotechnology, Inc.), and a mouse Akt siRNA (sc-43610, Santa Cruz Biotechnology, Inc.), respectively.

Techniques: Activation Assay, Transfection, Control, Cell Culture, Staining, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: Control of Adipogenesis by the Autocrine Interplays between Angiotensin 1–7/Mas Receptor and Angiotensin II/AT1 Receptor Signaling Pathways

doi: 10.1074/jbc.m113.459792

Figure Lengend Snippet: FIGURE4.AutocrineinterplaysbetweenAng(1–7)andAngIIinthedifferentiationof3T3-L1preadipocytes.ThecellsweretransfectedwithcontrolsiRNA or ACE2 siRNA, and cultured until confluence. This was followed by 7–8 days of differentiation, with () or without () 1 M Ang(1–7) or 10 M ZD7155 (selective AT1 receptor antagonist) or 1 M AngII plus PD123319 (selective AT2 receptor antagonist). A and B, the concentrations of released Ang(1–7) and AngII in the culture media (24 h incubation) at different differentiation stages (day 0–6). The concentrations at day 0 from the control siRNA-transfected cells were regarded as the control). C, representative images of differently cultured 3T3-L1 cells (day 7–8). Scale bars, 60 m. D, number of adipocytes (lipid droplet-laden cells) per field of view (40). The data is presented as mean S.E. (error bars) (n 6–7 images from 3–4 different culture batches). E and F, representative immunoblots of aP2, FAS, and actin (day 7–8) and the statistics (mean S.E., n 4–5) showing the optical densities of the blots normalized to actin density. G and H, Western blot analyses of ACE2 (90 kDa) and actin expression in untreated 3T3-L1 cells at different differentiation stages (day 0–6) (expression level atday0ascontrol).Thetoppanelshowstherepresentativeimmunoblots,andthebottompanelshowsthestatistics(meanS.E.,n4–5)oftheopticaldensity of the ACE2 normalized to that of actin. I, Western blot analyses of the phosphorylated and total ERK1/2 (pERK1/2 and ERK1/2, 42–44 kDa) in AT2 siRNA-transfected cells (1 or 4 h after induction of differentiation). The top panels show the representative immunoblots, and the bottom panel shows the statistics (mean S.E., n 4–5) of the optical density ratio between pERK1/2 and ERK1/2. Student’s t test was used: *, p 0.05; **, p 0.01; ***, p 0.001 versus control; #, p 0.05; ##, p 0.01 between indicated pairs.

Article Snippet: All other chemicals and reagents were purchased from Sigma-Aldrich. siRNA Silencing—Gene silencing of Mas receptor (Mas1), ACE2 (Ace2), AT1 (Agtr1), AT2 (Agtr2), ERK1 (Mapk3), ERK2 (Mapk1), or Akt (Akt1 and Akt2) in 3T3-L1 cells was achieved using a mouse MAS1 siRNA (sc62601, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), a mouse ACE2 siRNA (sc41401, Santa Cruz Biotechnology, Inc.), a mouse AT1 siRNA (sc29751, Santa Cruz Biotechnology, Inc.), a mouse AT2 siRNA (sc29753, Santa Cruz Biotechnology, Inc.), mouse ERK1 and ERK2 siRNAs (sc-29308 and sc-35336, Santa Cruz Biotechnology, Inc.), and a mouse Akt siRNA (sc-43610, Santa Cruz Biotechnology, Inc.), respectively.

Techniques: Cell Culture, Incubation, Control, Transfection, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: Control of Adipogenesis by the Autocrine Interplays between Angiotensin 1–7/Mas Receptor and Angiotensin II/AT1 Receptor Signaling Pathways

doi: 10.1074/jbc.m113.459792

Figure Lengend Snippet: FIGURE 5. Involvement of Mas receptor in 3T3-L1 adipogenesis. The cells were transfected with control siRNA or Mas receptor siRNA and cultured until confluence. Differentiation was then induced for 7–8 days, without () (control) or with () 1 M Ang(1–7), 1 M AngII, 10 M ZD7155, or 10 M PD123319. A and B, Western blot analyses of PPAR, FAS, ACC, aP2, and actin (day 7–8). The representative immunoblots are shown in A, and the statistics (mean S.E. (error bars), n 4–5) of the optical densities of the blots normalized to actin density are shown in B. C, Western blot analyses of Mas receptor (37 kDa) and actin in the untreated cells at different stages of differentiation (12 h to day 6). Protein level at 12 h without Ang(1–7) treatment was regarded as the control. The top panel shows the representative immunoblots, and the bottom panel shows the statistics (mean S.E., n 4–5) of the optical density ratio between the blot of Mas receptor and that of actin. Student’s t test was used: *, p 0.05; **, p 0.01; ***, p 0.001 versus control; #, p 0.05; ##, p 0.01 between indicated pairs.

Article Snippet: All other chemicals and reagents were purchased from Sigma-Aldrich. siRNA Silencing—Gene silencing of Mas receptor (Mas1), ACE2 (Ace2), AT1 (Agtr1), AT2 (Agtr2), ERK1 (Mapk3), ERK2 (Mapk1), or Akt (Akt1 and Akt2) in 3T3-L1 cells was achieved using a mouse MAS1 siRNA (sc62601, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), a mouse ACE2 siRNA (sc41401, Santa Cruz Biotechnology, Inc.), a mouse AT1 siRNA (sc29751, Santa Cruz Biotechnology, Inc.), a mouse AT2 siRNA (sc29753, Santa Cruz Biotechnology, Inc.), mouse ERK1 and ERK2 siRNAs (sc-29308 and sc-35336, Santa Cruz Biotechnology, Inc.), and a mouse Akt siRNA (sc-43610, Santa Cruz Biotechnology, Inc.), respectively.

Techniques: Transfection, Control, Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Control of Adipogenesis by the Autocrine Interplays between Angiotensin 1–7/Mas Receptor and Angiotensin II/AT1 Receptor Signaling Pathways

doi: 10.1074/jbc.m113.459792

Figure Lengend Snippet: FIGURE 7. Effect of PI3K/Akt and MAPK kinase/ERK signaling pathways on Ang(1–7)-induced adipogenesis of 3T3-L1 cells. The cells were induced to differentiate for 7–8 days without (control) or with 1 M Ang(1–7), 10 M LY294002 (selective inhibitor of PI3K), Ang(1–7) plus LY29400, 50 M PD98059 (selectiveinhibitorofMAPKkinase),Ang(1–7)plusPD98059,100nMPMA,orAng(1–7)plusPMA.A,representativeimagesofdifferentlyculturedcells(day7–8). Scale bars, 60 m. B, the number of adipocytes (lipid droplet-containing cells) per field of view (40) was counted on day 7 and is presented as mean S.E. (error bars) (n 6–7 images from 3–4 different culture batches). C–G, Western blot analyses of aP2, FAS, PPAR, ACC, and actin expression in 3T3-L1 cells (day 7–8). Representative immunoblots are shown in C, and the statistics (mean S.E., n 5–6) of the optical density ratio normalized to the actin blot are shown in D–G. H–J, 3T3-L1 cells were transfected with control siRNA or Akt siRNA or ERK1/2 siRNA and cultured until confluence. Differentiation was then induced for 7–8 days, without () (control) or with () 1 M Ang(1–7). Representative immunoblots of aP2, FAS, and actin expression in 3T3-L1 cells (day 7–8) are shown in H, and the statistics (mean S.E., n 4–5) of the optical density ratio normalized to the actin blot are shown in I and J. Student’s t test was used: *, p 0.05; **, p 0.01 versus control; #, p 0.05; ##, p 0.01; ###, p 0.001 between indicated pairs.

Article Snippet: All other chemicals and reagents were purchased from Sigma-Aldrich. siRNA Silencing—Gene silencing of Mas receptor (Mas1), ACE2 (Ace2), AT1 (Agtr1), AT2 (Agtr2), ERK1 (Mapk3), ERK2 (Mapk1), or Akt (Akt1 and Akt2) in 3T3-L1 cells was achieved using a mouse MAS1 siRNA (sc62601, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), a mouse ACE2 siRNA (sc41401, Santa Cruz Biotechnology, Inc.), a mouse AT1 siRNA (sc29751, Santa Cruz Biotechnology, Inc.), a mouse AT2 siRNA (sc29753, Santa Cruz Biotechnology, Inc.), mouse ERK1 and ERK2 siRNAs (sc-29308 and sc-35336, Santa Cruz Biotechnology, Inc.), and a mouse Akt siRNA (sc-43610, Santa Cruz Biotechnology, Inc.), respectively.

Techniques: Protein-Protein interactions, Control, Western Blot, Expressing, Transfection, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Control of Adipogenesis by the Autocrine Interplays between Angiotensin 1–7/Mas Receptor and Angiotensin II/AT1 Receptor Signaling Pathways

doi: 10.1074/jbc.m113.459792

Figure Lengend Snippet: FIGURE 8. Ang(1–7) exerts its stimulatory effect on adipogenesis in the early stage of differentiation. 3T3-L1 cells were induced to differentiate without (control) or with exposure to 1 M Ang(1–7) for different periods of time: from day 0 to 6 (d0-d6), from day 1 to 6 (d1–d6), from day 2 to 6 (d2–d6), from day 4 to6(d4–d6),orfromday0to1(d0–d1).A,representativeimagesofdifferentlycultured3T3-L1cells(day6).Scalebars,60m.B,thenumberofadipocytes(lipid droplet-containing cells) per field of view (40) was counted on day 6 and is presented as mean S.E. (error bars) (n 6–8 images from 3–4 different culture batches). C, number of 3T3-L1 cells per 12 wells was counted at days 0, 1, and 2 (mean S.E., n 4; the cell number at day 0 was regarded as the control). D–F, Western blot analyses of aP2, FAS, and actin expression in differently cultured cells (day 6). Representative immunoblots are shown in D, and the statistics (mean S.E., n 4–5) of the optical density ratio normalized to the actin blot are shown in E and F. G–I, 3T3-L1 cells were transfected with control siRNA or Mas receptor siRNA at different times. Shown is transfection of Mas receptor siRNA 2 days before the initiation of differentiation (d(2)) and at day 0 (d0), day 2 (d2), and day 4 (d4). Differentiation was induced at day 0 without () (control) or with () 1 M Ang(1–7) for 7–8 days. Representative immunoblots of aP2, FAS, and actin expression in 3T3-L1 cells (day 7–8) are shown in G, and the statistics (mean S.E., n 4–5) of the optical density ratio normalized to the actin blot are shown in H and I. Student’s t test was used: *, p 0.05; **, p 0.01 versus control; #, p 0.05; ##, p 0.01 between indicated pairs.

Article Snippet: All other chemicals and reagents were purchased from Sigma-Aldrich. siRNA Silencing—Gene silencing of Mas receptor (Mas1), ACE2 (Ace2), AT1 (Agtr1), AT2 (Agtr2), ERK1 (Mapk3), ERK2 (Mapk1), or Akt (Akt1 and Akt2) in 3T3-L1 cells was achieved using a mouse MAS1 siRNA (sc62601, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), a mouse ACE2 siRNA (sc41401, Santa Cruz Biotechnology, Inc.), a mouse AT1 siRNA (sc29751, Santa Cruz Biotechnology, Inc.), a mouse AT2 siRNA (sc29753, Santa Cruz Biotechnology, Inc.), mouse ERK1 and ERK2 siRNAs (sc-29308 and sc-35336, Santa Cruz Biotechnology, Inc.), and a mouse Akt siRNA (sc-43610, Santa Cruz Biotechnology, Inc.), respectively.

Techniques: Control, Western Blot, Expressing, Cell Culture, Transfection