Journal: iScience

Article Title: The role of long noncoding RNA Nron in atherosclerosis development and plaque stability

doi: 10.1016/j.isci.2022.103978

Figure Lengend Snippet:

Article Snippet: Primary antibodies specific for F4/80 (ab100790, Abcam), α-SMA (ab240654, Abcam) or VEGFA (ab1316, Abcam) was used.

Techniques: Virus, Recombinant, Plasmid Preparation, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Silver Staining, In Vitro, TUNEL Assay, Software

Journal: Frontiers in Pharmacology

Article Title: The role of the SGK3/TOPK signaling pathway in the transition from acute kidney injury to chronic kidney disease

doi: 10.3389/fphar.2023.1169054

Figure Lengend Snippet: The SGK3/TOPK signaling pathway is upregulated in CD206 + M2 macrophages while downregulated in TECs during the AKI-to-CKD transition. (A) Scr and BUN from the CON and 5/6 Nx groups after 5/6 nephrectomy surgery. *** p < 0.001 vs. the control group (n = 4). (B) Scr and BUN were assessed at 0, 3, 7, and 14 days after UUO surgery. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the control group (n = 4). (C) H&E staining and tubular damage score demonstrating tubular damage; Masson’s trichrome staining and graphic presentation showing fibrotic areas of the CON and 5/6 Nx groups after 5/6 nephrectomy surgery. ** p < 0.01 and *** p < 0.001 vs. the control group (n = 4). Scale bar, 50 μm. (D) H&E staining and tubular damage score demonstrating tubular damage; Masson’s trichrome staining and graphic presentation showing fibrotic areas of 0, 3, 7, and 14 days after UUO surgery. ** p < 0.01 and *** p < 0.001 vs. the 0 D group (n = 4). Scale bar, 50 μm. (E) Western blotting analysis for collagen I and a-SMA protein levels in the CON and 5/6 Nx groups after 5/6 nephrectomy surgery. * p < 0.05 and ** p < 0.01 vs. the control group (n = 3). (F) Western blotting analysis for collagen I and a-SMA protein levels in mice at 0, 3, 7, and 14 days after UUO surgery. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the 0D group (n = 3). (G) Immunohistochemical staining for F4/80, α-SMA, SGK3, and p-TOPK in CON, 5/6 Nx group kidney sections after 5/6 nephrectomy surgery and in 0, 3, 7, and 14 days of kidney sections after UUO surgery. Scale bar, 50 μm. (H, I) Representative immunofluorescent images showed staining with specific antibodies to SGK3 (red) or p-TOPK (red), CD206 (blue) and F4/80 (green) in renal sections of CON, and 5/6 Nx groups after 5/6 nephrectomy surgery and in kidney sections of 0, 3, 7, and 14 days mice after UUO surgery. Scale bar, 10 μm.

Article Snippet: Polyclonal chicken anti-F4/80 (ab186037) and polyclonal anti-p-TOPK (ab184953) antibodies for immunofluorescence analysis were provided by Abcam (Cambridge, England). pcDNA3.1 (+)/mMOCK (MOCK) and pcDNA3.1 (+)/mTOPK-T9E (TOPK-T9E) plasmids were obtained from Qiuhong Duan (Department of Biochemistry and Molecular Biology, School of Basic Medicine, Huazhong University of Science and Technology, Wuhan, Hubei 430,022, China). pcDNA3.1/mSGK3-S486D (SGK3-S486D), and pcDNA3.1/mSGK3 wild-type (SGK3 wild-type) plasmids were gifts from David Pearce (Department of Medicine and Molecular and Cellular Pharmacology, University of California, San Francisco, CA, 94,107-2140, United States).

Techniques: Staining, Western Blot, Immunohistochemical staining

Journal: Nature immunology

Article Title: Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation

doi: 10.1038/s41590-020-0681-x

Figure Lengend Snippet: Colonic epithelial cells from either wild-type or Cyld−/− mice were transduced with a lentivirus encoding either V5-tagged wild-type Cyld or Cyld(C601A). The cells were then stimulated overnight by transfection with LTA. a, Cells were lysed and immunoblotted with anti-V5, anti-Cyld and anti-IL-18. b, The IL-18 concentration of the supernatant was measured by ELISA (n=8 mice per group, NSP=0.0946, ***P<0.0001, ***P<0.0001, NSP=0.0578, left to right). c, CYLD mRNA expression from the colonic mucosa of patients with UC (n=23 samples from human patients with UC) was measured by real-time PCR and normalized to that of control samples from healthy individuals. The IL-18 concentration was measured from colon mucosal tissue extracts by ELISA. A regression curve was generated by plotting relative CYLD mRNA expression against IL-18 concentration from the corresponding patient’s samples. d, The Il18 mRNA levels were measured using real-time PCR of the RNA from the colonic mucosa of patients. A regression curve was generated by plotting relative Il18 mRNA against relative CYLD mRNA of patients. The statistics (b) are given as mean ± s.d., with P values determined by Student’s t-test (two tail). The data are representative of three or more independent experiments. The statistics (c,d) were measured by the Pearson correlation coefficient. The data are representative of three or more independent experiments.

Article Snippet: IL-18 cytokine concentrations by ELISA IL-18 concentrations from serum and colon tissue, colonic epithelial cell culture supernatants of mice and colon tissue lysates of humans were measured using commercially available kits mouse IL-18 platinum ELISA kit (eBioscience) and human IL-18 PicoKine ELISA kit (Bosterbio) according to the manufacturers’ protocols.

Techniques: Transduction, Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Generated

Journal: Nature immunology

Article Title: Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation

doi: 10.1038/s41590-020-0681-x

Figure Lengend Snippet: (a–c) (a) Body weight change (nsP=0.9666, ***P<0.0001), (b) diarrhea score (***P<0.0001), and (c) colon length (nsP=0.7766, ***P<0.0001) of wild-type vs. Cyld-/- mice (n = 10 mice per group) given intrarectal administration of TNBS. (d) H&E-stained colon sections from TNBS treated wild-type and Cyld-/- mice (scale bars, 50 μm) and (e) Histology scores of the H&E stained sections (n=10 mice per group, nsP=0.7895, ***P<0.0001). (f) Colon tissues from untreated and TNBS treated wild-type and Cyld–/– mice (n=10 mice per group) were cultured and supernatant IL-18 concentrations measured by ELISA and normalized by colon weight (nsP=0.0573, ***P<0.0001). (g) mRNA from colonic mucosa of age- and sex-matched TNBS treated wild-type and Cyld–/– mice (n=10 mice per group) were isolated, and the expression of Il18 was quantified by real-time PCR (nsP=0.7252). (h) Colonic mucosal extracts from TNBS treated wild-type and Cyld–/– mice was subjected to SDS-PAGE. The amounts of pro-IL-18 and mature IL-18 were assessed by immunoblotting with anti-IL-18 antibody. (i) Densitometric analysis of mature IL-18 expression from wild-type and Cyld–/– mice (n=5 mice per group) colon tissue after immunoblotting (***P=0.0009). a,b,c,e,f,g,i Statistics are mean ± SD and P values were determined by Student’s t test (two tail). Data are from one experiment representative of three independent experiments with similar results. Uncropped blots (h) are shown in the Source Data.

Article Snippet: IL-18 cytokine concentrations by ELISA IL-18 concentrations from serum and colon tissue, colonic epithelial cell culture supernatants of mice and colon tissue lysates of humans were measured using commercially available kits mouse IL-18 platinum ELISA kit (eBioscience) and human IL-18 PicoKine ELISA kit (Bosterbio) according to the manufacturers’ protocols.

Techniques: Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Real-time Polymerase Chain Reaction, SDS Page, Western Blot

Journal: Nature immunology

Article Title: Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation

doi: 10.1038/s41590-020-0681-x

Figure Lengend Snippet: (a) Body weight change (***P<0.0001) and (b) diarrhea score (***P<0.0001) of wild-type, Cyld-/-, Il18-/-, and Cyld-/-Il18-/- mice (n=8 mice per group) infected (inf) with C. rodentium. (c), Body weight change (***P<0.0001), (d) diarrhea score (***P<0.0001), and (e) colon length (***P<0.0001, **P=0.0039, ***P<0.0001, ***P<0.0001) of wild-type, Cyld-/-, Il18-/- and Cyld-/-Il18-/- mice (n=8 mice per group) infected (inf) with C. rodentium and along with rIL-18. (f) H&E-stained colonic sections from C. rodentium–infected and rIL-18 -treated wild-type, Cyld-/-, Il18-/- and Cyld-/-Il18-/- mice (scale bars, 50 μm) and g, histology scores of the sections (n=8 mice per group, ***P=0.0002, ***P=0.0001). Statistics are mean ± SD, with P values determined by Student’s t test (two tail). Data are from one experiment representative of three independent experiments with similar results.

Article Snippet: IL-18 cytokine concentrations by ELISA IL-18 concentrations from serum and colon tissue, colonic epithelial cell culture supernatants of mice and colon tissue lysates of humans were measured using commercially available kits mouse IL-18 platinum ELISA kit (eBioscience) and human IL-18 PicoKine ELISA kit (Bosterbio) according to the manufacturers’ protocols.

Techniques: Recombinant, Infection, Staining

Journal: Nature immunology

Article Title: Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation

doi: 10.1038/s41590-020-0681-x

Figure Lengend Snippet: a, Serum was collected from age-matched uninfected and infected wild-type and Cyld−/− mice (n=8 mice per group). The concentration of IL-18 was measured by ELISA (NSP=0.2103, **P=0.0016). b, Colon tissues from uninfected and infected wild-type and Cyld−/− mice (n=8 mice per group) were cultured for 24h, supernatants were collected, and the IL-18 concentration was measured by ELISA and normalized to colon weight (NSP=0.3657, ***P<0.0001). c, The expression of Il18 was quantified (NSP=0.9792) by real-time PCR on mRNA from colons of age- and sex-matched infected wild-type and Cyld−/− mice (n=8 mice per group). d, Colonic mucosal lysates from uninfected and infected wild-type and Cyld−/− mice were subjected to SDS-PAGE. The amounts of pro-IL-18 and mature IL-18 were assessed by immunoblotting with anti-IL-18. e–g, Body weight change (NSP=0.0520, ***P=0.0001; e), diarrhea score (NSP=0.8683, ***P<0.0001; f) and colon length (***P<0.0001; g) of Cyldfl/fl versus epithelial-specific Cyld−/− (IEC- Cyld(γ9)) mice (n=10 mice per group) infected (inf) with C. rodentium, treated with anti-IL-18 or control IgG. h, The mean fluorescence intensity showing the translocation of FITC-dextran assayed using serum 4h after administration (n=10 mice per group, ***P<0.0001). i,j, H&E-stained colonic sections from infected Cyldfl/fl and IEC-Cyld(Δ9) mice treated with control IgG or anti-IL-18 (scale bars, 50 μm; i) and the histology scores of the sections (n=10 mice per group, ***P<0.0001, **P=0.0011; j). The statistics are shown as mean ± s.d., with P values determined by Student’s t-test (two tail). The data are from one experiment representative of three independent experiments with similar results. Uncropped blots (d) are shown in the source data.

Article Snippet: IL-18 cytokine concentrations by ELISA IL-18 concentrations from serum and colon tissue, colonic epithelial cell culture supernatants of mice and colon tissue lysates of humans were measured using commercially available kits mouse IL-18 platinum ELISA kit (eBioscience) and human IL-18 PicoKine ELISA kit (Bosterbio) according to the manufacturers’ protocols.

Techniques: Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, SDS Page, Western Blot, Fluorescence, Translocation Assay, Staining

Journal: Nature immunology

Article Title: Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation

doi: 10.1038/s41590-020-0681-x

Figure Lengend Snippet: (a–c) (a) Body weight change (nsP=0.6251, **P=0.0029), (b) diarrhea score (***P=0.0005), and (c) colon length (nsP=0.6433, **P=0.0046) of Cyldfl/fl vs. IEC-Cyld (δ9) (Epithelial specific cyld knockout mice) mice (n = 5 mice per group) infected (inf) with C. rodentium. (d, e) H&E-stained colonic sections from C. rodentium–infected Cyldfl/fl and IEC-Cyld (δ9) mice (scale bars, 50 μm) and histology scores (n = 5 mice per group, nsP=0.99, **P=0.0028). (f) CFU from the organ culture (n = 5 mice per group, *P=0.0241, **P=0.0029, **P=0.0056). (g) Colon tissues from uninfected and infected Cyldfl/fl and IEC-Cyld (δ9) mice were cultured, IL-18 was measured from the supernatant by ELISA and normalized by colon weight (n = 5 mice per group, nsP=0.2755, **P=0.0013). (h) Colonic mucosal lysates from infected Cyldfl/fl and IEC-Cyld (δ9) was subjected to SDS-PAGE. The amounts of pro-IL-18 and mature IL-18 were assessed by immunoblotting with anti–IL-18 antibody. a,b,c,e,f,g Statistics are mean ± SD and P values were determined by Student’s t test (Two tail). Data are from one experiment representative of three independent experiments with similar results. Uncropped blots (h) are shown in the Source Data.

Article Snippet: IL-18 cytokine concentrations by ELISA IL-18 concentrations from serum and colon tissue, colonic epithelial cell culture supernatants of mice and colon tissue lysates of humans were measured using commercially available kits mouse IL-18 platinum ELISA kit (eBioscience) and human IL-18 PicoKine ELISA kit (Bosterbio) according to the manufacturers’ protocols.

Techniques: Knock-Out, Infection, Staining, Organ Culture, Cell Culture, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot

Journal: Nature immunology

Article Title: Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation

doi: 10.1038/s41590-020-0681-x

Figure Lengend Snippet: a, mRNA from colonic mucosa of age- and sex-matched C. rodentium-infected wild-type, Cyld−/−, Il18−/− and Cyld−/−Il18−/− mice (n=8 mice per group) was isolated, and the expression of Il18 was quantified by real-time PCR (NSP=0.3390). b, Colon tissues from C. rodentium–infected wild-type, Cyld−/−, Il18−/− and Cyld−/−Il18−/− mice were cultured for 24h (n=8 mice per group), and the IL-18 concentration in the supernatant was measured by ELISA and normalized to colon weight (***P<0.0001). c, mRNA from colonic mucosa of age- and sex-matched C. rodentium-infected wild-type, Cyld−/−, Nlrp6−/− and Cyld−/−Nlrp6−/− mice was isolated (n=8 mice per group), and the level of Il18 was quantified by real-time PCR (NSP=0.9748, NSP=0.2241, NSP=0.5841, left to right). d, Colon tissues from C. rodentium-infected wild-type, Cyld−/−, Nlrp6−/− and Cyld−/−Nlrp6−/− mice (n=8 mice per group) were cultured for 24h, and the IL-18 concentration in the supernatant was measured by ELISA and normalized to colon weight (***P=0.0001; NS, not significant). The statistics are given as mean ± s.d., with P values determined by Student’s t-test (two tail). The data are from one experiment representative of three independent experiments with similar results.

Article Snippet: IL-18 cytokine concentrations by ELISA IL-18 concentrations from serum and colon tissue, colonic epithelial cell culture supernatants of mice and colon tissue lysates of humans were measured using commercially available kits mouse IL-18 platinum ELISA kit (eBioscience) and human IL-18 PicoKine ELISA kit (Bosterbio) according to the manufacturers’ protocols.

Techniques: Infection, Isolation, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Human adipose tissue-derived small extracellular vesicles promote soft tissue repair through modulating M1-to-M2 polarization of macrophages

doi: 10.1186/s13287-023-03306-7

Figure Lengend Snippet: Macrophage polarization induced by sEV-AT. a Schematic view of the treatment process. M0 macrophages were induced into M1 macrophages by LPS. Then, the culture medium was removed, and h-sEV-AT was added to polarize M1 macrophages to M2 macrophages for 4 days. b Representative images of M1 macrophages (F4/80 + iNOS + ) and M2 macrophages (F4/80 + CD206 + ) with immunofluorescence staining (green: F4/80, red: iNOS/CD206, blue: DAPI). Scale bar = 50 µm. c The percentage of M1 macrophages or M2 macrophages in total cells per field of view (scale bar = 50 µm) was analyzed by ImageJ software ( n = 3). d The ratio of M2/M1 per field of view (scale bar = 50 µm) was analyzed ( n = 3). e The expression of M1 macrophage marker genes (IL-6, IL-1β, TNFα, and iNOS) and M2 macrophage marker genes (Arg1, TGF-β1, IL-10, and CD206) was measured by qRT-PCR ( n = 3). The significance was tested with an unpaired two-tailed Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Primary antibodies against F4/80 (1:200, Abcam, ab240946) and iNOS (1:200, Santa Cruz, sc-7271) or CD206 (1:200, Santa Cruz, sc-58986) were used in this study to analyze M1 and M2 macrophages.

Techniques: Immunofluorescence, Staining, Software, Expressing, Marker, Quantitative RT-PCR, Two Tailed Test

Journal: Stem Cell Research & Therapy

Article Title: Human adipose tissue-derived small extracellular vesicles promote soft tissue repair through modulating M1-to-M2 polarization of macrophages

doi: 10.1186/s13287-023-03306-7

Figure Lengend Snippet: Macrophage phenotypes in adipose tissue regeneration. a Representative images of M1 macrophages (F4/80 + iNOS + ) and M2 macrophages (F4/80 + CD206 + ) at 4 weeks with immunofluorescence staining (green: F4/80, red: iNOS/CD206, blue: DAPI). Scale bar = 200 µm. b The percentage of M1 macrophages and M2 macrophages in total macrophages per field of view (scale bar = 200 µm) was analyzed by ImageJ software ( n = 4). c The ratio of M2/M1 per field of view (scale bar = 200 µm) was analyzed ( n = 4). The significance was tested with an unpaired two-tailed Student’s t test. **** p < 0.0001

Article Snippet: Primary antibodies against F4/80 (1:200, Abcam, ab240946) and iNOS (1:200, Santa Cruz, sc-7271) or CD206 (1:200, Santa Cruz, sc-58986) were used in this study to analyze M1 and M2 macrophages.

Techniques: Immunofluorescence, Staining, Software, Two Tailed Test

Journal: Stem Cell Research & Therapy

Article Title: Human adipose tissue-derived small extracellular vesicles promote soft tissue repair through modulating M1-to-M2 polarization of macrophages

doi: 10.1186/s13287-023-03306-7

Figure Lengend Snippet: Macrophage phenotypes in skin wound healing. a Representative longitudinal section of the wounds at day 8 with immunofluorescence staining (green: F4/80, red: iNOS/CD206, blue: DAPI). The white dotted lines pointed out the edge of wound areas. The white dotted boxes represented areas that were magnified in the right-magnified panels. Scale bar = 50 µm. b The percentage of M1 macrophages and M2 macrophages in total macrophages per field of view (scale bar = 50 µm) was analyzed ( n = 4). c The ratio of M2/M1 per field of view (scale bar = 50 µm) was analyzed by ImageJ software ( n = 4). The significance was tested with an unpaired two-tailed Student’s t test. *** p < 0.001, **** p < 0.0001

Article Snippet: Primary antibodies against F4/80 (1:200, Abcam, ab240946) and iNOS (1:200, Santa Cruz, sc-7271) or CD206 (1:200, Santa Cruz, sc-58986) were used in this study to analyze M1 and M2 macrophages.

Techniques: Immunofluorescence, Staining, Software, Two Tailed Test