Journal: Journal of Functional Foods

Article Title: Membrane vesicles from the probiotic Nissle 1917 and gut resident Escherichia coli strains distinctly modulate human dendritic cells and subsequent T cell responses

doi: 10.1016/j.jff.2019.103495

Figure Lengend Snippet: Fig. 6. Quantification of the Th17 and Treg subsets induced by MV-stimulated DCs. (A-B) Flow cytometric analysis of CD4+ T cells co-incubated for 3 days with untreated mo-DCs (iDC) or mo-DCs primed with MVs of the indicated strains (10 μg/ml) at a DC:T cell ratio of 1:5 with specific markers for: (A) Th17 population (CD4+ CD196+) and (B) Treg (CD25+(High) FoxP3+) population. Representative dot plots of CD4+ cells co-incubated with iDCs (control) or with EcN MV- stimulated DCS are shown for each cell subset analysis. The percentage of positive cells is indicated in each quadrant or box. (C) Data (mean ± SEM) from three- independent experiments performed in triplicate are expressed as fold-change increase in the percentage of positive T cells for Th17 or Treg specific markers induced by MV-stimulated DCs with respect control iDCs induced values (set as 1, dot line). Statistical differences were evaluated by one-way ANOVA followed by Bonferroni’s test (normal distribution) *p < 0.05 versus control DCs.

Article Snippet: For surface marker staining, lymphocytes were first stained with anti-CD4 FITC (clone VIT4), anti-CD25 APC (clone 4E3) and anti-CD196 PE (clone REA190) mAb or with the corresponding isotype-matched conjugated irrelevant antibody (Miltenyi Biotec, Bergish Gladbach, Germany).

Techniques: Incubation, Control

Journal: Kidney international

Article Title: Decoy receptor 2 mediation of the senescent phenotype of tubular cells by interacting with peroxiredoxin 1 presents a novel mechanism of renal fibrosis in diabetic nephropathy.

doi: 10.1016/j.kint.2020.03.026

Figure Lengend Snippet: Figure 1 | Expression Q7Q8 of decoy receptor 2 (DcR2) in streptozotocin (STZ)-induced diabetic nephropathy in mice and the association of DcR2 with renal fibrosis. (a) Fasting glucose, (b) serum creatinine, (c) blood urea nitrogen (BUN), and (d) urinary albumin to creatinine ratio (ACR) in each group. *P < 0.05 versus the control group. (e) Quantitative real-time polymerase chain reaction analyzed the expression of DcR2 mRNA levels in each group (n ¼ 6–7 for each group). (f) Representative immunostaining micrographs showed tubular DcR2 expression, and (g) the percentage of positive renal tubular epithelial cells (RTECs) was quantified (n ¼ 6 for each group). Bars ¼ 80 mm. (h) Western Q9 blotting detected the renal expression of DcR2, and (i) the relative levels to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified in different groups (n ¼ 6 for each group). *P < 0.05 versus control; **P < 0.05 versus STZ at 4 weeks. (j) The percentage of positive RTECs was quantified according to tubulointerstitial fibrosis (TIF) scores (n ¼ 6 for each group). Data are expressed as the means SD for each group. *P < 0.05 versus TIF1; **P < 0.05 versus TIF2. OD Q10 , optical density. To optimize Q11 viewing of this image, please see the online version of this article at www.kidney-international.org.

Article Snippet: Tissue sections and cells were incubated with anti-DcR2 antibody followed by Alexa-555 conjugated goat anti-rabbit antibody (ab150078; Abcam), DR5 (sc-7192, Santa Cruz) followed by Alexa647 conjugated monkey anti-goat antibody (ab150135; Abcam), anti-PRDX1 (ab16745; Abcam), cyclin D1 (ab16663; Abcam), CDK6 (ab151247; Abcam), p16 (ab54210; Abcam), and anti-TRAIL antibody (550912; BD Pharmingen, San Diego, CA) overnight at 4 C. Then they were incubated with Alexa-488 conjugated goat antimouse antibody (ab150117; Abcam) at 37 C for 1 hour and costained with 40,6-diamidino-2-phenylindole (C1006; Biyuntian Biotechnology, Shanghai, China).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Immunostaining, Western Blot

Journal: Kidney international

Article Title: Decoy receptor 2 mediation of the senescent phenotype of tubular cells by interacting with peroxiredoxin 1 presents a novel mechanism of renal fibrosis in diabetic nephropathy.

doi: 10.1016/j.kint.2020.03.026

Figure Lengend Snippet: Figure 2 | Decoy receptor 2 (DcR2) aggravates renal fibrosis in streptozotocin (STZ)-induced diabetic nephropathy. (a) Fasting glucose, (b) serum creatinine, (c) blood urea nitrogen (BUN), and (d) urinary albumin to creatinine ratio (ACR) in each group (n ¼ 9 for each group). (e) Masson trichrome staining showed renal fibrosis with blue color, and the percentage of fibrotic area in different groups after quantitative analysis (n ¼ 9 for each group). Bars ¼ 40 mm. (f) Representative immunostaining micrographs show fibrotic markers a-smooth muscle actin (a-SMA), collagen I, and fibronectin expression in different groups (n ¼ 9 for each group). Bars ¼ 40 mm. (g) Western blotting showed renal expression of a-SMA, collagen I, and fibronectin in different groups, and (h,j), and the relative levels to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified (n ¼ 6 for each group). Data are expressed as the means SD for each group. *P < 0.05 versus control; **P < 0.05 versus STZ. OD, optical density. To optimize viewing of this image, please see the online version of this article at www. kidney-international.org. Q12

Article Snippet: Tissue sections and cells were incubated with anti-DcR2 antibody followed by Alexa-555 conjugated goat anti-rabbit antibody (ab150078; Abcam), DR5 (sc-7192, Santa Cruz) followed by Alexa647 conjugated monkey anti-goat antibody (ab150135; Abcam), anti-PRDX1 (ab16745; Abcam), cyclin D1 (ab16663; Abcam), CDK6 (ab151247; Abcam), p16 (ab54210; Abcam), and anti-TRAIL antibody (550912; BD Pharmingen, San Diego, CA) overnight at 4 C. Then they were incubated with Alexa-488 conjugated goat antimouse antibody (ab150117; Abcam) at 37 C for 1 hour and costained with 40,6-diamidino-2-phenylindole (C1006; Biyuntian Biotechnology, Shanghai, China).

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

Journal: Kidney international

Article Title: Decoy receptor 2 mediation of the senescent phenotype of tubular cells by interacting with peroxiredoxin 1 presents a novel mechanism of renal fibrosis in diabetic nephropathy.

doi: 10.1016/j.kint.2020.03.026

Figure Lengend Snippet: Figure 3 | Decoy receptor 2 (DcR2) mediates the senescent phenotype of renal tubular epithelial cell (RTEC) in streptozotocin (STZ) diabetic nephropathy. (a) Quantitative real-time polymerase chain reaction analyzed the p16 and (b) p21 mRNA levels in each (continued)

Article Snippet: Tissue sections and cells were incubated with anti-DcR2 antibody followed by Alexa-555 conjugated goat anti-rabbit antibody (ab150078; Abcam), DR5 (sc-7192, Santa Cruz) followed by Alexa647 conjugated monkey anti-goat antibody (ab150135; Abcam), anti-PRDX1 (ab16745; Abcam), cyclin D1 (ab16663; Abcam), CDK6 (ab151247; Abcam), p16 (ab54210; Abcam), and anti-TRAIL antibody (550912; BD Pharmingen, San Diego, CA) overnight at 4 C. Then they were incubated with Alexa-488 conjugated goat antimouse antibody (ab150117; Abcam) at 37 C for 1 hour and costained with 40,6-diamidino-2-phenylindole (C1006; Biyuntian Biotechnology, Shanghai, China).

Techniques: Real-time Polymerase Chain Reaction

Journal: Kidney international

Article Title: Decoy receptor 2 mediation of the senescent phenotype of tubular cells by interacting with peroxiredoxin 1 presents a novel mechanism of renal fibrosis in diabetic nephropathy.

doi: 10.1016/j.kint.2020.03.026

Figure Lengend Snippet: Figure 4 | Decoy receptor 2 (DcR2) induces renal tubular epithelial cell (RTEC) senescence leading to fibrosis in high glucose (HG). (a,b) Expression of DcR2 and p16 mRNA was detected at different time points after HG (30 mmol/l) treatment (n ¼ 6 for each group). (c) Representative Western blot shows DcR2 and p16 expression, and (d,e) the relative levels to glyceraldehyde-3-phosphate (continued)

Article Snippet: Tissue sections and cells were incubated with anti-DcR2 antibody followed by Alexa-555 conjugated goat anti-rabbit antibody (ab150078; Abcam), DR5 (sc-7192, Santa Cruz) followed by Alexa647 conjugated monkey anti-goat antibody (ab150135; Abcam), anti-PRDX1 (ab16745; Abcam), cyclin D1 (ab16663; Abcam), CDK6 (ab151247; Abcam), p16 (ab54210; Abcam), and anti-TRAIL antibody (550912; BD Pharmingen, San Diego, CA) overnight at 4 C. Then they were incubated with Alexa-488 conjugated goat antimouse antibody (ab150117; Abcam) at 37 C for 1 hour and costained with 40,6-diamidino-2-phenylindole (C1006; Biyuntian Biotechnology, Shanghai, China).

Techniques: Expressing, Western Blot

Journal: Kidney international

Article Title: Decoy receptor 2 mediation of the senescent phenotype of tubular cells by interacting with peroxiredoxin 1 presents a novel mechanism of renal fibrosis in diabetic nephropathy.

doi: 10.1016/j.kint.2020.03.026

Figure Lengend Snippet: Figure 5 | Proteomics identification, functional analysis, and validation of decoy receptor 2 (DcR2)-interacting proteins. (a) Venn diagram showing DcR2-interacting proteins in normal control and diabetic nephropathy (DN) groups. (b) Venn diagram indicating DcR2- interacting proteins in control and high glucose (HG) groups. (c) Gene Ontology analysis of cellular component, biological processes, and molecular function in the comparison between DN and normal control and (d) between HG and control. (e) Representative immunofluorescence staining for DcR2 (red) and peroxiredoxin 1 (PRDX1) (green) of renal tissues in patients with DN and streptozotocin (STZ) models (n ¼ 6 for each group). Bars ¼ 80 mm. (f,g) Reciprocal co-immunoprecipitation showing that the interaction of DcR2 and dimeric PRDX1 (44 kDa) in STZ-DN (n ¼ 6 for each group). Arrows indicate specific bands. (h) Immunoprecipitation (IP) assays of renal tubular epithelial cells co-transfected with Flag-DcR2 or Myc-PRDX1, followed by immunoblotting with anti-Myc or anti-Flag, respectively (n ¼ 3 for each group). DAPI, 40,6-diamidino-2-phenylindole. To optimize viewing of this image, please see the online version of this article at www.kidney- international.org.

Article Snippet: Tissue sections and cells were incubated with anti-DcR2 antibody followed by Alexa-555 conjugated goat anti-rabbit antibody (ab150078; Abcam), DR5 (sc-7192, Santa Cruz) followed by Alexa647 conjugated monkey anti-goat antibody (ab150135; Abcam), anti-PRDX1 (ab16745; Abcam), cyclin D1 (ab16663; Abcam), CDK6 (ab151247; Abcam), p16 (ab54210; Abcam), and anti-TRAIL antibody (550912; BD Pharmingen, San Diego, CA) overnight at 4 C. Then they were incubated with Alexa-488 conjugated goat antimouse antibody (ab150117; Abcam) at 37 C for 1 hour and costained with 40,6-diamidino-2-phenylindole (C1006; Biyuntian Biotechnology, Shanghai, China).

Techniques: Functional Assay, Biomarker Discovery, Control, Comparison, Staining, Immunoprecipitation, Transfection, Western Blot

Journal: Kidney international

Article Title: Decoy receptor 2 mediation of the senescent phenotype of tubular cells by interacting with peroxiredoxin 1 presents a novel mechanism of renal fibrosis in diabetic nephropathy.

doi: 10.1016/j.kint.2020.03.026

Figure Lengend Snippet: Figure 7 | Interaction of decoy receptor 2 (DcR2) and peroxiredoxin 1 (PRDX1) mediates the senescent phenotype of renal tubular epithelial cell (RTEC). (a) Representative immunofluorescence staining for green fluorescent protein ([GFP], green) and p16 (continued)

Article Snippet: Tissue sections and cells were incubated with anti-DcR2 antibody followed by Alexa-555 conjugated goat anti-rabbit antibody (ab150078; Abcam), DR5 (sc-7192, Santa Cruz) followed by Alexa647 conjugated monkey anti-goat antibody (ab150135; Abcam), anti-PRDX1 (ab16745; Abcam), cyclin D1 (ab16663; Abcam), CDK6 (ab151247; Abcam), p16 (ab54210; Abcam), and anti-TRAIL antibody (550912; BD Pharmingen, San Diego, CA) overnight at 4 C. Then they were incubated with Alexa-488 conjugated goat antimouse antibody (ab150117; Abcam) at 37 C for 1 hour and costained with 40,6-diamidino-2-phenylindole (C1006; Biyuntian Biotechnology, Shanghai, China).

Techniques: Staining

Journal: Kidney international

Article Title: Decoy receptor 2 mediation of the senescent phenotype of tubular cells by interacting with peroxiredoxin 1 presents a novel mechanism of renal fibrosis in diabetic nephropathy.

doi: 10.1016/j.kint.2020.03.026

Figure Lengend Snippet: Figure 8 | Decoy receptor 2 (DcR2) regulates the level of peroxiredoxin 1 (PRDX1) phosphorylation through protein tyrosine kinase (PTK). (a) Quantitative real-time polymerase chain reaction analyzed the expression of PRDX1 mRNA in high glucose (HG)–induced renal tubular epithelial cells at different time points (n ¼ 4 for each group). (b) Western blotting showed DcR2 and PRDX1 expression (continued)

Article Snippet: Tissue sections and cells were incubated with anti-DcR2 antibody followed by Alexa-555 conjugated goat anti-rabbit antibody (ab150078; Abcam), DR5 (sc-7192, Santa Cruz) followed by Alexa647 conjugated monkey anti-goat antibody (ab150135; Abcam), anti-PRDX1 (ab16745; Abcam), cyclin D1 (ab16663; Abcam), CDK6 (ab151247; Abcam), p16 (ab54210; Abcam), and anti-TRAIL antibody (550912; BD Pharmingen, San Diego, CA) overnight at 4 C. Then they were incubated with Alexa-488 conjugated goat antimouse antibody (ab150117; Abcam) at 37 C for 1 hour and costained with 40,6-diamidino-2-phenylindole (C1006; Biyuntian Biotechnology, Shanghai, China).

Techniques: Phospho-proteomics, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Kidney international

Article Title: Decoy receptor 2 mediation of the senescent phenotype of tubular cells by interacting with peroxiredoxin 1 presents a novel mechanism of renal fibrosis in diabetic nephropathy.

doi: 10.1016/j.kint.2020.03.026

Figure Lengend Snippet: Figure 9 | Schematic presentation of decoy receptor 2 (DcR2)- mediated senescent phenotype of tubular cell and renal fibrosis in diabetic nephropathy. DcR2 was specifically expressed in renal tubules and interacted with peroxiredoxin 1 (PRDX1) in the progression of diabetic nephropathy. Subsequently, the interaction of DcR2 and PRDX1 regulated the expression of cell cycle–related proteins, which led to cell cycle arrest, cell senescence, and senescence-associated secretory phenotype (SASP). These effects ultimately contributed to renal fibrosis.

Article Snippet: Tissue sections and cells were incubated with anti-DcR2 antibody followed by Alexa-555 conjugated goat anti-rabbit antibody (ab150078; Abcam), DR5 (sc-7192, Santa Cruz) followed by Alexa647 conjugated monkey anti-goat antibody (ab150135; Abcam), anti-PRDX1 (ab16745; Abcam), cyclin D1 (ab16663; Abcam), CDK6 (ab151247; Abcam), p16 (ab54210; Abcam), and anti-TRAIL antibody (550912; BD Pharmingen, San Diego, CA) overnight at 4 C. Then they were incubated with Alexa-488 conjugated goat antimouse antibody (ab150117; Abcam) at 37 C for 1 hour and costained with 40,6-diamidino-2-phenylindole (C1006; Biyuntian Biotechnology, Shanghai, China).

Techniques: Expressing

Journal: Rheumatology (Oxford, England)

Article Title: Concurrent gene therapy strategies effectively destroy synoviocytes of patients with rheumatoid arthritis.

doi: 10.1093/rheumatology/kel448

Figure Lengend Snippet: FIG. 6. Down-regulation of TRAIL-R4 expression further sensitized RA synovio- cytes to Ad5hTRAIL. Panel A is a flow cytometric analysis of TRAIL receptor composition of synoviocytes following a siRNA approach. C represents isotype control staining. Numbers above the each peak represent TRAIL receptor subtype. Peak labelled as 4 (far right) received siRNA-A while the rest of the samples received DcR2 siRNA. 104 cells are gated for each histogram. Panel B is the cell viability assay following either DcR2 siRNA or siRNA-A approach. Both DcR2 and siRNA treated samples are also infected with an Ad5hTRAIL virus at an MOI of 10 000 DNA particles/cell. Panels A and B are composed of data from a single patient.

Article Snippet: Down-regulation of TRAIL-R4 expression by a DcR2 siRNA approach A DcR2 siRNA kit (sc-35185), siRNA transfection medium (sc-36868) and siRNA transfection reagent (sc-29528) were used to perform DcR2 siRNA experiments as described by the manufacturer (Santa Cruz Biotechnology).

Techniques: Expressing, Control, Staining, Viability Assay, Infection, Virus

Journal:

Article Title: Tumour necrosis factor related apoptosis inducing ligand (TRAIL) induces hepatic steatosis in viral hepatitis and after alcohol intake

doi: 10.1136/gut.2004.056929

Figure Lengend Snippet: Viral hepatitis sensitised for tumour necrosis factor related apoptosis inducing ligand (TRAIL) mediated apoptosis and steatosis. (A) Viral hepatitis was induced by intravenous application of 1×1010 pfu/g of AdlacZ into Balb/c mice. After 40 hours whole cell extracts of the livers were isolated and investigated for protein expression of TRAIL, DR5, DcR1, DcR2, and β-actin by western blot experiments. (B) Adenoviral vectors AdGFP, AdFasL, and Ad-TRAIL (1×107 pfu/g) were administered into the tail vein of Balb/c mice 24 hours after pretreatment with 5×107 pfu/g of AdlacZ. Twenty four hours after the last injection, apoptosis of hepatocytes was analysed by TUNEL assay (magnification 200×) and steatosis by fat red staining (magnification 200×). Figure shows representative data of four independent experiments. (C) Adenoviral vectors AdGFP, AdFasL, and AdTRAIL (1×107 pfu/g) were administered into the tail vein of Balb/c mice 24 hours after pretreatment with 5×107 pfu/g of AdlacZ. Twenty four hours after the last injection, livers were harvested for caspase 8 assays. FasL and TRAIL significantly (*) induced caspase 8 activity. C 8 inhi., caspase 8 inhibitor. (D) In animals with adenoviral hepatitis, one hour prior to AdTRAIL application, anti-TRAIL antibody N2B2 (250 μg / mouse) or isotype control antibody (250 μg/mouse) was injected into the tail vein. Hepatic steatosis was assessed by fat red staining. Administration of TRAIL neutralising antibody significantly (*) inhibited TRAIL mediated hepatic steatosis. Figure shows representative data of four independent experiments (magnification 600×).

Article Snippet: The following primary antibodies were used: AF837 against DR5 human and mouse (R&D, Wiesbaden, Germany), AF630 against DcR1 mouse (R&D), anti-DcR2 against DcR2 mouse (ProSci, Poway, California, USA), C-11 against β-actin (Santa Cruz, Santa Cruz, California, USA), rabbit polyclonal FasL antibody Ab-1 (Oncogene, Calbiochem, California, USA), and H-257 against TRAIL human and mouse (Santa-Cruz).

Techniques: Isolation, Expressing, Western Blot, Injection, TUNEL Assay, Staining, Activity Assay, Control

Journal: Oncotarget

Article Title: Hypoxia regulates TRAIL sensitivity of colorectal cancer cells through mitochondrial autophagy

doi: 10.18632/oncotarget.9206

Figure Lengend Snippet: A. DLD1 cells were grown under normoxic (21% O 2 ) or hypoxic (0.5% O 2 ) conditions for 18 h. Subsequently, cells were lysed and relative expression levels of the indicated proteins were measured using an antibody-based array (see Material and Methods section). Proteins were quantified using densitometry and normalized to reference spots. Data shown are representative of two experiments performed. AU, arbitrary units. B. DLD-1, HCT-8 and HCT116 cells were grown for 18 h under normoxic (21% O 2 ) or hypoxic (0.5% O 2 ) conditions and subsequently analyzed for TRAIL-R1, TRAIL-R2, TRAIL-R3 and TRAIL-R4 cell surface expression using flow cytometry. Data shown are representative of three experiments performed. C. TRAIL-R1/−R2 signaling complexes were induced in DLD1 cells grown for 18 h under normoxic (21% O 2 ) or hypoxic (0.5% O 2 ) conditions by stimulation with Fc-FLAG-scTRAIL (1 μg/mL) for 90 min. Proteins associated with Fc-FLAG-scTRAIL were immunoprecipitated using protein G agarose and were analyzed together with the corresponding lysates by Western blotting for the presence of TRAIL-R1 and TRAIL-R2 and the DISC components FADD, FLIP and caspase-8. Data shown are representative of two experiments performed. D. DLD1 cells were grown for 18 h under normoxic (21% O 2 ) or hypoxic (0.5% O 2 ) conditions and subsequently challenged with 256 ng/mL TRAIL for the indicated periods of time. Caspase-8 activity was measured using the fluorogenic substrate (Ac-IETD) 2 -R110. Values are means ± SEM from three experiments. AU, arbitrary units.

Article Snippet: Anti-TRAIL-R1, anti-TRAIL-R2, anti-TRAIL-R3 and anti-TRAIL-R4 antibodies were from ProSci (Poway, CA, USA), anti-HIF1α was from Cayman Chemical (Ann Arbor, MI, USA) and anti-tubulin from Dunnlab (Asbach, Germany).

Techniques: Expressing, Ab Array, Flow Cytometry, Immunoprecipitation, Western Blot, Activity Assay