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  • 99
    ATCC madin darby canine kidney mdck cells
    Overexpression and endogenous or exogenous expression of BI-1 or BI-1 ∆C in <t>Madin-Darby</t> Canine Kidney <t>(MDCK)</t> cells. ( A ) Scheme of the lentiviral constructs for the expression of wild type BI-1 or nonfunctional mutant BI-1 ΔC in MDCK cells. BI-1 or BI-1 ΔC was inserted into the lentiviral pEF vector. The pEF lentivirus containing wild type BI-1 or the non-functional mutant BI-1 ΔC was produced and used to infect MDCK cells. ( B ) Illustration of specific primer sets which was used to analyze endogenous or exogenous expression of BI-1 . ( C ) The endogenous or exogenous expression of BI-1 was confirmed by RT-PCR analysis in indicated cells; and the expression of HA was confirmed by western blot analysis in indicated cells. The expression of GAPDH and Actin was used as loading control. ( D ) The illustration of whole experimental protocol used in this study. (Abbreviations: chimeric Rous sarcoma virus (RSV) sequence, Rev response elements (RRE), central polypurine tract (cPPT), elongation factor 1 alpha (EF-1α) promoter region, a copGFP (copepod GFP) tag, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), cells growth media (CGM), virus growth media (VGM), and hemagglutinin assay (HA assay)).
    Madin Darby Canine Kidney Mdck Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1403 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore flow cytometry kidneys
    Substantial portion of circulating ICAM-1 + neutrophils during the recovery phase of IRI are positive for THP. ( A ) Flow <t>cytometry</t> show the increased percentage of THP + ICAM-1 + neutrophils on day 2 after IRI. Neutrophils were gated first. ( B ) Percentage of THP + ICAM-1 + neutrophils after IRI. ( C ) THP was increased in peripheral blood leukocytes during the recovery phase of IRI (n=4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, THP = Tamm-Horsfall protein. * P
    Flow Cytometry Kidneys, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Randox kidney function
    Substantial portion of circulating ICAM-1 + neutrophils during the recovery phase of IRI are positive for THP. ( A ) Flow <t>cytometry</t> show the increased percentage of THP + ICAM-1 + neutrophils on day 2 after IRI. Neutrophils were gated first. ( B ) Percentage of THP + ICAM-1 + neutrophils after IRI. ( C ) THP was increased in peripheral blood leukocytes during the recovery phase of IRI (n=4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, THP = Tamm-Horsfall protein. * P
    Kidney Function, supplied by Randox, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amarin Corporation kidney function
    Substantial portion of circulating ICAM-1 + neutrophils during the recovery phase of IRI are positive for THP. ( A ) Flow <t>cytometry</t> show the increased percentage of THP + ICAM-1 + neutrophils on day 2 after IRI. Neutrophils were gated first. ( B ) Percentage of THP + ICAM-1 + neutrophils after IRI. ( C ) THP was increased in peripheral blood leukocytes during the recovery phase of IRI (n=4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, THP = Tamm-Horsfall protein. * P
    Kidney Function, supplied by Amarin Corporation, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Siemens Healthineers kidney function
    Substantial portion of circulating ICAM-1 + neutrophils during the recovery phase of IRI are positive for THP. ( A ) Flow <t>cytometry</t> show the increased percentage of THP + ICAM-1 + neutrophils on day 2 after IRI. Neutrophils were gated first. ( B ) Percentage of THP + ICAM-1 + neutrophils after IRI. ( C ) THP was increased in peripheral blood leukocytes during the recovery phase of IRI (n=4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, THP = Tamm-Horsfall protein. * P
    Kidney Function, supplied by Siemens Healthineers, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Novo Nordisk kidney function
    Substantial portion of circulating ICAM-1 + neutrophils during the recovery phase of IRI are positive for THP. ( A ) Flow <t>cytometry</t> show the increased percentage of THP + ICAM-1 + neutrophils on day 2 after IRI. Neutrophils were gated first. ( B ) Percentage of THP + ICAM-1 + neutrophils after IRI. ( C ) THP was increased in peripheral blood leukocytes during the recovery phase of IRI (n=4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, THP = Tamm-Horsfall protein. * P
    Kidney Function, supplied by Novo Nordisk, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Roche creatinine reflecting kidney function
    Substantial portion of circulating ICAM-1 + neutrophils during the recovery phase of IRI are positive for THP. ( A ) Flow <t>cytometry</t> show the increased percentage of THP + ICAM-1 + neutrophils on day 2 after IRI. Neutrophils were gated first. ( B ) Percentage of THP + ICAM-1 + neutrophils after IRI. ( C ) THP was increased in peripheral blood leukocytes during the recovery phase of IRI (n=4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, THP = Tamm-Horsfall protein. * P
    Creatinine Reflecting Kidney Function, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abbott Laboratories kidney function tests
    Substantial portion of circulating ICAM-1 + neutrophils during the recovery phase of IRI are positive for THP. ( A ) Flow <t>cytometry</t> show the increased percentage of THP + ICAM-1 + neutrophils on day 2 after IRI. Neutrophils were gated first. ( B ) Percentage of THP + ICAM-1 + neutrophils after IRI. ( C ) THP was increased in peripheral blood leukocytes during the recovery phase of IRI (n=4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, THP = Tamm-Horsfall protein. * P
    Kidney Function Tests, supplied by Abbott Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Allon Therapeutics m kidney function influences warfarin responsiveness
    Substantial portion of circulating ICAM-1 + neutrophils during the recovery phase of IRI are positive for THP. ( A ) Flow <t>cytometry</t> show the increased percentage of THP + ICAM-1 + neutrophils on day 2 after IRI. Neutrophils were gated first. ( B ) Percentage of THP + ICAM-1 + neutrophils after IRI. ( C ) THP was increased in peripheral blood leukocytes during the recovery phase of IRI (n=4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, THP = Tamm-Horsfall protein. * P
    M Kidney Function Influences Warfarin Responsiveness, supplied by Allon Therapeutics, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio-Rad kidney iri
    <t>FTY-DC</t> require S1pr1 on BMDC to protect kidneys from <t>IRI.</t> (A) Protocol for experimental setup. FTY720 is a ligand for four out of five S1P receptors. Plasma creatinine (PCr, mg/dL) was measured 24 h after IRI. We tested if S1pr1 or S1pr3 were requited for FTY720 dependent regulatory DC phenotype. BMDCs were propagated from either C57BL/6 WT, CD11cCreS1pr1 fl / fl ( S1pr1 −/− DC), or S1pr3 −/− and treated with FTY720. FTY- S1pr1 −/− DC do not protect kidneys from IRI. As demonstrated in our earlier published studies and again confirmed, transfer of S1pr3 −/− DC with or without FTY720 significantly protect kidney from IRI. These studies suggest only S1pr1 are necessary for FTY720 dependent regulatory DC phenotype. Next, we tested if the route of delivery was important for FTY-DC induced protection from kidney IRI. (B) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. FTY-DC were injected either intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.) or as i.v. using Veh-DC that were acutely treated with FTY720 (overnight along with LPS). These studies suggest that FTY-DC only protect kidneys from IRI if injected via i.v. and the FTY-DCs must be propagated in presence of FTY720 at start of the BMDC culture. ** p ≤ 0.01. (C) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. In all studies presented we injected FTY-DC 1 day before kidney IRI. We also tested if FTY-DC could be injected after injury. Mice were treated with either NC, Veh-DC or FTY-DC 4 h after bilateral IRI. Mice injected with Veh-DC had higher PCr compared to NC mice and FTY-DC treated mice were significantly protected. (D) Protocol for experimental setup. Plasma creatinine (mg/dL) was measured 24 h after IRI. C57BL/6 FTY-DC induce protection in BALB/c mice and protect kidneys from ischemic injury. Data represent means ± SEM, Unpaired t -test [D], *** p ≤ 0.001; ** p ≤ 0.01 and *** p ≤ 0.001, one-way ANOVA followed by Tukey's post-test.
    Kidney Iri, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Ikaria long term fish intake preserves kidney function
    <t>FTY-DC</t> require S1pr1 on BMDC to protect kidneys from <t>IRI.</t> (A) Protocol for experimental setup. FTY720 is a ligand for four out of five S1P receptors. Plasma creatinine (PCr, mg/dL) was measured 24 h after IRI. We tested if S1pr1 or S1pr3 were requited for FTY720 dependent regulatory DC phenotype. BMDCs were propagated from either C57BL/6 WT, CD11cCreS1pr1 fl / fl ( S1pr1 −/− DC), or S1pr3 −/− and treated with FTY720. FTY- S1pr1 −/− DC do not protect kidneys from IRI. As demonstrated in our earlier published studies and again confirmed, transfer of S1pr3 −/− DC with or without FTY720 significantly protect kidney from IRI. These studies suggest only S1pr1 are necessary for FTY720 dependent regulatory DC phenotype. Next, we tested if the route of delivery was important for FTY-DC induced protection from kidney IRI. (B) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. FTY-DC were injected either intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.) or as i.v. using Veh-DC that were acutely treated with FTY720 (overnight along with LPS). These studies suggest that FTY-DC only protect kidneys from IRI if injected via i.v. and the FTY-DCs must be propagated in presence of FTY720 at start of the BMDC culture. ** p ≤ 0.01. (C) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. In all studies presented we injected FTY-DC 1 day before kidney IRI. We also tested if FTY-DC could be injected after injury. Mice were treated with either NC, Veh-DC or FTY-DC 4 h after bilateral IRI. Mice injected with Veh-DC had higher PCr compared to NC mice and FTY-DC treated mice were significantly protected. (D) Protocol for experimental setup. Plasma creatinine (mg/dL) was measured 24 h after IRI. C57BL/6 FTY-DC induce protection in BALB/c mice and protect kidneys from ischemic injury. Data represent means ± SEM, Unpaired t -test [D], *** p ≤ 0.001; ** p ≤ 0.01 and *** p ≤ 0.001, one-way ANOVA followed by Tukey's post-test.
    Long Term Fish Intake Preserves Kidney Function, supplied by Ikaria, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Collaborative Drug Discovery Inc kidney cortical collecting duct
    <t>FTY-DC</t> require S1pr1 on BMDC to protect kidneys from <t>IRI.</t> (A) Protocol for experimental setup. FTY720 is a ligand for four out of five S1P receptors. Plasma creatinine (PCr, mg/dL) was measured 24 h after IRI. We tested if S1pr1 or S1pr3 were requited for FTY720 dependent regulatory DC phenotype. BMDCs were propagated from either C57BL/6 WT, CD11cCreS1pr1 fl / fl ( S1pr1 −/− DC), or S1pr3 −/− and treated with FTY720. FTY- S1pr1 −/− DC do not protect kidneys from IRI. As demonstrated in our earlier published studies and again confirmed, transfer of S1pr3 −/− DC with or without FTY720 significantly protect kidney from IRI. These studies suggest only S1pr1 are necessary for FTY720 dependent regulatory DC phenotype. Next, we tested if the route of delivery was important for FTY-DC induced protection from kidney IRI. (B) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. FTY-DC were injected either intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.) or as i.v. using Veh-DC that were acutely treated with FTY720 (overnight along with LPS). These studies suggest that FTY-DC only protect kidneys from IRI if injected via i.v. and the FTY-DCs must be propagated in presence of FTY720 at start of the BMDC culture. ** p ≤ 0.01. (C) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. In all studies presented we injected FTY-DC 1 day before kidney IRI. We also tested if FTY-DC could be injected after injury. Mice were treated with either NC, Veh-DC or FTY-DC 4 h after bilateral IRI. Mice injected with Veh-DC had higher PCr compared to NC mice and FTY-DC treated mice were significantly protected. (D) Protocol for experimental setup. Plasma creatinine (mg/dL) was measured 24 h after IRI. C57BL/6 FTY-DC induce protection in BALB/c mice and protect kidneys from ischemic injury. Data represent means ± SEM, Unpaired t -test [D], *** p ≤ 0.001; ** p ≤ 0.01 and *** p ≤ 0.001, one-way ANOVA followed by Tukey's post-test.
    Kidney Cortical Collecting Duct, supplied by Collaborative Drug Discovery Inc, used in various techniques. Bioz Stars score: 88/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    NanoString Technologies Inc kidney organoids
    <t>FTY-DC</t> require S1pr1 on BMDC to protect kidneys from <t>IRI.</t> (A) Protocol for experimental setup. FTY720 is a ligand for four out of five S1P receptors. Plasma creatinine (PCr, mg/dL) was measured 24 h after IRI. We tested if S1pr1 or S1pr3 were requited for FTY720 dependent regulatory DC phenotype. BMDCs were propagated from either C57BL/6 WT, CD11cCreS1pr1 fl / fl ( S1pr1 −/− DC), or S1pr3 −/− and treated with FTY720. FTY- S1pr1 −/− DC do not protect kidneys from IRI. As demonstrated in our earlier published studies and again confirmed, transfer of S1pr3 −/− DC with or without FTY720 significantly protect kidney from IRI. These studies suggest only S1pr1 are necessary for FTY720 dependent regulatory DC phenotype. Next, we tested if the route of delivery was important for FTY-DC induced protection from kidney IRI. (B) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. FTY-DC were injected either intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.) or as i.v. using Veh-DC that were acutely treated with FTY720 (overnight along with LPS). These studies suggest that FTY-DC only protect kidneys from IRI if injected via i.v. and the FTY-DCs must be propagated in presence of FTY720 at start of the BMDC culture. ** p ≤ 0.01. (C) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. In all studies presented we injected FTY-DC 1 day before kidney IRI. We also tested if FTY-DC could be injected after injury. Mice were treated with either NC, Veh-DC or FTY-DC 4 h after bilateral IRI. Mice injected with Veh-DC had higher PCr compared to NC mice and FTY-DC treated mice were significantly protected. (D) Protocol for experimental setup. Plasma creatinine (mg/dL) was measured 24 h after IRI. C57BL/6 FTY-DC induce protection in BALB/c mice and protect kidneys from ischemic injury. Data represent means ± SEM, Unpaired t -test [D], *** p ≤ 0.001; ** p ≤ 0.01 and *** p ≤ 0.001, one-way ANOVA followed by Tukey's post-test.
    Kidney Organoids, supplied by NanoString Technologies Inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Johnson & Johnson grantham j j kidney
    <t>FTY-DC</t> require S1pr1 on BMDC to protect kidneys from <t>IRI.</t> (A) Protocol for experimental setup. FTY720 is a ligand for four out of five S1P receptors. Plasma creatinine (PCr, mg/dL) was measured 24 h after IRI. We tested if S1pr1 or S1pr3 were requited for FTY720 dependent regulatory DC phenotype. BMDCs were propagated from either C57BL/6 WT, CD11cCreS1pr1 fl / fl ( S1pr1 −/− DC), or S1pr3 −/− and treated with FTY720. FTY- S1pr1 −/− DC do not protect kidneys from IRI. As demonstrated in our earlier published studies and again confirmed, transfer of S1pr3 −/− DC with or without FTY720 significantly protect kidney from IRI. These studies suggest only S1pr1 are necessary for FTY720 dependent regulatory DC phenotype. Next, we tested if the route of delivery was important for FTY-DC induced protection from kidney IRI. (B) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. FTY-DC were injected either intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.) or as i.v. using Veh-DC that were acutely treated with FTY720 (overnight along with LPS). These studies suggest that FTY-DC only protect kidneys from IRI if injected via i.v. and the FTY-DCs must be propagated in presence of FTY720 at start of the BMDC culture. ** p ≤ 0.01. (C) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. In all studies presented we injected FTY-DC 1 day before kidney IRI. We also tested if FTY-DC could be injected after injury. Mice were treated with either NC, Veh-DC or FTY-DC 4 h after bilateral IRI. Mice injected with Veh-DC had higher PCr compared to NC mice and FTY-DC treated mice were significantly protected. (D) Protocol for experimental setup. Plasma creatinine (mg/dL) was measured 24 h after IRI. C57BL/6 FTY-DC induce protection in BALB/c mice and protect kidneys from ischemic injury. Data represent means ± SEM, Unpaired t -test [D], *** p ≤ 0.001; ** p ≤ 0.01 and *** p ≤ 0.001, one-way ANOVA followed by Tukey's post-test.
    Grantham J J Kidney, supplied by Johnson & Johnson, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    LGC Standards GmbH porcine kidney cell lines sk 6
    Drugs affecting cytoskeleton and membrane cholesterol prevent stimulation of pDC by infected cells. A. <t>SK-6</t> cells were infected with VRPΔE rns for 24 h, washed and then treated with a DMSO control, nocodazole (5 µM), MβCD (20 mM) or latrunculin (1 µM) for 2 h at 37°C. The inhibitors were then removed and the cells washed three times to avoid effects on pDC. The treated cells were co-cultured with pDC and IFN-α measured after 20 h of incubation. B. Uninfected SK-6 cells were treated as in A and co-cultured with pDC in presence of CpG. The mean values of three replicates with standard deviation are shown. The data is representative of three independent experiments.
    Porcine Kidney Cell Lines Sk 6, supplied by LGC Standards GmbH, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher h uman embryonic kidney cells
    Drugs affecting cytoskeleton and membrane cholesterol prevent stimulation of pDC by infected cells. A. <t>SK-6</t> cells were infected with VRPΔE rns for 24 h, washed and then treated with a DMSO control, nocodazole (5 µM), MβCD (20 mM) or latrunculin (1 µM) for 2 h at 37°C. The inhibitors were then removed and the cells washed three times to avoid effects on pDC. The treated cells were co-cultured with pDC and IFN-α measured after 20 h of incubation. B. Uninfected SK-6 cells were treated as in A and co-cultured with pDC in presence of CpG. The mean values of three replicates with standard deviation are shown. The data is representative of three independent experiments.
    H Uman Embryonic Kidney Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Schiff Nutrition International kidney periodic acid schiff sections
    CD40 ASO treatment mitigated <t>DOX</t> nephropathy . CD40 antisense oligonucleotide (ASO) and control ASO treatments were initiated 2 weeks following initiation of DOX nephropathy and after 8 weeks of ASO treatments renal functional assessments of ( a ) proteinuria (graphed as the ratio of the urine albumin to creatinine) and ( b ) plasma fluorescein isothicyanate conjugated inulin (FITC)-inulin elimination (assessed by a single measurement of residual plasma FITC-Inulin two hours following FITC-inulin injection) were performed. Additionally, kidneys were harvested and whole kidney ( c ) CD40 mRNA and protein, ( d ) CCL5, MCP-1 and NGAL mRNA, and ( e ) CCL5 protein were measured. ( f–i ) Periodic <t>acid-Schiff</t> staining of representative kidney sections from ( f ) phosphate buffered saline (PBS), ( g ) control ASO, ( h ) 12.5 mg/kg CD40 ASO, and ( i ) 25 mg/kg CD40 ASO groups. Mean ± SEM, * P
    Kidney Periodic Acid Schiff Sections, supplied by Schiff Nutrition International, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Microbix 293 human kidney cell line
    Functional characterization of sCAR fusion proteins. (A) Inhibition of Ad binding. 3 H-labeled Ad was preincubated with different amounts of either sCAR-His 6 or sCAR-EGF, and then 3 H-Ad/sCAR-ligand complex samples (10 5 cpm) were mixed with <t>293</t> cells (10 6 cells per aliquot) and allowed to bind at 4°C. Cell-bound radioactivities were determined as described in Materials and Methods. Data are presented as the percentage of input 3 H-Ad bound after washing and calculated as the cumulative mean ± SD of triplicate determinations. Error bars depicting SDs are smaller than the symbols. (B) Inhibition of Ad-mediated gene transfer. Recombinant Ad vector AdCMVLuc, expressing the firefly luciferase reporter gene, was preincubated with various amounts of either sCAR-His 6 or sCAR-EGF. Monolayers of 293 cells were then exposed to AdCMVLuc/sCAR-ligand complexes and assayed for luciferase activity as described in Materials and Methods. Gene transfer indices were calculated from the ratio of the mean luciferase activity documented in cells infected with either AdCMVLuc/sCAR-EGF or AdCMVLuc/sCAR-His 6 to those treated with AdCMVLuc alone. Each point represents the cumulative mean ± SD of triplicate determinations. Some error bars depicting SDs are smaller than the symbols.
    293 Human Kidney Cell Line, supplied by Microbix, used in various techniques. Bioz Stars score: 85/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC 293t embryonic kidney cells
    GPR85 is a functional receptor for CXCL14 activity. ( A ) Confocal microscopy of NAF6 cells treated with 100 ng/ml biotin or biotin-CXCL14 at 4°C and stained with an antibody against Cy3-streptavidin. An isotype-matched IgG was used as a control. Cell nuclei were counterstained with DAPI. ( B ) Schematic of the procedure used to detect biotin-CXCL14–binding proteins using HuProt human proteome microarrays containing 18,583 affinity-purified N-terminally GST-tagged proteins. ( C ) Representative CXCL14-binding membrane proteins in the proteome microarrays. ( D ) Western blot validation using a streptavidin-agarose pull-down assay of proteome microarray determination that CXCL14 binds directly to GPR85. ( E ) Mobilization of [Ca 2+ ] i in NAF6 cells that were transfected with control siRNA (NC) or GPR85-3 siRNA and then treated with 100 ng/ml HBSS, rhCXCL14, or rhCXCL12. The black arrows denote the times at which stimulation was initiated. ( F ) 125 I-CXCL14 binding properties between <t>293T-NC</t> and 293T-GPR85 cells. Data are shown as mean ± SD. n = 4 repetitions. ** P
    293t Embryonic Kidney Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioChain Institute human adult normal kidney tissues
    GPR85 is a functional receptor for CXCL14 activity. ( A ) Confocal microscopy of NAF6 cells treated with 100 ng/ml biotin or biotin-CXCL14 at 4°C and stained with an antibody against Cy3-streptavidin. An isotype-matched IgG was used as a control. Cell nuclei were counterstained with DAPI. ( B ) Schematic of the procedure used to detect biotin-CXCL14–binding proteins using HuProt human proteome microarrays containing 18,583 affinity-purified N-terminally GST-tagged proteins. ( C ) Representative CXCL14-binding membrane proteins in the proteome microarrays. ( D ) Western blot validation using a streptavidin-agarose pull-down assay of proteome microarray determination that CXCL14 binds directly to GPR85. ( E ) Mobilization of [Ca 2+ ] i in NAF6 cells that were transfected with control siRNA (NC) or GPR85-3 siRNA and then treated with 100 ng/ml HBSS, rhCXCL14, or rhCXCL12. The black arrows denote the times at which stimulation was initiated. ( F ) 125 I-CXCL14 binding properties between <t>293T-NC</t> and 293T-GPR85 cells. Data are shown as mean ± SD. n = 4 repetitions. ** P
    Human Adult Normal Kidney Tissues, supplied by BioChain Institute, used in various techniques. Bioz Stars score: 85/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore kidney function blood urea nitrogen bun levels
    GPR85 is a functional receptor for CXCL14 activity. ( A ) Confocal microscopy of NAF6 cells treated with 100 ng/ml biotin or biotin-CXCL14 at 4°C and stained with an antibody against Cy3-streptavidin. An isotype-matched IgG was used as a control. Cell nuclei were counterstained with DAPI. ( B ) Schematic of the procedure used to detect biotin-CXCL14–binding proteins using HuProt human proteome microarrays containing 18,583 affinity-purified N-terminally GST-tagged proteins. ( C ) Representative CXCL14-binding membrane proteins in the proteome microarrays. ( D ) Western blot validation using a streptavidin-agarose pull-down assay of proteome microarray determination that CXCL14 binds directly to GPR85. ( E ) Mobilization of [Ca 2+ ] i in NAF6 cells that were transfected with control siRNA (NC) or GPR85-3 siRNA and then treated with 100 ng/ml HBSS, rhCXCL14, or rhCXCL12. The black arrows denote the times at which stimulation was initiated. ( F ) 125 I-CXCL14 binding properties between <t>293T-NC</t> and 293T-GPR85 cells. Data are shown as mean ± SD. n = 4 repetitions. ** P
    Kidney Function Blood Urea Nitrogen Bun Levels, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human embryo kidney cells hek293t
    USP35 acts as a functional DUB and interacts with ABIN-2 A. and B. H1299 (A) and LNCaP (B) cells were transfected with HA-ubiquitin alone or together with USP35 expression plasmids. Cell lysates were immunoprecipitated with anti-HA and analyzed by immunoblotting with anti-HA. Lower panel shows the input levels of the indicated proteins. C. H1299 cells were transiently transfected with empty vector and plasmid expressing Myc-USP35. Cell lysates were immunoprecipitated with anti-Myc antibody and then seperated by SDS-PAGE. After staining with Coomassie blue R-250, a specific protein band was subsequently excised to mass spectrometric analysis. D. and E. <t>HEK293T</t> (D) and H1299 (E) cells were cotransfected with Flag-ABIN-2 and Myc-USP35. Cell lysates were immunoprecipitated with anti-Flag, and subjected to subsequent immunoblotting with anti-Myc or anti-Flag. Lower panel shows the input levels of the indicated proteins. F. and G. Whole cell lysates from LNCaP (F) and HeLa cells (G) were immunoprecipitated with anti-ABIN-2 or normal IgG, and then immunoblotted with anti-USP35 or anti-ABIN-2. Left panel shows the input levels of the indicated proteins. All results are representative of three independent experiments.
    Human Embryo Kidney Cells Hek293t, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Overexpression and endogenous or exogenous expression of BI-1 or BI-1 ∆C in Madin-Darby Canine Kidney (MDCK) cells. ( A ) Scheme of the lentiviral constructs for the expression of wild type BI-1 or nonfunctional mutant BI-1 ΔC in MDCK cells. BI-1 or BI-1 ΔC was inserted into the lentiviral pEF vector. The pEF lentivirus containing wild type BI-1 or the non-functional mutant BI-1 ΔC was produced and used to infect MDCK cells. ( B ) Illustration of specific primer sets which was used to analyze endogenous or exogenous expression of BI-1 . ( C ) The endogenous or exogenous expression of BI-1 was confirmed by RT-PCR analysis in indicated cells; and the expression of HA was confirmed by western blot analysis in indicated cells. The expression of GAPDH and Actin was used as loading control. ( D ) The illustration of whole experimental protocol used in this study. (Abbreviations: chimeric Rous sarcoma virus (RSV) sequence, Rev response elements (RRE), central polypurine tract (cPPT), elongation factor 1 alpha (EF-1α) promoter region, a copGFP (copepod GFP) tag, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), cells growth media (CGM), virus growth media (VGM), and hemagglutinin assay (HA assay)).

    Journal: International Journal of Molecular Sciences

    Article Title: Bax Inhibitor-1 Acts as an Anti-Influenza Factor by Inhibiting ROS Mediated Cell Death and Augmenting Heme-Oxygenase 1 Expression in Influenza Virus Infected Cells

    doi: 10.3390/ijms19030712

    Figure Lengend Snippet: Overexpression and endogenous or exogenous expression of BI-1 or BI-1 ∆C in Madin-Darby Canine Kidney (MDCK) cells. ( A ) Scheme of the lentiviral constructs for the expression of wild type BI-1 or nonfunctional mutant BI-1 ΔC in MDCK cells. BI-1 or BI-1 ΔC was inserted into the lentiviral pEF vector. The pEF lentivirus containing wild type BI-1 or the non-functional mutant BI-1 ΔC was produced and used to infect MDCK cells. ( B ) Illustration of specific primer sets which was used to analyze endogenous or exogenous expression of BI-1 . ( C ) The endogenous or exogenous expression of BI-1 was confirmed by RT-PCR analysis in indicated cells; and the expression of HA was confirmed by western blot analysis in indicated cells. The expression of GAPDH and Actin was used as loading control. ( D ) The illustration of whole experimental protocol used in this study. (Abbreviations: chimeric Rous sarcoma virus (RSV) sequence, Rev response elements (RRE), central polypurine tract (cPPT), elongation factor 1 alpha (EF-1α) promoter region, a copGFP (copepod GFP) tag, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), cells growth media (CGM), virus growth media (VGM), and hemagglutinin assay (HA assay)).

    Article Snippet: Madin Darby Canine Kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC CCL-3, Manassas, VA, USA) and maintained in a minimum essential medium (MEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), and 100 U/mL penicillin/streptomycin (Gibco).

    Techniques: Over Expression, Expressing, Construct, Mutagenesis, Plasmid Preparation, Functional Assay, Produced, Reverse Transcription Polymerase Chain Reaction, Western Blot, Sequencing, Hemagglutination Assay

    Overexpression and endogenous or exogenous expression of BI-1 or BI-1 ∆C in Madin-Darby Canine Kidney (MDCK) cells. ( A ) Scheme of the lentiviral constructs for the expression of wild type BI-1 or nonfunctional mutant BI-1 ΔC in MDCK cells. BI-1 or BI-1 ΔC was inserted into the lentiviral pEF vector. The pEF lentivirus containing wild type BI-1 or the non-functional mutant BI-1 ΔC was produced and used to infect MDCK cells. ( B ) Illustration of specific primer sets which was used to analyze endogenous or exogenous expression of BI-1 . ( C ) The endogenous or exogenous expression of BI-1 was confirmed by RT-PCR analysis in indicated cells; and the expression of HA was confirmed by western blot analysis in indicated cells. The expression of GAPDH and Actin was used as loading control. ( D ) The illustration of whole experimental protocol used in this study. (Abbreviations: chimeric Rous sarcoma virus (RSV) sequence, Rev response elements (RRE), central polypurine tract (cPPT), elongation factor 1 alpha (EF-1α) promoter region, a copGFP (copepod GFP) tag, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), cells growth media (CGM), virus growth media (VGM), and hemagglutinin assay (HA assay)).

    Journal: International Journal of Molecular Sciences

    Article Title: Bax Inhibitor-1 Acts as an Anti-Influenza Factor by Inhibiting ROS Mediated Cell Death and Augmenting Heme-Oxygenase 1 Expression in Influenza Virus Infected Cells

    doi: 10.3390/ijms19030712

    Figure Lengend Snippet: Overexpression and endogenous or exogenous expression of BI-1 or BI-1 ∆C in Madin-Darby Canine Kidney (MDCK) cells. ( A ) Scheme of the lentiviral constructs for the expression of wild type BI-1 or nonfunctional mutant BI-1 ΔC in MDCK cells. BI-1 or BI-1 ΔC was inserted into the lentiviral pEF vector. The pEF lentivirus containing wild type BI-1 or the non-functional mutant BI-1 ΔC was produced and used to infect MDCK cells. ( B ) Illustration of specific primer sets which was used to analyze endogenous or exogenous expression of BI-1 . ( C ) The endogenous or exogenous expression of BI-1 was confirmed by RT-PCR analysis in indicated cells; and the expression of HA was confirmed by western blot analysis in indicated cells. The expression of GAPDH and Actin was used as loading control. ( D ) The illustration of whole experimental protocol used in this study. (Abbreviations: chimeric Rous sarcoma virus (RSV) sequence, Rev response elements (RRE), central polypurine tract (cPPT), elongation factor 1 alpha (EF-1α) promoter region, a copGFP (copepod GFP) tag, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), cells growth media (CGM), virus growth media (VGM), and hemagglutinin assay (HA assay)).

    Article Snippet: Cells, Virus, Reagents, and Lentiviral Infection Madin Darby Canine Kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC CCL-3, Manassas, VA, USA) and maintained in a minimum essential medium (MEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), and 100 U/mL penicillin/streptomycin (Gibco).

    Techniques: Over Expression, Expressing, Construct, Mutagenesis, Plasmid Preparation, Functional Assay, Produced, Reverse Transcription Polymerase Chain Reaction, Western Blot, Sequencing, Hemagglutination Assay

    Substantial portion of circulating ICAM-1 + neutrophils during the recovery phase of IRI are positive for THP. ( A ) Flow cytometry show the increased percentage of THP + ICAM-1 + neutrophils on day 2 after IRI. Neutrophils were gated first. ( B ) Percentage of THP + ICAM-1 + neutrophils after IRI. ( C ) THP was increased in peripheral blood leukocytes during the recovery phase of IRI (n=4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, THP = Tamm-Horsfall protein. * P

    Journal: Journal of Korean Medical Science

    Article Title: Fate of Neutrophils during the Recovery Phase of Ischemia/Reperfusion Induced Acute Kidney Injury

    doi: 10.3346/jkms.2017.32.10.1616

    Figure Lengend Snippet: Substantial portion of circulating ICAM-1 + neutrophils during the recovery phase of IRI are positive for THP. ( A ) Flow cytometry show the increased percentage of THP + ICAM-1 + neutrophils on day 2 after IRI. Neutrophils were gated first. ( B ) Percentage of THP + ICAM-1 + neutrophils after IRI. ( C ) THP was increased in peripheral blood leukocytes during the recovery phase of IRI (n=4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, THP = Tamm-Horsfall protein. * P

    Article Snippet: Flow cytometry Kidneys were minced and then treated with collagenase type IV (10 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) and DNAse (Sigma-Aldrich).

    Techniques: Flow Cytometry, Cytometry

    Effect of JAM-C blocking antibody on the number of circulating ICAM-1 + neutrophils, kidney neutrophils, functional, and histological recovery following IRI. ( A ) Upper: Flow cytometry show that treatment with anti-mouse JAM-C blocking Ab resulted in increase of circulating ICAM-1 + neutrophils following IRI. Lower: percentage of circulating ICMA-1 + Gr-1 + neutrophils out of total neutrophils (n = 4–5 per group). ( B ) Treatment with anti-mouse JAM-C blocking Ab resulted in decreased number of infiltrating neutrophils on day 3 after IRI. Upper: representative pictures of Gr-1 immunohistochemistry, × 100, lower: mean number of Gr-1 + neutrophils in kidneys on day 3 of IRI (n = 4–5 per group). ( C ) Treatment with anti-mouse JAM-C blocking Ab resulted in faster functional recovery. ( D ) Treatment with anti-mouse JAM-C blocking Ab resulted in faster histological recovery. Upper: representative pictures of PAS stained kidney tissues, × 100, lower: semiquantitative scoring of tubular injury (n = 5–6 per group). JAM-C = junctional adhesion molecule-C, ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, Ab = antibody, PAS = periodic acid-Schiff. * P

    Journal: Journal of Korean Medical Science

    Article Title: Fate of Neutrophils during the Recovery Phase of Ischemia/Reperfusion Induced Acute Kidney Injury

    doi: 10.3346/jkms.2017.32.10.1616

    Figure Lengend Snippet: Effect of JAM-C blocking antibody on the number of circulating ICAM-1 + neutrophils, kidney neutrophils, functional, and histological recovery following IRI. ( A ) Upper: Flow cytometry show that treatment with anti-mouse JAM-C blocking Ab resulted in increase of circulating ICAM-1 + neutrophils following IRI. Lower: percentage of circulating ICMA-1 + Gr-1 + neutrophils out of total neutrophils (n = 4–5 per group). ( B ) Treatment with anti-mouse JAM-C blocking Ab resulted in decreased number of infiltrating neutrophils on day 3 after IRI. Upper: representative pictures of Gr-1 immunohistochemistry, × 100, lower: mean number of Gr-1 + neutrophils in kidneys on day 3 of IRI (n = 4–5 per group). ( C ) Treatment with anti-mouse JAM-C blocking Ab resulted in faster functional recovery. ( D ) Treatment with anti-mouse JAM-C blocking Ab resulted in faster histological recovery. Upper: representative pictures of PAS stained kidney tissues, × 100, lower: semiquantitative scoring of tubular injury (n = 5–6 per group). JAM-C = junctional adhesion molecule-C, ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, Ab = antibody, PAS = periodic acid-Schiff. * P

    Article Snippet: Flow cytometry Kidneys were minced and then treated with collagenase type IV (10 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) and DNAse (Sigma-Aldrich).

    Techniques: Blocking Assay, Functional Assay, Flow Cytometry, Cytometry, Immunohistochemistry, Staining

    Circulating ICAM-1 + neutrophils increased during the recovery phase of IRI. ( A ) Flow cytometry showed the increase of ICMA-1 + Gr-1 + reverse transmigrated neutrophils in peripheral blood on day 2 after IRI. ( B ) The number of circulating ICMA-1 + Gr-1 + neutrophils out of 10 6 kidney cells (n = 4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury. * P

    Journal: Journal of Korean Medical Science

    Article Title: Fate of Neutrophils during the Recovery Phase of Ischemia/Reperfusion Induced Acute Kidney Injury

    doi: 10.3346/jkms.2017.32.10.1616

    Figure Lengend Snippet: Circulating ICAM-1 + neutrophils increased during the recovery phase of IRI. ( A ) Flow cytometry showed the increase of ICMA-1 + Gr-1 + reverse transmigrated neutrophils in peripheral blood on day 2 after IRI. ( B ) The number of circulating ICMA-1 + Gr-1 + neutrophils out of 10 6 kidney cells (n = 4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury. * P

    Article Snippet: Flow cytometry Kidneys were minced and then treated with collagenase type IV (10 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) and DNAse (Sigma-Aldrich).

    Techniques: Flow Cytometry, Cytometry

    FTY-DC require S1pr1 on BMDC to protect kidneys from IRI. (A) Protocol for experimental setup. FTY720 is a ligand for four out of five S1P receptors. Plasma creatinine (PCr, mg/dL) was measured 24 h after IRI. We tested if S1pr1 or S1pr3 were requited for FTY720 dependent regulatory DC phenotype. BMDCs were propagated from either C57BL/6 WT, CD11cCreS1pr1 fl / fl ( S1pr1 −/− DC), or S1pr3 −/− and treated with FTY720. FTY- S1pr1 −/− DC do not protect kidneys from IRI. As demonstrated in our earlier published studies and again confirmed, transfer of S1pr3 −/− DC with or without FTY720 significantly protect kidney from IRI. These studies suggest only S1pr1 are necessary for FTY720 dependent regulatory DC phenotype. Next, we tested if the route of delivery was important for FTY-DC induced protection from kidney IRI. (B) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. FTY-DC were injected either intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.) or as i.v. using Veh-DC that were acutely treated with FTY720 (overnight along with LPS). These studies suggest that FTY-DC only protect kidneys from IRI if injected via i.v. and the FTY-DCs must be propagated in presence of FTY720 at start of the BMDC culture. ** p ≤ 0.01. (C) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. In all studies presented we injected FTY-DC 1 day before kidney IRI. We also tested if FTY-DC could be injected after injury. Mice were treated with either NC, Veh-DC or FTY-DC 4 h after bilateral IRI. Mice injected with Veh-DC had higher PCr compared to NC mice and FTY-DC treated mice were significantly protected. (D) Protocol for experimental setup. Plasma creatinine (mg/dL) was measured 24 h after IRI. C57BL/6 FTY-DC induce protection in BALB/c mice and protect kidneys from ischemic injury. Data represent means ± SEM, Unpaired t -test [D], *** p ≤ 0.001; ** p ≤ 0.01 and *** p ≤ 0.001, one-way ANOVA followed by Tukey's post-test.

    Journal: Frontiers in Immunology

    Article Title: FTY720 Regulates Mitochondria Biogenesis in Dendritic Cells to Prevent Kidney Ischemic Reperfusion Injury

    doi: 10.3389/fimmu.2020.01278

    Figure Lengend Snippet: FTY-DC require S1pr1 on BMDC to protect kidneys from IRI. (A) Protocol for experimental setup. FTY720 is a ligand for four out of five S1P receptors. Plasma creatinine (PCr, mg/dL) was measured 24 h after IRI. We tested if S1pr1 or S1pr3 were requited for FTY720 dependent regulatory DC phenotype. BMDCs were propagated from either C57BL/6 WT, CD11cCreS1pr1 fl / fl ( S1pr1 −/− DC), or S1pr3 −/− and treated with FTY720. FTY- S1pr1 −/− DC do not protect kidneys from IRI. As demonstrated in our earlier published studies and again confirmed, transfer of S1pr3 −/− DC with or without FTY720 significantly protect kidney from IRI. These studies suggest only S1pr1 are necessary for FTY720 dependent regulatory DC phenotype. Next, we tested if the route of delivery was important for FTY-DC induced protection from kidney IRI. (B) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. FTY-DC were injected either intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.) or as i.v. using Veh-DC that were acutely treated with FTY720 (overnight along with LPS). These studies suggest that FTY-DC only protect kidneys from IRI if injected via i.v. and the FTY-DCs must be propagated in presence of FTY720 at start of the BMDC culture. ** p ≤ 0.01. (C) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. In all studies presented we injected FTY-DC 1 day before kidney IRI. We also tested if FTY-DC could be injected after injury. Mice were treated with either NC, Veh-DC or FTY-DC 4 h after bilateral IRI. Mice injected with Veh-DC had higher PCr compared to NC mice and FTY-DC treated mice were significantly protected. (D) Protocol for experimental setup. Plasma creatinine (mg/dL) was measured 24 h after IRI. C57BL/6 FTY-DC induce protection in BALB/c mice and protect kidneys from ischemic injury. Data represent means ± SEM, Unpaired t -test [D], *** p ≤ 0.001; ** p ≤ 0.01 and *** p ≤ 0.001, one-way ANOVA followed by Tukey's post-test.

    Article Snippet: DiscussionIn the current study we demonstrated that immunosuppression and protection from kidney IRI induced by adoptive transfer of FTY-DC is dependent on the recipient spleen, DC-S1P1 and functional viability of transferred DC mitochondria.

    Techniques: Polymerase Chain Reaction, Injection, Mouse Assay

    Pretreatment with FTY-DCs protects kidneys from ischemia reperfusion injury. Mice were i.v. injected with 0.5 × 10 6 DCs (Veh-DC or FTY-DC) and as control no cells (NC) 1 day before bilateral kidney IRI. (A) Protocol for experimental setup and plasma creatinine. (B,C) Quantification of acute tubular injury (ATN). Renal histology (H E). Scale bar, 100 μm. (D–F) Flow cytometry of kidney tissue gated on neutrophils (CD45 + Mac-1 + Gr1 + ; PMNs) from mice either treated with NC, Veh-DC or FTY-DC. (G) Gene expression of kidney S1pr1, Ngal, Kim1 , and Il6 . Data represent means ± SEM, * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 one-way ANOVA followed by Tukey's post-test.

    Journal: Frontiers in Immunology

    Article Title: FTY720 Regulates Mitochondria Biogenesis in Dendritic Cells to Prevent Kidney Ischemic Reperfusion Injury

    doi: 10.3389/fimmu.2020.01278

    Figure Lengend Snippet: Pretreatment with FTY-DCs protects kidneys from ischemia reperfusion injury. Mice were i.v. injected with 0.5 × 10 6 DCs (Veh-DC or FTY-DC) and as control no cells (NC) 1 day before bilateral kidney IRI. (A) Protocol for experimental setup and plasma creatinine. (B,C) Quantification of acute tubular injury (ATN). Renal histology (H E). Scale bar, 100 μm. (D–F) Flow cytometry of kidney tissue gated on neutrophils (CD45 + Mac-1 + Gr1 + ; PMNs) from mice either treated with NC, Veh-DC or FTY-DC. (G) Gene expression of kidney S1pr1, Ngal, Kim1 , and Il6 . Data represent means ± SEM, * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 one-way ANOVA followed by Tukey's post-test.

    Article Snippet: DiscussionIn the current study we demonstrated that immunosuppression and protection from kidney IRI induced by adoptive transfer of FTY-DC is dependent on the recipient spleen, DC-S1P1 and functional viability of transferred DC mitochondria.

    Techniques: Mouse Assay, Injection, Flow Cytometry, Expressing

    FTY-DC require recipient spleen to protects kidneys from IRI. Protocol for experimental setup. (A) Plasma Creatinine (PCr). Mice were i.v. injected with 0.5 × 10 6 DCs (Veh-DC or FTY-DC) 7 days after splenectomy (Splnx) and 1 day before bilateral kidney IRI. (B,C) Quantification of tubular injury. Renal histology (H E). (D,E) Flow cytometry of kidney tissue gated on neutrophils from Sham (no Splnx) mice either treated with Veh-DC or FTY-DC. Data represent means ± SEM, *** p ≤ 0.001, ** p ≤ 0.01; one-way ANOVA followed by Tukey's post-test.

    Journal: Frontiers in Immunology

    Article Title: FTY720 Regulates Mitochondria Biogenesis in Dendritic Cells to Prevent Kidney Ischemic Reperfusion Injury

    doi: 10.3389/fimmu.2020.01278

    Figure Lengend Snippet: FTY-DC require recipient spleen to protects kidneys from IRI. Protocol for experimental setup. (A) Plasma Creatinine (PCr). Mice were i.v. injected with 0.5 × 10 6 DCs (Veh-DC or FTY-DC) 7 days after splenectomy (Splnx) and 1 day before bilateral kidney IRI. (B,C) Quantification of tubular injury. Renal histology (H E). (D,E) Flow cytometry of kidney tissue gated on neutrophils from Sham (no Splnx) mice either treated with Veh-DC or FTY-DC. Data represent means ± SEM, *** p ≤ 0.001, ** p ≤ 0.01; one-way ANOVA followed by Tukey's post-test.

    Article Snippet: DiscussionIn the current study we demonstrated that immunosuppression and protection from kidney IRI induced by adoptive transfer of FTY-DC is dependent on the recipient spleen, DC-S1P1 and functional viability of transferred DC mitochondria.

    Techniques: Polymerase Chain Reaction, Mouse Assay, Injection, Flow Cytometry

    Inhibition of FTY-DC mitochondrial function abrogates protection from kidney IRI. Protocol for experimental setup. (A) Plasma Creatinine (PCr). Mice were i.v. injected with 0.5 × 10 6 DCs (Veh-DC or FTY-DC) and as control no cells (NC) 1 day before bilateral kidney IRI. A group of mice were injected with FTY-DC that were treated with rotenone and antimycin A (Rot/AA). (B) Protocol for experimental setup. Plasma Creatinine (PCr). Mice were i.v. injected with 0.5X10 6 DCs (FTY-DC) and as control no cells (NC) 1 day before bilateral kidney IRI. Two additional group of mice were injected with FTY-DC that were treated with cytochalasin D (Cyto D) or carbenoxolone (CBX). (C) Immunofluorescence of CD11cCrePham fl / fl FTY-DC (green, mitochondria) and labeled with phalloidin (red, actin) that were treated with Cyto D or CBX. Scale bar, 20 μm. (D,E) Mice were i.v. injected with 0.5 × 10 6 DCs (Veh-DC or FTY-DC) or (FTY-DC treated with Rot/AA or Cyto D) and as control no cells (NC) 1 day before splenocytes were harvested and treated ex vivo with LPS (25 or 100 ng/ml) for 6 or 24 h and supernatant was analyzed by Elisa for TNFα. (F) Mice were i.v. injected with various amounts of isolated mitochondria (0–100 μg/mouse). Spleen was harvested 1 day after mitochondria injections and single cells suspensions were treated ex vivo with 100 ng/ml LPS for 6 h and supernatant was analyzed by Elisa for TNFα. (G) Spleen from (0 μg mito) treated mouse was harvested and incubated with various amounts of isolated mitochondria (0–15 μg/well) for 1 day before stimulating with 100 ng/ml LPS for additional 6 h and supernatant was analyzed for TNFα. Data represent means ± SEM, * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001, one-way ANOVA followed by Tukey's post-test.

    Journal: Frontiers in Immunology

    Article Title: FTY720 Regulates Mitochondria Biogenesis in Dendritic Cells to Prevent Kidney Ischemic Reperfusion Injury

    doi: 10.3389/fimmu.2020.01278

    Figure Lengend Snippet: Inhibition of FTY-DC mitochondrial function abrogates protection from kidney IRI. Protocol for experimental setup. (A) Plasma Creatinine (PCr). Mice were i.v. injected with 0.5 × 10 6 DCs (Veh-DC or FTY-DC) and as control no cells (NC) 1 day before bilateral kidney IRI. A group of mice were injected with FTY-DC that were treated with rotenone and antimycin A (Rot/AA). (B) Protocol for experimental setup. Plasma Creatinine (PCr). Mice were i.v. injected with 0.5X10 6 DCs (FTY-DC) and as control no cells (NC) 1 day before bilateral kidney IRI. Two additional group of mice were injected with FTY-DC that were treated with cytochalasin D (Cyto D) or carbenoxolone (CBX). (C) Immunofluorescence of CD11cCrePham fl / fl FTY-DC (green, mitochondria) and labeled with phalloidin (red, actin) that were treated with Cyto D or CBX. Scale bar, 20 μm. (D,E) Mice were i.v. injected with 0.5 × 10 6 DCs (Veh-DC or FTY-DC) or (FTY-DC treated with Rot/AA or Cyto D) and as control no cells (NC) 1 day before splenocytes were harvested and treated ex vivo with LPS (25 or 100 ng/ml) for 6 or 24 h and supernatant was analyzed by Elisa for TNFα. (F) Mice were i.v. injected with various amounts of isolated mitochondria (0–100 μg/mouse). Spleen was harvested 1 day after mitochondria injections and single cells suspensions were treated ex vivo with 100 ng/ml LPS for 6 h and supernatant was analyzed by Elisa for TNFα. (G) Spleen from (0 μg mito) treated mouse was harvested and incubated with various amounts of isolated mitochondria (0–15 μg/well) for 1 day before stimulating with 100 ng/ml LPS for additional 6 h and supernatant was analyzed for TNFα. Data represent means ± SEM, * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001, one-way ANOVA followed by Tukey's post-test.

    Article Snippet: DiscussionIn the current study we demonstrated that immunosuppression and protection from kidney IRI induced by adoptive transfer of FTY-DC is dependent on the recipient spleen, DC-S1P1 and functional viability of transferred DC mitochondria.

    Techniques: Inhibition, Polymerase Chain Reaction, Mouse Assay, Injection, Immunofluorescence, Labeling, Ex Vivo, Enzyme-linked Immunosorbent Assay, Isolation, Incubation

    Drugs affecting cytoskeleton and membrane cholesterol prevent stimulation of pDC by infected cells. A. SK-6 cells were infected with VRPΔE rns for 24 h, washed and then treated with a DMSO control, nocodazole (5 µM), MβCD (20 mM) or latrunculin (1 µM) for 2 h at 37°C. The inhibitors were then removed and the cells washed three times to avoid effects on pDC. The treated cells were co-cultured with pDC and IFN-α measured after 20 h of incubation. B. Uninfected SK-6 cells were treated as in A and co-cultured with pDC in presence of CpG. The mean values of three replicates with standard deviation are shown. The data is representative of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

    doi: 10.1371/journal.ppat.1003412

    Figure Lengend Snippet: Drugs affecting cytoskeleton and membrane cholesterol prevent stimulation of pDC by infected cells. A. SK-6 cells were infected with VRPΔE rns for 24 h, washed and then treated with a DMSO control, nocodazole (5 µM), MβCD (20 mM) or latrunculin (1 µM) for 2 h at 37°C. The inhibitors were then removed and the cells washed three times to avoid effects on pDC. The treated cells were co-cultured with pDC and IFN-α measured after 20 h of incubation. B. Uninfected SK-6 cells were treated as in A and co-cultured with pDC in presence of CpG. The mean values of three replicates with standard deviation are shown. The data is representative of three independent experiments.

    Article Snippet: Cell preparation and cell culture The porcine kidney cell lines SK-6 , PK-15 (LGC Standards-ATCC, Molsheim, France) and the porcine immortalized endothelial cells PEDSV.15 (obtained from Dr. Jörg Seebach, University of Geneva, Switzerland) were propagated in Earle's minimal essential medium (MEM) substituted with 7% horse serum and in Dulbecco's minimal essential medium (DMEM) supplemented with 5% horse serum, nonessential amino acids and 1 mM Na-pyruvate, respectively.

    Techniques: Infection, Cell Culture, Incubation, Standard Deviation

    E rns disables stimulation of pDC by infected cells. A and B. IFN-α induction in pDC after stimulation with SK-6 or SK-6(E rns ) cells infected with CSFV (A) or VRPΔE rns (B). C. Lack of inhibitory activity in supernatants (SN) of SK-6(E rns ) cells. Supernatants harvested from SK-6 or SK-6(E rns ) cell cultures were added to co-cultures of enriched pDCs with MOCK- or VRPΔE rns -infected SK-6 cells at 50% (V/V) D. N pro has no impact on stimulation of pDC by infected cells. SK-6 or SK-6(E rns ) cells were infected with wild type VRPΔE rns or VRPΔE rns with the D 136 N mutation in N pro (VRPΔE rns D 136 N) abrogating the functional activity of N pro in preventing IRF3 and IRF7-mediated IFN type I responses. E and F. E rns also inhibits the activation of pDC by SK-6 cells infected with TGEV (E) and FMDV (F). SK-6 or SK-6(E rns ) cells were infected with TGEV (MOI of 10 TCID 50 /cell), or FMDV (MOI of 2.5 TCID 50 /cell), cultured for 90 min followed by addition of enriched pDC. For A to F, IFN-α in the supernatants was quantified by ELISA after 20 h of co-culture. The mean values of three replicates with standard deviation are shown. In A and B, the results are representative of five independent experiments. In C to F, the data is representative of two independent experiments.

    Journal: PLoS Pathogens

    Article Title: Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

    doi: 10.1371/journal.ppat.1003412

    Figure Lengend Snippet: E rns disables stimulation of pDC by infected cells. A and B. IFN-α induction in pDC after stimulation with SK-6 or SK-6(E rns ) cells infected with CSFV (A) or VRPΔE rns (B). C. Lack of inhibitory activity in supernatants (SN) of SK-6(E rns ) cells. Supernatants harvested from SK-6 or SK-6(E rns ) cell cultures were added to co-cultures of enriched pDCs with MOCK- or VRPΔE rns -infected SK-6 cells at 50% (V/V) D. N pro has no impact on stimulation of pDC by infected cells. SK-6 or SK-6(E rns ) cells were infected with wild type VRPΔE rns or VRPΔE rns with the D 136 N mutation in N pro (VRPΔE rns D 136 N) abrogating the functional activity of N pro in preventing IRF3 and IRF7-mediated IFN type I responses. E and F. E rns also inhibits the activation of pDC by SK-6 cells infected with TGEV (E) and FMDV (F). SK-6 or SK-6(E rns ) cells were infected with TGEV (MOI of 10 TCID 50 /cell), or FMDV (MOI of 2.5 TCID 50 /cell), cultured for 90 min followed by addition of enriched pDC. For A to F, IFN-α in the supernatants was quantified by ELISA after 20 h of co-culture. The mean values of three replicates with standard deviation are shown. In A and B, the results are representative of five independent experiments. In C to F, the data is representative of two independent experiments.

    Article Snippet: Cell preparation and cell culture The porcine kidney cell lines SK-6 , PK-15 (LGC Standards-ATCC, Molsheim, France) and the porcine immortalized endothelial cells PEDSV.15 (obtained from Dr. Jörg Seebach, University of Geneva, Switzerland) were propagated in Earle's minimal essential medium (MEM) substituted with 7% horse serum and in Dulbecco's minimal essential medium (DMEM) supplemented with 5% horse serum, nonessential amino acids and 1 mM Na-pyruvate, respectively.

    Techniques: Infection, Activity Assay, Mutagenesis, Functional Assay, Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Standard Deviation

    pDC stimulation by infected cells is cell contact-dependent. A and B. VRPΔE rns - (A) or CSFV- (B) infected SK-6 cells were co-cultured with enriched pDC for 24 h either in contact or separation using transwell inserts of 0.4 or 1 µm. The mean IFN-α values of three replicates measured by ELISA are shown with the standard deviation, calculated from a set of data which is representative of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

    doi: 10.1371/journal.ppat.1003412

    Figure Lengend Snippet: pDC stimulation by infected cells is cell contact-dependent. A and B. VRPΔE rns - (A) or CSFV- (B) infected SK-6 cells were co-cultured with enriched pDC for 24 h either in contact or separation using transwell inserts of 0.4 or 1 µm. The mean IFN-α values of three replicates measured by ELISA are shown with the standard deviation, calculated from a set of data which is representative of three independent experiments.

    Article Snippet: Cell preparation and cell culture The porcine kidney cell lines SK-6 , PK-15 (LGC Standards-ATCC, Molsheim, France) and the porcine immortalized endothelial cells PEDSV.15 (obtained from Dr. Jörg Seebach, University of Geneva, Switzerland) were propagated in Earle's minimal essential medium (MEM) substituted with 7% horse serum and in Dulbecco's minimal essential medium (DMEM) supplemented with 5% horse serum, nonessential amino acids and 1 mM Na-pyruvate, respectively.

    Techniques: Infection, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Stimulation of pDC by infected cells is not mediated by virions. A and B. Blocking of direct CSFV (A) or VRPΔE rns (B) activation of pDC by addition of neutralizing antibodies. Results for different dilutions of anti-E2 glycoprotein mAb and of anti-CSFV serum are shown. C and D. The same antibodies (anti-E2 at 1∶40 dilution) did not reduce activation of pDC by CSFV- (C) or VRPΔE rns -infected (D) cells. The results are representative of four (A, B) or three (C–F) independent experiments. E. Cells transfected with self-replicating RNA unable to form virions stimulate pDC. In vitro transcribed viral RNA lacking the E rns gene (ΔE rns RNA) or all genes encoding the structural proteins of CSFV (ΔApa RNA) were co-cultured with enriched pDC for 20 h and IFN-α was determined. VRP- and CSFV-infected SK-6 cells were used as control (labeled VRP and CSFV). F. Stimulation of enriched pDC with either VRPΔE rns , VRPΔE2, SK-6 cells infected with VRPΔE rns or SK-6 cells infected with VRPΔE2. For all experiments the mean of triplicate cultures with standard deviations are shown.

    Journal: PLoS Pathogens

    Article Title: Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

    doi: 10.1371/journal.ppat.1003412

    Figure Lengend Snippet: Stimulation of pDC by infected cells is not mediated by virions. A and B. Blocking of direct CSFV (A) or VRPΔE rns (B) activation of pDC by addition of neutralizing antibodies. Results for different dilutions of anti-E2 glycoprotein mAb and of anti-CSFV serum are shown. C and D. The same antibodies (anti-E2 at 1∶40 dilution) did not reduce activation of pDC by CSFV- (C) or VRPΔE rns -infected (D) cells. The results are representative of four (A, B) or three (C–F) independent experiments. E. Cells transfected with self-replicating RNA unable to form virions stimulate pDC. In vitro transcribed viral RNA lacking the E rns gene (ΔE rns RNA) or all genes encoding the structural proteins of CSFV (ΔApa RNA) were co-cultured with enriched pDC for 20 h and IFN-α was determined. VRP- and CSFV-infected SK-6 cells were used as control (labeled VRP and CSFV). F. Stimulation of enriched pDC with either VRPΔE rns , VRPΔE2, SK-6 cells infected with VRPΔE rns or SK-6 cells infected with VRPΔE2. For all experiments the mean of triplicate cultures with standard deviations are shown.

    Article Snippet: Cell preparation and cell culture The porcine kidney cell lines SK-6 , PK-15 (LGC Standards-ATCC, Molsheim, France) and the porcine immortalized endothelial cells PEDSV.15 (obtained from Dr. Jörg Seebach, University of Geneva, Switzerland) were propagated in Earle's minimal essential medium (MEM) substituted with 7% horse serum and in Dulbecco's minimal essential medium (DMEM) supplemented with 5% horse serum, nonessential amino acids and 1 mM Na-pyruvate, respectively.

    Techniques: Infection, Blocking Assay, Activation Assay, Transfection, In Vitro, Cell Culture, Labeling

    CSFV-infected cells are powerful inducers of INF-α response by pDC. Enriched pDC were either stimulated with CSFV-infected SK-6 cells, VRPΔE rns -infected SK-6 cells or directly by CSFV or VRPΔE rns infection. IFN-α was quantified by ELISA after 22 h of culture. A and B. Cell number-dependent IFN-α induction in pDC by CSFV- or VRP-infected SK-6 cells, respectively. The number of infected cells (# of inf. cells) or mock treated cells added to 200'000 CD172a + enriched pDC per microwell is indicated. The last bars, labeled CSFV (A) and VRPΔE rns (B), show the IFN-α levels induced by direct virus stimulation. In C–F, the results of 12–15 independent experiments with standard deviations, fold increase calculated from the means of the compared groups and p values of paired students t-test are shown (n.s.: not significant). C. IFN-α induced by direct stimulation with either CSFV or VRPΔE rns . D. IFN-α induced by stimulation with either CSFV or CSFV-infected SK-6 cells. E. IFN-α induced by stimulation with either VRPΔE rns or VRPΔE rns -infected SK-6 cells. F IFN-α induced by stimulation with cells infected with either CSFV or VRPΔE rns . The purity of pDC 2–5%.

    Journal: PLoS Pathogens

    Article Title: Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

    doi: 10.1371/journal.ppat.1003412

    Figure Lengend Snippet: CSFV-infected cells are powerful inducers of INF-α response by pDC. Enriched pDC were either stimulated with CSFV-infected SK-6 cells, VRPΔE rns -infected SK-6 cells or directly by CSFV or VRPΔE rns infection. IFN-α was quantified by ELISA after 22 h of culture. A and B. Cell number-dependent IFN-α induction in pDC by CSFV- or VRP-infected SK-6 cells, respectively. The number of infected cells (# of inf. cells) or mock treated cells added to 200'000 CD172a + enriched pDC per microwell is indicated. The last bars, labeled CSFV (A) and VRPΔE rns (B), show the IFN-α levels induced by direct virus stimulation. In C–F, the results of 12–15 independent experiments with standard deviations, fold increase calculated from the means of the compared groups and p values of paired students t-test are shown (n.s.: not significant). C. IFN-α induced by direct stimulation with either CSFV or VRPΔE rns . D. IFN-α induced by stimulation with either CSFV or CSFV-infected SK-6 cells. E. IFN-α induced by stimulation with either VRPΔE rns or VRPΔE rns -infected SK-6 cells. F IFN-α induced by stimulation with cells infected with either CSFV or VRPΔE rns . The purity of pDC 2–5%.

    Article Snippet: Cell preparation and cell culture The porcine kidney cell lines SK-6 , PK-15 (LGC Standards-ATCC, Molsheim, France) and the porcine immortalized endothelial cells PEDSV.15 (obtained from Dr. Jörg Seebach, University of Geneva, Switzerland) were propagated in Earle's minimal essential medium (MEM) substituted with 7% horse serum and in Dulbecco's minimal essential medium (DMEM) supplemented with 5% horse serum, nonessential amino acids and 1 mM Na-pyruvate, respectively.

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Labeling

    The RNase activity of E rns is required for its inhibitory function. A and C. Deletion of histidine 346 abolishes the RNase activity of the viral E rns in virus-infected cells (A) and in stable cell lines expressing the glycoprotein (C). Confluent monolayers of SK-6 cells infected for 24 h with CSFV vA187-1 (CSFV WT) or with the mutant vA187-E rns Δ346 virus (CSFV-E rns Δ346) (A), and of the cell lines SK-6, SK6(E rns ), SK-6LV(E rns ) and SK-6LV(E rns Δ346) (C) were washed 5 times with phosphate buffered saline and cultured for another 16 h in serum-free MEM. The culture supernatants were then collected and the cell extracts obtained by hypotonic lysis with H 2 O. The Dy-781-O1-RNA probe was incubated with MEM containing RNase A, with TrisHCl, and with cell extract or supernatant of cells infected with CSFV WT, CSFV-E rns Δ346 or mock (A) or of SK-6, SK6(E rns ), SK-6LV(E rns ) and SK-6LV(E rns Δ346) cells (C), and separated by urea/polyacrylamide gel electrophoresis. The star indicates the position of undigested RNA probe. B. CSFV with inactive RNase function of E rns (CSFV-E rns Δ346) is a better stimulator of pDC. Enriched pDC were activated by virus (hatched bars) or by co-culture with infected SK-6 cells. The IFN-α responses to wild type CSFV and CSFV-E rns Δ346 are compared. D to F. SK-6 cells expressing RNase active E rns prevent IFN-α induction by VRPΔE rns infection, while VRPΔE rns -infected SK-6 cells expressing E rns Δ346 re-gain their ability to stimulate pDC. SK-6 cells were transduced by lentiviral gene delivery to express wild type E rns or E rns Δ346 with 75% transduction efficiency (E). The SK-6(E rns ), and the LV-transduced SK-6LV(E rns ) and SK-6LV(E rns Δ346) were infected with VRPΔE rns and then tested for their ability to induced IFN-α in enriched pDC (D). The mean values of three replicates with standard deviations are shown calculated from data which is representative of three independent experiments. All E rns expressing cells lines were highly susceptible to infection by VRPΔE rns as determined by viral NS3 expression (F).

    Journal: PLoS Pathogens

    Article Title: Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

    doi: 10.1371/journal.ppat.1003412

    Figure Lengend Snippet: The RNase activity of E rns is required for its inhibitory function. A and C. Deletion of histidine 346 abolishes the RNase activity of the viral E rns in virus-infected cells (A) and in stable cell lines expressing the glycoprotein (C). Confluent monolayers of SK-6 cells infected for 24 h with CSFV vA187-1 (CSFV WT) or with the mutant vA187-E rns Δ346 virus (CSFV-E rns Δ346) (A), and of the cell lines SK-6, SK6(E rns ), SK-6LV(E rns ) and SK-6LV(E rns Δ346) (C) were washed 5 times with phosphate buffered saline and cultured for another 16 h in serum-free MEM. The culture supernatants were then collected and the cell extracts obtained by hypotonic lysis with H 2 O. The Dy-781-O1-RNA probe was incubated with MEM containing RNase A, with TrisHCl, and with cell extract or supernatant of cells infected with CSFV WT, CSFV-E rns Δ346 or mock (A) or of SK-6, SK6(E rns ), SK-6LV(E rns ) and SK-6LV(E rns Δ346) cells (C), and separated by urea/polyacrylamide gel electrophoresis. The star indicates the position of undigested RNA probe. B. CSFV with inactive RNase function of E rns (CSFV-E rns Δ346) is a better stimulator of pDC. Enriched pDC were activated by virus (hatched bars) or by co-culture with infected SK-6 cells. The IFN-α responses to wild type CSFV and CSFV-E rns Δ346 are compared. D to F. SK-6 cells expressing RNase active E rns prevent IFN-α induction by VRPΔE rns infection, while VRPΔE rns -infected SK-6 cells expressing E rns Δ346 re-gain their ability to stimulate pDC. SK-6 cells were transduced by lentiviral gene delivery to express wild type E rns or E rns Δ346 with 75% transduction efficiency (E). The SK-6(E rns ), and the LV-transduced SK-6LV(E rns ) and SK-6LV(E rns Δ346) were infected with VRPΔE rns and then tested for their ability to induced IFN-α in enriched pDC (D). The mean values of three replicates with standard deviations are shown calculated from data which is representative of three independent experiments. All E rns expressing cells lines were highly susceptible to infection by VRPΔE rns as determined by viral NS3 expression (F).

    Article Snippet: Cell preparation and cell culture The porcine kidney cell lines SK-6 , PK-15 (LGC Standards-ATCC, Molsheim, France) and the porcine immortalized endothelial cells PEDSV.15 (obtained from Dr. Jörg Seebach, University of Geneva, Switzerland) were propagated in Earle's minimal essential medium (MEM) substituted with 7% horse serum and in Dulbecco's minimal essential medium (DMEM) supplemented with 5% horse serum, nonessential amino acids and 1 mM Na-pyruvate, respectively.

    Techniques: Activity Assay, Infection, Stable Transfection, Expressing, Mutagenesis, Cell Culture, Lysis, Incubation, Polyacrylamide Gel Electrophoresis, Co-Culture Assay, Transduction

    Viral protein expression in pDC and monocytes after co-culture with VRP-infected cells. Enriched pDC were either infected with CSFV, VRPΔE rns or co-cultured with CSFV- or VRPΔE rns -infected SK-6 cells for 20 h, and then analyzed by three-color FCM to determine the NS3 expression in pDC (defined as CD4 + CD172a low , left side dot plots) and monocytes (defined as CD4 − CD172a high , right side dot plots). For the CD4/NS3 and CD172a/NS3 dot plots, the blue dots represent NS3 staining of infected cultures overlaid on red dots representing the NS3 staining on non-infected cultures. The percentage of NS3 cells is shown in red for the infected cultures. On the left side the mean IFN-α values and viral titers measured in the cultures are shown. The results are representative of triplicate cultures repeated in three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

    doi: 10.1371/journal.ppat.1003412

    Figure Lengend Snippet: Viral protein expression in pDC and monocytes after co-culture with VRP-infected cells. Enriched pDC were either infected with CSFV, VRPΔE rns or co-cultured with CSFV- or VRPΔE rns -infected SK-6 cells for 20 h, and then analyzed by three-color FCM to determine the NS3 expression in pDC (defined as CD4 + CD172a low , left side dot plots) and monocytes (defined as CD4 − CD172a high , right side dot plots). For the CD4/NS3 and CD172a/NS3 dot plots, the blue dots represent NS3 staining of infected cultures overlaid on red dots representing the NS3 staining on non-infected cultures. The percentage of NS3 cells is shown in red for the infected cultures. On the left side the mean IFN-α values and viral titers measured in the cultures are shown. The results are representative of triplicate cultures repeated in three independent experiments.

    Article Snippet: Cell preparation and cell culture The porcine kidney cell lines SK-6 , PK-15 (LGC Standards-ATCC, Molsheim, France) and the porcine immortalized endothelial cells PEDSV.15 (obtained from Dr. Jörg Seebach, University of Geneva, Switzerland) were propagated in Earle's minimal essential medium (MEM) substituted with 7% horse serum and in Dulbecco's minimal essential medium (DMEM) supplemented with 5% horse serum, nonessential amino acids and 1 mM Na-pyruvate, respectively.

    Techniques: Expressing, Co-Culture Assay, Infection, Cell Culture, Staining

    CD40 ASO treatment mitigated DOX nephropathy . CD40 antisense oligonucleotide (ASO) and control ASO treatments were initiated 2 weeks following initiation of DOX nephropathy and after 8 weeks of ASO treatments renal functional assessments of ( a ) proteinuria (graphed as the ratio of the urine albumin to creatinine) and ( b ) plasma fluorescein isothicyanate conjugated inulin (FITC)-inulin elimination (assessed by a single measurement of residual plasma FITC-Inulin two hours following FITC-inulin injection) were performed. Additionally, kidneys were harvested and whole kidney ( c ) CD40 mRNA and protein, ( d ) CCL5, MCP-1 and NGAL mRNA, and ( e ) CCL5 protein were measured. ( f–i ) Periodic acid-Schiff staining of representative kidney sections from ( f ) phosphate buffered saline (PBS), ( g ) control ASO, ( h ) 12.5 mg/kg CD40 ASO, and ( i ) 25 mg/kg CD40 ASO groups. Mean ± SEM, * P

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: CD40 Generation 2.5 Antisense Oligonucleotide Treatment Attenuates Doxorubicin-induced Nephropathy and Kidney Inflammation

    doi: 10.1038/mtna.2015.40

    Figure Lengend Snippet: CD40 ASO treatment mitigated DOX nephropathy . CD40 antisense oligonucleotide (ASO) and control ASO treatments were initiated 2 weeks following initiation of DOX nephropathy and after 8 weeks of ASO treatments renal functional assessments of ( a ) proteinuria (graphed as the ratio of the urine albumin to creatinine) and ( b ) plasma fluorescein isothicyanate conjugated inulin (FITC)-inulin elimination (assessed by a single measurement of residual plasma FITC-Inulin two hours following FITC-inulin injection) were performed. Additionally, kidneys were harvested and whole kidney ( c ) CD40 mRNA and protein, ( d ) CCL5, MCP-1 and NGAL mRNA, and ( e ) CCL5 protein were measured. ( f–i ) Periodic acid-Schiff staining of representative kidney sections from ( f ) phosphate buffered saline (PBS), ( g ) control ASO, ( h ) 12.5 mg/kg CD40 ASO, and ( i ) 25 mg/kg CD40 ASO groups. Mean ± SEM, * P

    Article Snippet: Kidney periodic acid-Schiff sections revealed marked improvement in DOX-mediated pathology as demonstrated by an improvement of glomerular nephropathy, interstitial and mesangial expansion and granular tubular casts in CD40 ASO-treated mice ( , ) versus controls ( , ).

    Techniques: Allele-specific Oligonucleotide, Functional Assay, Injection, Staining

    Functional characterization of sCAR fusion proteins. (A) Inhibition of Ad binding. 3 H-labeled Ad was preincubated with different amounts of either sCAR-His 6 or sCAR-EGF, and then 3 H-Ad/sCAR-ligand complex samples (10 5 cpm) were mixed with 293 cells (10 6 cells per aliquot) and allowed to bind at 4°C. Cell-bound radioactivities were determined as described in Materials and Methods. Data are presented as the percentage of input 3 H-Ad bound after washing and calculated as the cumulative mean ± SD of triplicate determinations. Error bars depicting SDs are smaller than the symbols. (B) Inhibition of Ad-mediated gene transfer. Recombinant Ad vector AdCMVLuc, expressing the firefly luciferase reporter gene, was preincubated with various amounts of either sCAR-His 6 or sCAR-EGF. Monolayers of 293 cells were then exposed to AdCMVLuc/sCAR-ligand complexes and assayed for luciferase activity as described in Materials and Methods. Gene transfer indices were calculated from the ratio of the mean luciferase activity documented in cells infected with either AdCMVLuc/sCAR-EGF or AdCMVLuc/sCAR-His 6 to those treated with AdCMVLuc alone. Each point represents the cumulative mean ± SD of triplicate determinations. Some error bars depicting SDs are smaller than the symbols.

    Journal: Journal of Virology

    Article Title: Ectodomain of Coxsackievirus and Adenovirus Receptor Genetically Fused to Epidermal Growth Factor Mediates Adenovirus Targeting to Epidermal Growth Factor Receptor-Positive Cells

    doi:

    Figure Lengend Snippet: Functional characterization of sCAR fusion proteins. (A) Inhibition of Ad binding. 3 H-labeled Ad was preincubated with different amounts of either sCAR-His 6 or sCAR-EGF, and then 3 H-Ad/sCAR-ligand complex samples (10 5 cpm) were mixed with 293 cells (10 6 cells per aliquot) and allowed to bind at 4°C. Cell-bound radioactivities were determined as described in Materials and Methods. Data are presented as the percentage of input 3 H-Ad bound after washing and calculated as the cumulative mean ± SD of triplicate determinations. Error bars depicting SDs are smaller than the symbols. (B) Inhibition of Ad-mediated gene transfer. Recombinant Ad vector AdCMVLuc, expressing the firefly luciferase reporter gene, was preincubated with various amounts of either sCAR-His 6 or sCAR-EGF. Monolayers of 293 cells were then exposed to AdCMVLuc/sCAR-ligand complexes and assayed for luciferase activity as described in Materials and Methods. Gene transfer indices were calculated from the ratio of the mean luciferase activity documented in cells infected with either AdCMVLuc/sCAR-EGF or AdCMVLuc/sCAR-His 6 to those treated with AdCMVLuc alone. Each point represents the cumulative mean ± SD of triplicate determinations. Some error bars depicting SDs are smaller than the symbols.

    Article Snippet: The 293 human kidney cell line transformed with Ad5 DNA was purchased from Microbix (Toronto, Ontario, Canada).

    Techniques: Functional Assay, Inhibition, Binding Assay, Labeling, Recombinant, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Infection

    GPR85 is a functional receptor for CXCL14 activity. ( A ) Confocal microscopy of NAF6 cells treated with 100 ng/ml biotin or biotin-CXCL14 at 4°C and stained with an antibody against Cy3-streptavidin. An isotype-matched IgG was used as a control. Cell nuclei were counterstained with DAPI. ( B ) Schematic of the procedure used to detect biotin-CXCL14–binding proteins using HuProt human proteome microarrays containing 18,583 affinity-purified N-terminally GST-tagged proteins. ( C ) Representative CXCL14-binding membrane proteins in the proteome microarrays. ( D ) Western blot validation using a streptavidin-agarose pull-down assay of proteome microarray determination that CXCL14 binds directly to GPR85. ( E ) Mobilization of [Ca 2+ ] i in NAF6 cells that were transfected with control siRNA (NC) or GPR85-3 siRNA and then treated with 100 ng/ml HBSS, rhCXCL14, or rhCXCL12. The black arrows denote the times at which stimulation was initiated. ( F ) 125 I-CXCL14 binding properties between 293T-NC and 293T-GPR85 cells. Data are shown as mean ± SD. n = 4 repetitions. ** P

    Journal: The Journal of Clinical Investigation

    Article Title: HIC1 deletion promotes breast cancer progression by activating tumor cell/fibroblast crosstalk

    doi: 10.1172/JCI99974

    Figure Lengend Snippet: GPR85 is a functional receptor for CXCL14 activity. ( A ) Confocal microscopy of NAF6 cells treated with 100 ng/ml biotin or biotin-CXCL14 at 4°C and stained with an antibody against Cy3-streptavidin. An isotype-matched IgG was used as a control. Cell nuclei were counterstained with DAPI. ( B ) Schematic of the procedure used to detect biotin-CXCL14–binding proteins using HuProt human proteome microarrays containing 18,583 affinity-purified N-terminally GST-tagged proteins. ( C ) Representative CXCL14-binding membrane proteins in the proteome microarrays. ( D ) Western blot validation using a streptavidin-agarose pull-down assay of proteome microarray determination that CXCL14 binds directly to GPR85. ( E ) Mobilization of [Ca 2+ ] i in NAF6 cells that were transfected with control siRNA (NC) or GPR85-3 siRNA and then treated with 100 ng/ml HBSS, rhCXCL14, or rhCXCL12. The black arrows denote the times at which stimulation was initiated. ( F ) 125 I-CXCL14 binding properties between 293T-NC and 293T-GPR85 cells. Data are shown as mean ± SD. n = 4 repetitions. ** P

    Article Snippet: MDA-MB-231, MCF7, and T47D human BrCa cells, MCF 10A human breast epithelial cells, and 293T embryonic kidney cells were obtained from ATCC and grown according to standard protocols.

    Techniques: Functional Assay, Activity Assay, Confocal Microscopy, Staining, Binding Assay, Affinity Purification, Western Blot, Pull Down Assay, Microarray, Transfection

    MTG1 is a mitochondrial protein that interacts with the mitoribosome large subunit (mtLSU). ( A ) Immunoblot analyses of the steady-state levels of MTG1 and indicated mtLSU and mtSSU proteins in HEK293T, 143B and 143B-206 rho° cells. voltage-dependent anion channel (VDAC) was used as loading control. ( B ) Sucrose gradient sedimentation analyses of mtSSU (mS27) and mtLSU proteins (bL12 and uL16) in mitochondrial extracts from 143B WT and 206 rho° cells, prepared in the presence of 5 mM EDTA. ( C and D ) Sucrose gradient sedimentation analyses of MTG1 and indicated MRPs in mitochondrial extracts from HEK293T cells, prepared in the presence of either (C) 5 mM EDTA or (D) 10 mM MgCl 2 . ( E ) Sucrose gradient sedimentation analyses of MTG1 and indicated MRPs in whole-cell extracts (WCE) from HEK293T cells, prepared in the presence of 10 mM MgCl 2. Transparent red, purple and green colors mark the fractions where the 55S monosome, 39S mtLSU and 28S mtSSU sediment, respectively.

    Journal: Nucleic Acids Research

    Article Title: MTG1 couples mitoribosome large subunit assembly with intersubunit bridge formation

    doi: 10.1093/nar/gky672

    Figure Lengend Snippet: MTG1 is a mitochondrial protein that interacts with the mitoribosome large subunit (mtLSU). ( A ) Immunoblot analyses of the steady-state levels of MTG1 and indicated mtLSU and mtSSU proteins in HEK293T, 143B and 143B-206 rho° cells. voltage-dependent anion channel (VDAC) was used as loading control. ( B ) Sucrose gradient sedimentation analyses of mtSSU (mS27) and mtLSU proteins (bL12 and uL16) in mitochondrial extracts from 143B WT and 206 rho° cells, prepared in the presence of 5 mM EDTA. ( C and D ) Sucrose gradient sedimentation analyses of MTG1 and indicated MRPs in mitochondrial extracts from HEK293T cells, prepared in the presence of either (C) 5 mM EDTA or (D) 10 mM MgCl 2 . ( E ) Sucrose gradient sedimentation analyses of MTG1 and indicated MRPs in whole-cell extracts (WCE) from HEK293T cells, prepared in the presence of 10 mM MgCl 2. Transparent red, purple and green colors mark the fractions where the 55S monosome, 39S mtLSU and 28S mtSSU sediment, respectively.

    Article Snippet: HEK293T embryonic kidney cells (CRL‐3216) and 143B osteosarcoma cells (CRL‐8303) were obtained from ATCC.

    Techniques: Sedimentation

    MTG1 is essential for the formation of functional mtLSU and monosomes. ( A ) Immunoblot analysis of the steady‐state levels of MRPs in HEK293T (WT), MTG1 -KO clone 1F5 treated with siNT and the 1F5 clone treated for 5 days with si MTG1 . VDAC was used as a loading control. Two exposures of the anti-MTG1 immunoblot (short and long) are presented. The lower panel shows the densitometry values normalized by the signal of VDAC and expressed relative to the WT. Data represent the mean ± SD of three independent repetitions; one-way ANOVA with a Tukey’s multiple comparisons test: * P

    Journal: Nucleic Acids Research

    Article Title: MTG1 couples mitoribosome large subunit assembly with intersubunit bridge formation

    doi: 10.1093/nar/gky672

    Figure Lengend Snippet: MTG1 is essential for the formation of functional mtLSU and monosomes. ( A ) Immunoblot analysis of the steady‐state levels of MRPs in HEK293T (WT), MTG1 -KO clone 1F5 treated with siNT and the 1F5 clone treated for 5 days with si MTG1 . VDAC was used as a loading control. Two exposures of the anti-MTG1 immunoblot (short and long) are presented. The lower panel shows the densitometry values normalized by the signal of VDAC and expressed relative to the WT. Data represent the mean ± SD of three independent repetitions; one-way ANOVA with a Tukey’s multiple comparisons test: * P

    Article Snippet: HEK293T embryonic kidney cells (CRL‐3216) and 143B osteosarcoma cells (CRL‐8303) were obtained from ATCC.

    Techniques: Functional Assay

    MTG1 catalyzes a mtLSU late-assembly step during the hierarchical incorporation of MRPs. Knockdown (KD) of mitoribosome AFs and mitoribosome subunits in HEK293T cells using siRNAs for 8–9 days, verified by immunoblotting of whole-cell lysates. ( A ). ( B ) Representative image of immunoblot analysis of the steady-state levels of MRPs after silencing of target proteins. siRNA-NT is a non-targeting silencing control. Antibodies are listed on the right side, and VDAC was used as a loading control. ( C ) Following analysis in panel (B), the densitometric data obtained on the abundance of MRPs and AFs accumulated after silencing of each target protein were used for cluster analysis (see STAR Methods). The heat map, generated with the R studio software, represents a log 2 scale of the normalized average levels of ratio to control (NT) in three independent repetitions of immunoblotting analyses. VDAC was used as a loading control. Two-way ANOVA followed by a Dunnett’s multiple comparisons test: * P

    Journal: Nucleic Acids Research

    Article Title: MTG1 couples mitoribosome large subunit assembly with intersubunit bridge formation

    doi: 10.1093/nar/gky672

    Figure Lengend Snippet: MTG1 catalyzes a mtLSU late-assembly step during the hierarchical incorporation of MRPs. Knockdown (KD) of mitoribosome AFs and mitoribosome subunits in HEK293T cells using siRNAs for 8–9 days, verified by immunoblotting of whole-cell lysates. ( A ). ( B ) Representative image of immunoblot analysis of the steady-state levels of MRPs after silencing of target proteins. siRNA-NT is a non-targeting silencing control. Antibodies are listed on the right side, and VDAC was used as a loading control. ( C ) Following analysis in panel (B), the densitometric data obtained on the abundance of MRPs and AFs accumulated after silencing of each target protein were used for cluster analysis (see STAR Methods). The heat map, generated with the R studio software, represents a log 2 scale of the normalized average levels of ratio to control (NT) in three independent repetitions of immunoblotting analyses. VDAC was used as a loading control. Two-way ANOVA followed by a Dunnett’s multiple comparisons test: * P

    Article Snippet: HEK293T embryonic kidney cells (CRL‐3216) and 143B osteosarcoma cells (CRL‐8303) were obtained from ATCC.

    Techniques: Generated, Software

    MTG1 is required for efficient mitochondrial translation and OXPHOS function in HEK293T cells. ( A ) Schematics of the first exon of the MTG1 locus and the sequences recognition sites of two different TALEN pairs. The genotyping of a three-allele compound heterozygous MTG1 -KO clone, 1F5, is depicted. ( B ) Immunoblot analysis of the steady‐state levels of MTG1 in HEK293T (WT), MTG1 -KO clone 1F5 and the 1F5 clone treated for 5 days with si MTG1 . VDAC was used as a loading control. Two exposures of the anti-MTG1 immunoblot (short and long) are presented. The lower panel shows the densitometry values normalized by the signal of VDAC and expressed relative to the WT; one-way ANOVA with a Tukey’s multiple comparisons test: ** P

    Journal: Nucleic Acids Research

    Article Title: MTG1 couples mitoribosome large subunit assembly with intersubunit bridge formation

    doi: 10.1093/nar/gky672

    Figure Lengend Snippet: MTG1 is required for efficient mitochondrial translation and OXPHOS function in HEK293T cells. ( A ) Schematics of the first exon of the MTG1 locus and the sequences recognition sites of two different TALEN pairs. The genotyping of a three-allele compound heterozygous MTG1 -KO clone, 1F5, is depicted. ( B ) Immunoblot analysis of the steady‐state levels of MTG1 in HEK293T (WT), MTG1 -KO clone 1F5 and the 1F5 clone treated for 5 days with si MTG1 . VDAC was used as a loading control. Two exposures of the anti-MTG1 immunoblot (short and long) are presented. The lower panel shows the densitometry values normalized by the signal of VDAC and expressed relative to the WT; one-way ANOVA with a Tukey’s multiple comparisons test: ** P

    Article Snippet: HEK293T embryonic kidney cells (CRL‐3216) and 143B osteosarcoma cells (CRL‐8303) were obtained from ATCC.

    Techniques:

    USP35 acts as a functional DUB and interacts with ABIN-2 A. and B. H1299 (A) and LNCaP (B) cells were transfected with HA-ubiquitin alone or together with USP35 expression plasmids. Cell lysates were immunoprecipitated with anti-HA and analyzed by immunoblotting with anti-HA. Lower panel shows the input levels of the indicated proteins. C. H1299 cells were transiently transfected with empty vector and plasmid expressing Myc-USP35. Cell lysates were immunoprecipitated with anti-Myc antibody and then seperated by SDS-PAGE. After staining with Coomassie blue R-250, a specific protein band was subsequently excised to mass spectrometric analysis. D. and E. HEK293T (D) and H1299 (E) cells were cotransfected with Flag-ABIN-2 and Myc-USP35. Cell lysates were immunoprecipitated with anti-Flag, and subjected to subsequent immunoblotting with anti-Myc or anti-Flag. Lower panel shows the input levels of the indicated proteins. F. and G. Whole cell lysates from LNCaP (F) and HeLa cells (G) were immunoprecipitated with anti-ABIN-2 or normal IgG, and then immunoblotted with anti-USP35 or anti-ABIN-2. Left panel shows the input levels of the indicated proteins. All results are representative of three independent experiments.

    Journal: Oncotarget

    Article Title: USP35 activated by miR let-7a inhibits cell proliferation and NF-κB activation through stabilization of ABIN-2

    doi:

    Figure Lengend Snippet: USP35 acts as a functional DUB and interacts with ABIN-2 A. and B. H1299 (A) and LNCaP (B) cells were transfected with HA-ubiquitin alone or together with USP35 expression plasmids. Cell lysates were immunoprecipitated with anti-HA and analyzed by immunoblotting with anti-HA. Lower panel shows the input levels of the indicated proteins. C. H1299 cells were transiently transfected with empty vector and plasmid expressing Myc-USP35. Cell lysates were immunoprecipitated with anti-Myc antibody and then seperated by SDS-PAGE. After staining with Coomassie blue R-250, a specific protein band was subsequently excised to mass spectrometric analysis. D. and E. HEK293T (D) and H1299 (E) cells were cotransfected with Flag-ABIN-2 and Myc-USP35. Cell lysates were immunoprecipitated with anti-Flag, and subjected to subsequent immunoblotting with anti-Myc or anti-Flag. Lower panel shows the input levels of the indicated proteins. F. and G. Whole cell lysates from LNCaP (F) and HeLa cells (G) were immunoprecipitated with anti-ABIN-2 or normal IgG, and then immunoblotted with anti-USP35 or anti-ABIN-2. Left panel shows the input levels of the indicated proteins. All results are representative of three independent experiments.

    Article Snippet: Cell lines Human prostate cancer cells LNCaP, PC3 and non-malignant prostate epithelial cells RWPE-1, breast cancer cells MCF7, BT474 and non-malignant breast epithelial cells MCF10A, cervical cancer cells HeLa, lung cancer cells H1299, A549 and human embryo kidney cells HEK293T were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

    Techniques: Functional Assay, Transfection, Expressing, Immunoprecipitation, Plasmid Preparation, SDS Page, Staining