Journal: Veterinary Sciences

Article Title: Feline Uroepithelial Cell Culture as a Novel Model of Idiopathic Cystitis: Investigations on the Effects of Norepinephrine on Inflammatory Response, Oxidative Stress, and Barrier Function

doi: 10.3390/vetsci10020132

Figure Lengend Snippet: Immunofluorescent staining of uroepithelial cell culture with eFluor labeled pan-cytokeratin antibody on day 1 after plating. Blue color indicates diamidino phenylindole (DAPI) labeled cell nuclei, while red color shows epithelial cells detected with eFluor labeled pan-cytokeratin antibody. (bar = 3.5 µm).

Article Snippet: The cells were washed three times with PBS and then they were incubated in a blocking solution containing 3% bovine serum albumin (BSA) in PBS at room temperature for 30 min. To confirm the epithelial origin of the cells, eFluor labeled pan-cytokeratin antibody (Cat. No. 41-9003-82, Thermo Fisher, Waltham, MA, USA) was used.

Techniques: Staining, Cell Culture, Labeling

Journal: Veterinary Sciences

Article Title: Feline Uroepithelial Cell Culture as a Novel Model of Idiopathic Cystitis: Investigations on the Effects of Norepinephrine on Inflammatory Response, Oxidative Stress, and Barrier Function

doi: 10.3390/vetsci10020132

Figure Lengend Snippet: Immunofluorescent staining of uroepithelial cell culture on day 6 after plating. Blue color indicates diamidino phenylindole (DAPI) labeled cell nuclei, red color shows epithelial cells detected with eFluor labeled pan-cytokeratin antibody, and green color refers to differentiated uroepithelial cells stained with fluorescein isothiocyanate (FITC) labeled uroplakin III antibody. (bar = 3.5 µm).

Article Snippet: The cells were washed three times with PBS and then they were incubated in a blocking solution containing 3% bovine serum albumin (BSA) in PBS at room temperature for 30 min. To confirm the epithelial origin of the cells, eFluor labeled pan-cytokeratin antibody (Cat. No. 41-9003-82, Thermo Fisher, Waltham, MA, USA) was used.

Techniques: Staining, Cell Culture, Labeling

Journal: Cell host & microbe

Article Title: Hepatitis C virus subverts liver-specific miR-122 to protect the viral genome from exoribonuclease Xrn2

doi: 10.1016/j.chom.2014.07.006

Figure Lengend Snippet: (A) Effects on RNA abundance. Cells were mock-transfected (Mock), or transfected with control (NC) or Xrn2 siRNAs. One day later, cells were infected with HCV as indicated. RNA abundances were measured by Northern blot analysis three days after infection. The Northern blot is representative of at least three independent replicates (top). Quantitation of HCV RNA abundance (bottom). Error bars represent standard error of the mean. P value was determined by Student’s t-test. (B) siRNA-mediated depletion of Xrn2. Abundances of HCV NS5A and core proteins, and GAPDH were examined. Immunoblot shown is representative of at least three independent replicates. (C) Overexpression of Xrn2. 3XFLAG-Xrn2 or 3XFLAG plasmids were transfected one day prior to infection with HCV. HCV and actin RNA abundances were measured by Northern blot analysis three days after infection. The Northern blot shown represents at least three independent replicates (top). Quantitation of HCV RNA abundance (bottom). Error bars represent standard error of the mean. P value was determined by Student’s t-test. (D) Effects of Xrn2 depletion on HCV IRES-mediated translation, measured by luciferase assay as described in the text. The data shown represent at least three independent replicates. Error bars represent standard error of the mean. P-value was determined by Student’s t-test. (E and F) Polysomal profile from Xrn2-depleted cells (top). siRNAs were transfected one day prior to infection. Three days after infection, cell lysates from HCV-infected, siXrn2-treated or siNC-treated samples were separated in 10–60% sucrose gradients. 40S and 60S ribosomal subunits and polysomes are indicated in the A260nm profile. Northern blot analyses of the sucrose gradient fractions (bottom). The Northern blots shown represent at least three independent replicates.

Article Snippet: In-vitro assays were carried out in Xrn2 buffer (20 mM HEPES (pH 7.5), 100 mM KCl, 4 mM MgCl2, 5% (v/v) glycerol, 1 mM DTT) with 0.766 µg of human Xrn2 (OriGene TP323980) and ~2 pmol RNA substrate in a volume of 10 µl at 30 °C for 20 min to 60 min.

Techniques: Transfection, Infection, Northern Blot, Quantitation Assay, Western Blot, Over Expression, Luciferase

Journal: Cell host & microbe

Article Title: Hepatitis C virus subverts liver-specific miR-122 to protect the viral genome from exoribonuclease Xrn2

doi: 10.1016/j.chom.2014.07.006

Figure Lengend Snippet: (A) Effects of Xrn2 depletion on rates of HCV RNA synthesis. siRNAs were transfected one day prior to infection. Abundances of total and newly synthesized HCV and actin RNAs were examined by Northern blot analysis three days after infection (top) (Norman and Sarnow, 2010). The Northern blot shown is representative of at least three independent replicates. Replication rates of HCV RNA correspond to the ratio of newly synthesized HCV RNA to input RNA. Error bars represent standard error of the mean. P-values were determined by Student’s t-test. (B) Effects of Xrn2 depletion on HCV RNA decay. Control (siNC) or Xrn2 (siXrn2) siRNAs were transfected one day prior to infection. Three days after infection cells were treated with 25 µM of the MK-0608 nucleoside analog of HCV NS5B, and RNAs were extracted at indicated times. HCV RNA levels were measured by Northern blot analysis. Northern blots shown represent at least three independent experiments. (C) One-phase decay graph of HCV RNA. Data from cells harvested at time 0 were set to 1. Data shown represent remaining HCV RNA following addition of MK-0608. Error bars represent standard error of the mean. Estimated half-life (t1/2) of HCV RNA under these conditions is indicated; ± 95% confidence intervals (CI) are 0.54–2.24 and 5.82–8.42 in hours for NC and Xrn2 respectively (see Fig. S7). The data shown represent the means of at least three independent replicates.

Article Snippet: In-vitro assays were carried out in Xrn2 buffer (20 mM HEPES (pH 7.5), 100 mM KCl, 4 mM MgCl2, 5% (v/v) glycerol, 1 mM DTT) with 0.766 µg of human Xrn2 (OriGene TP323980) and ~2 pmol RNA substrate in a volume of 10 µl at 30 °C for 20 min to 60 min.

Techniques: Transfection, Infection, Synthesized, Northern Blot

Journal: Cell host & microbe

Article Title: Hepatitis C virus subverts liver-specific miR-122 to protect the viral genome from exoribonuclease Xrn2

doi: 10.1016/j.chom.2014.07.006

Figure Lengend Snippet: A) Xrn2 association with HCV RNA. Representative Northern blot of co-immunoprecipitation reactions performed with antibodies against Hif1-α, Xrn2, and Rck. Lane 1 corresponds to 10% of input RNA. (B) Protection of viral RNA by miR-122 from Xrn2-mediated degradation. 3’-32P-labelled capped- (m7G-5’), monophosphorylated- (p-5’), and triphosphorylated- (ppp-5’) HCV reporter RNAs were incubated with recombinant human Xrn2 in the absence (top) or presence of miR-122 (middle) or miR-124 (bottom) for the indicated periods of time, and subsequently analyzed by denaturing gel electrophoresis. Quantitation of radiolabeled RNA is on the right. Results from at least three independent replicates are shown. Error bars represent standard error of the mean. Values were normalized to the 0-minute no-Xrn2 control.

Article Snippet: In-vitro assays were carried out in Xrn2 buffer (20 mM HEPES (pH 7.5), 100 mM KCl, 4 mM MgCl2, 5% (v/v) glycerol, 1 mM DTT) with 0.766 µg of human Xrn2 (OriGene TP323980) and ~2 pmol RNA substrate in a volume of 10 µl at 30 °C for 20 min to 60 min.

Techniques: Northern Blot, Immunoprecipitation, Incubation, Recombinant, Nucleic Acid Electrophoresis, Quantitation Assay

Journal: Cell host & microbe

Article Title: Hepatitis C virus subverts liver-specific miR-122 to protect the viral genome from exoribonuclease Xrn2

doi: 10.1016/j.chom.2014.07.006

Figure Lengend Snippet: (A) Indicated siRNAs were transfected one day prior to infection. 0.5 nM miR-122 or 0.5 nM miR-106b LNA were transfected one day post-infection, and total RNA was harvested three days after infection. HCV and γ-actin RNA abundance was measured by Northern blot analysis (top). The Northern blot shown represents three independent experiments. Quantitation of HCV RNA abundance (bottom). HCV RNA abundances were normalized to γ-actin levels. Data from cells transfected with control siRNAs were set to 1. Error bars represent standard error of the mean. P-values determined by Student’s t-test. (B) Model of protection of the 5’-end of the HCV genome by miR-122 from Xrn2-mediated 5’-3’ degradation. (a) In the presence of miR-122, Xrn2 produces few viral degradation products (thin arrow). (b) Loss of miR-122-mediated protection renders the 5’-triphosphate of viral genome susceptible to Xrn2 degradation. Helicases could remove miR-122 from the viral genome, resulting in larger amounts of degradation products (thick arrow). (c) Removal of the 5’ triphosphate moiety of HCV RNA by a pyrophosphohydrolase could generate 5’-monophosphates that would be degraded by Xrn2 (very thick arrow).

Article Snippet: In-vitro assays were carried out in Xrn2 buffer (20 mM HEPES (pH 7.5), 100 mM KCl, 4 mM MgCl2, 5% (v/v) glycerol, 1 mM DTT) with 0.766 µg of human Xrn2 (OriGene TP323980) and ~2 pmol RNA substrate in a volume of 10 µl at 30 °C for 20 min to 60 min.

Techniques: Transfection, Infection, Northern Blot, Quantitation Assay

Journal: bioRxiv

Article Title: Wilms Tumorigenesis in Human Kidney Organoids

doi: 10.1101/2021.02.02.429313

Figure Lengend Snippet: A: Quantification of subpopulations in pooled d21 un-edited, KO iPSC , KO d4-7 , KO d9-11 and KO d11-14 organoids by flow cytometry of indicated markers. Data is presented as mean % of positive cells +/- SD derived from n=5 (un-edited, KO iPSC , KO d4-7 ) or n=2 (KO d9-11 , KO d11-14 ) independent experiments. Two-sided student’s t-test; p -value: ns >0.05; * ≤0.05; ** ≤0.01; *** ≤0.001; **** ≤0.0001; SIX2-Ki-67: asterisks correspond to the SIX2-KI67 population. B: IF staining for the indicated markers in representative un-edited, KO iPSC , KO d4-7 , KO d9-11 and KO d11-14 d21 organoids. Scale bar: 100 µm. White boxed regions are shown in Figure S1J. C: Flow cytometry-based quantification of EpCAM high , EPCAM mid and EpCAM low populations (see gating shown in Figure S1I) in pooled organoids of indicated time-points and genotypes. Data is shown as mean % of positive cells +/- SD derived from n=3 (Day 7); n=4 (Day 9); n=2 (Day 10); n=5 (un-edited, KO iPSC , KO d4-7 ; Day 21) or n=2 (KO d9-11 , KO d11-14 ; Day 21) independent differentiation experiments. Two-sided student’s t-test; p -value: ns > 0.05; * ≤ 0.05; ** ≤ 0.01; asterisks are placed above the respective populations (EPCAM high and EPCAM mid ).

Article Snippet: Antibodies were: CDH1 (BD Biosciences #610181, 1:200); EPCAM-AF647 (Abcam #ab239273, 1:200); Hoechst 33342 (Thermo Fisher #H3570; 1:10000); HOXD11 (Sigma #SAB1403944; 1:300); IGF2 (Thermo Fisher #MA5-17096; 1:200); Ki-67 (SolA15, eBisoscience #14-5698-82, 1:200) (Ki-67 / Ki-67-FITC (SolA15, eBisoscience #14-5698-82, 1:200); LHX1 (OriGene #TA504528; 1:1000); LTL-Biotinylated (Vectorlabs B-1325, 1:500); LIN28A (Cell Signaling #3978, 1:600); LIN28B (Cell Signaling #4196, 1:250); NPHS1 (R&D Systems #AF4269, 1:60); PAX2 (Invitrogen #71-6000; 1:100); PAX8 (Proteintech #10336-1-AP; 1:100); PODXL (R&D Systems #AF1658, 1:500); SIX1 (Cell Signaling #12891; 1:200), SIX2 (Proteintech #11562-1-AP, 1:100); WT1 (Abcam #ab89901; 1:200); donkey-anti-rabbit AF 488 (Thermo Fisher #A-21206, 1:500); donkey-anti-rabbit AF 594 (Thermo Fisher #A-21207, 1:500); donkey-anti-rabbit AF 647 (Thermo Fisher #A-31573, 1:500); donkey-anti-goat AF 594 (Thermo Fisher #A-11058, 1:500); donkey-anti-goat AF 647 (Thermo Fisher #A-21447, 1:500); donkey-anti-sheep AF 488 (Thermo Fisher # A-11015, 1:500); donkey-anti-mouse AF 488 (Thermo Fisher #A-21202, 1:500); donkey-anti-mouse AF 594 (Thermo Fisher #A-21203, 1:500); donkey-anti-mouse AF 647 (Thermo Fisher #A-31571, 1:500); donkey-anti-rat AF 647 (Abcam #150155, 1:500); Streptavidin Fluorescent Dye 633-I (Abnova #U0295, 1:500).

Techniques: Flow Cytometry, Derivative Assay, Staining

Journal: Frontiers in Immunology

Article Title: Ischemia Reperfusion Injury Triggers CXCL13 Release and B-Cell Recruitment After Allogenic Kidney Transplantation

doi: 10.3389/fimmu.2020.01204

Figure Lengend Snippet: Serum CXCL13 levels in mouse ktx. Post ktx levels of serum CXCL13 at day 1 were significantly increased compared to baseline. A higher increase was observed in longer cold ischemia time (30 vs. 60 min cold ischemia time). Isogenic ktx with prolonged cold ischemia time of 60 min had significantly lower CXCL13 levels compared to allogenic ktx (A) . PAS stain at day 7 revealed enhanced cell infiltration in allogenic compared to isogenic ktx (B) . Double staining for CD3+ T-lymphocytes (green) and CD45R+ B-cells (red) was performed at day 7. More interstitial CD3+ T-lymphocytes were observed in allografts compared to isografts. Allografts exhibited scattered B-cells in interstitial tissue, but also clusters of CD45R+ cells. Isografts showed only few B cells in the interstitium at day 7 (B) (bar: 100 μm, n = 6 per group, one-way ANOVA * p < 0.05; ** p < 0.01; *** p < 0.001). BL, baseline.

Article Snippet: For further characterization of infiltrating leukocytes, immunostaining was performed with antibodies against Gr-1 (neutrophils) (Biorad, MCA 771G), F4/80 (monocytes/macrophages) (Acris Antibodies, BM4007), CD22 (Southern Biotech, 1580-01), and CD45R (B cells) (eBioscience, 14-0452-82), CD3 (T cells) (Dako, A0452), respectively.

Techniques: Staining, Double Staining