Journal: Frontiers in Microbiology

Article Title: Combining flagellin and human β-defensin-3 to combat bacterial infections

doi: 10.3389/fmicb.2014.00673

Figure Lengend Snippet: In vitro activity of F and FBD3 proteins-secretion of IL-8. (A,B) FACS analysis of Toll-Like-Receptor-5 on Colo-205 and Bjab cell lines. (C,D) In vitro activity of the F protein on Colo-205 and Bjab cells. Colo-205 or Bjab cells (10 5 cells/ml) were incubated with 20 ng (protein) of F for 4 h. The medium separated from the various samples was tested and the amount of the IL-8 was measured as described in Methods. One representative experiment out of two performed is shown (C) . Variations were ±10%. Colo-205 cells (10 5 cells/ml) were incubated with different concentrations of F (1.2 and 12 ng protein), FBD3 (2 or 20 ng fusion protein; comprising about 1.8 and 18 ng, respectively, of the F protein) and GnRH-Cherry for 4 h (20 ng protein). The medium from the different samples was analyzed and the amount of the IL-8 was measured by an Elisa assay as described in the “Methods.” One representative experiment out of four performed is shown (D) .

Article Snippet: After 4 h incubation the cell-free supernatants were analyzed according to the manufacturer’s instructions for human IL-8 content, using Human IL-8 ELIZA KIT II (BD Biosciences San Diego, CA, USA).

Techniques: In Vitro, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: BMC urology

Article Title: MicroRNA-148a-3p in pericyte-derived extracellular vesicles improves erectile function in diabetic mice by promoting cavernous neurovascular regeneration.

doi: 10.1186/s12894-023-01378-4

Figure Lengend Snippet: Fig. 1 MCP-derived extracellular vesicle (MCPs-EV) characterization and tracking analysis in MCECs. a Representative transmission electron micrograph (TEM) phase images for detecting isolated MCPs-EVs as indicated by the arrows. Scale bar = 100 nm. b Representative Western blot for three positive EV markers (CD9, CD63, and CD81) and one negative EV marker (GM130) in MCP lysate and MCPs-EVs. c DiD-labeled MCPs-EVs (red) were treated with MCECs for 6 h. Scale bar = 50 μm. MCPs, mouse corpus cavernous pericyte; MCECs, mouse cavernous endothelial cells; DiD, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt

Article Snippet: Tissues were cultured with the complement M199 medium (Gibco) containing 20% fetal bovine serum (FBS, Gibco), 0.5 mg/ mL of heparin (Sigma-Aldrich), 5 ng/mL of recombinant human vascular endothelial growth factor (VEGF, R&D Systems Inc., Minneapolis, MN, USA), and 1% penicillin/ streptomycin (Gibco) in a 5% CO2 atmosphere incubator at 37 °C.

Techniques: Derivative Assay, Transmission Assay, Isolation, Western Blot, Marker, Labeling

Journal: BMC urology

Article Title: MicroRNA-148a-3p in pericyte-derived extracellular vesicles improves erectile function in diabetic mice by promoting cavernous neurovascular regeneration.

doi: 10.1186/s12894-023-01378-4

Figure Lengend Snippet: Fig. 3 MCPs-EVs decreased apoptosis and increased proliferation of MCECs through miR-148a-3p under high-glucose (HG) conditions. a TUNEL (green) immunostaining in MCECs treated with PBS, MCPs-EVs-reagent control (MCPs-EVs-RC, 1 μg/mL), MCPs-EVs-miR-148a-3p inhibitor (MCPs-EVs-miR-148a-3p-i, 1 μg/mL) under normal-glucose (NG) and HG conditions for 3 days. Scale bar = 100 μm. b Number of apoptotic cells were quantified by ImageJ and the results are presented as mean ± SEM (n = 4). c PH3 (red) immunostaining in MCECs with the same treatment conditions as for TUNEL immunostaining. Nuclear were labeled with DAPI (blue). d Number of PH3-positive cells were quantified by ImageJ and the results are presented as mean ± SEM (n = 4). ***p < 0.001. TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; MCPs, mouse corpus cavernous pericyte; MCECs, mouse cavernous endothelial cells; PH3, phospho-Histone H3; ns, not significant; DAPI, 4.6-diamidino-2-phenylindole

Article Snippet: Tissues were cultured with the complement M199 medium (Gibco) containing 20% fetal bovine serum (FBS, Gibco), 0.5 mg/ mL of heparin (Sigma-Aldrich), 5 ng/mL of recombinant human vascular endothelial growth factor (VEGF, R&D Systems Inc., Minneapolis, MN, USA), and 1% penicillin/ streptomycin (Gibco) in a 5% CO2 atmosphere incubator at 37 °C.

Techniques: TUNEL Assay, Immunostaining, Control, Labeling, End Labeling

Journal: BMC urology

Article Title: MicroRNA-148a-3p in pericyte-derived extracellular vesicles improves erectile function in diabetic mice by promoting cavernous neurovascular regeneration.

doi: 10.1186/s12894-023-01378-4

Figure Lengend Snippet: Fig. 5 MCPs-EVs improve cavernous endothelial cell, pericytes, and neuronal cell content through miR-148a-3p in STZ-induced diabetic mice. a CD31 (green) and NG2 (red) immunostaining in cavernous tissue from age-matched nondiabetic controls, diabetic mice stimulated at 2 weeks after intracavernous PBS, MCPs-EVs-reagent control (MCPs-EVs-RC, 5 μg/20 μL), or MCPs-EVs-miR-148a-3p inhibitor (MCPs-EVs-miR-148a-3p-i, 5 μg/20 μL) injection; scale bar = 100 μm. b β (III)-tubulin (red) and nNOS (green) immunostaining in the same cavernous tissue section with the abovementioned CD31 staining groups; scale bar = 25 μm. Nuclear were labeled with DAPI (blue). c-f Quantitative analysis of cavernous endothelial cell, pericytes, and β (III)-tubulin- or nNOS-expressing neuronal cell contents using Image J. Data in graphs are presented as mean ± SEM (n = 4). The value expressed as ratios of the control group was set to 1. **p < 0.01; ***p < 0.001. STZ, streptozotocin; MCPs, mouse corpus cavernous pericytes; DAPI = 4,6-diamidino-2-phenylindole; ns, not significant

Article Snippet: Tissues were cultured with the complement M199 medium (Gibco) containing 20% fetal bovine serum (FBS, Gibco), 0.5 mg/ mL of heparin (Sigma-Aldrich), 5 ng/mL of recombinant human vascular endothelial growth factor (VEGF, R&D Systems Inc., Minneapolis, MN, USA), and 1% penicillin/ streptomycin (Gibco) in a 5% CO2 atmosphere incubator at 37 °C.

Techniques: Immunostaining, Control, Injection, Staining, Labeling, Expressing

Journal: BMC urology

Article Title: MicroRNA-148a-3p in pericyte-derived extracellular vesicles improves erectile function in diabetic mice by promoting cavernous neurovascular regeneration.

doi: 10.1186/s12894-023-01378-4

Figure Lengend Snippet: Fig. 6 PDK4 was identified as a target gene of miR-148a-3p. a Representative Western blots for PDK4 in MCECs treated with PBS, MCPs-EVs-reagent control (MCPs-EVs-RC, 1 μg/mL) and MCPs-EVs-miR-148a-3p inhibitor (MCPs-EVs-miR-148a-3p-i, 1 μg/mL) under normal-glucose (NG) and high-glucose (HG) conditions for 3 days. b Relative intensities of PDK4 and β-actin on Image J analysis. Data in graphs are presented as mean ± SEM (n = 4). Values expressed as ratios of the control group were set to 1. **p < 0.01; ***p < 0.001. c The binding sequences of miR-148a-3p on position 655-662 of PDK4 3’UTR. d Luciferase reporter assay was used to assess the binding capacity between miR-148a-3p and PDK4 in MCECs. Data in graphs are presented as mean ± SEM (n = 4). Values expressed as ratios of the NC mimics co-transfected with PDK4 3’UTR plasmid group was set to 1. ***p < 0.001. MCPs, mouse corpus cavernous pericyte; MCECs, mouse cavernous endothelial cells; NC mimics, negative control mimics; ns, not significant

Article Snippet: Tissues were cultured with the complement M199 medium (Gibco) containing 20% fetal bovine serum (FBS, Gibco), 0.5 mg/ mL of heparin (Sigma-Aldrich), 5 ng/mL of recombinant human vascular endothelial growth factor (VEGF, R&D Systems Inc., Minneapolis, MN, USA), and 1% penicillin/ streptomycin (Gibco) in a 5% CO2 atmosphere incubator at 37 °C.

Techniques: Western Blot, Control, Binding Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Negative Control