Journal: International Journal of Molecular Sciences

Article Title: Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production

doi: 10.3390/ijms18050968

Figure Lengend Snippet: Knockdown of Atg7 inhibited angiogenesis of brain microvascular endothelial cells. ( A ) Human brain microvascular endothelial cells (HBMEC) were stably transfected with Atg7-specific shRNA construct, Atg7 shRNA1, and Atg7 shRNA2, respectively. HBMEC stably transfected with non-silencing shRNA were served as the control. Then the protein levels of Atg7 were examined by western blot, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control. The relative expression level of Atg7 and Atg7/GAPDH were calculated by measuring the band intensity using ImageJ software. ** p < 0.01; ( B ) Tube formation assays were performed with HBMEC stably transfected with Atg7 shRNA1 and Atg7 shRNA2, respectively, with non-silencing shRNA as the control. Then the images were captured under an inverted microscope at indicated times. The representative images from three independent experiments were shown. Scale, 200 μm; ( C , D ) To quantify the results of tube formation assays in ( B ), the number of branch points were counted and the tube length were calculated. ** p < 0.01.

Article Snippet: Human brain microvascular endothelial cell line (HBMEC) was a kind gift from Dr. Kwang Sik Kim (Johns Hopkins University School of Medicine, Baltimore, MD, USA).

Techniques: Knockdown, Stable Transfection, Transfection, shRNA, Construct, Control, Western Blot, Expressing, Software, Inverted Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production

doi: 10.3390/ijms18050968

Figure Lengend Snippet: Atg7 knockdown reduced IL-6 production in brain endothelial cells. ( A ) The mRNA levels of IL-6 in the HBMEC transfected with Atg7 shRNA1 were determined by real time RT-PCR. HBMEC transfected with non-silencing shRNA were used as control. ** p < 0.01; ( B ) The concentration of IL-6 and vascular endothelial growth factor (VEGF) in the supernatant of HBMEC transfected with Atg7 shRNA1 were determined by ELISA. ** p < 0.01; ( C ) The mRNA levels of IL-6 and VEGF in the brain cortex from the three-month-old Atg7 endothelial-specific knockout mice were determined by real time RT-PCR, with littermate wild-type mice as control. * p < 0.05.

Article Snippet: Human brain microvascular endothelial cell line (HBMEC) was a kind gift from Dr. Kwang Sik Kim (Johns Hopkins University School of Medicine, Baltimore, MD, USA).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, shRNA, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production

doi: 10.3390/ijms18050968

Figure Lengend Snippet: Exogenous addition of IL-6 restored the impaired angiogenesis in brain endothelial cells with Atg7 knockdown. ( A ) Tube formation assays were performed with the indicated HBMEC in the absence (vehicle) or presence of IL-6 (10 ng/mL). The representative images from three independent experiments were presented. Scale, 200 μm; ( B , C ) To quantify the results in ( A ), the number of branch points ( B ) and the tube lengths were calculated ( C ). * p < 0.05, ** p < 0.01.

Article Snippet: Human brain microvascular endothelial cell line (HBMEC) was a kind gift from Dr. Kwang Sik Kim (Johns Hopkins University School of Medicine, Baltimore, MD, USA).

Techniques: Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production

doi: 10.3390/ijms18050968

Figure Lengend Snippet: Knockdown of Atg7 inhibited migration of brain microvascular endothelial cell, which was restored by exogenous IL-6. ( A ) The scratch wound assays were performed using the indicated transfected HBMEC in the absence (vehicle) or presence of IL-6 (10 ng/mL). The representative images at indicated times from three independent experiments were shown. Scale, 200 μm; ( B ) To quantify the results in ( A ), the wound recover rates were calculated by ImageJ software. * p < 0.05, ** p < 0.01.

Article Snippet: Human brain microvascular endothelial cell line (HBMEC) was a kind gift from Dr. Kwang Sik Kim (Johns Hopkins University School of Medicine, Baltimore, MD, USA).

Techniques: Knockdown, Migration, Transfection, Software

Journal: International Journal of Molecular Sciences

Article Title: Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production

doi: 10.3390/ijms18050968

Figure Lengend Snippet: Interleukin-6 promoted cell migration to rescue angiogenesis in the Atg7-knockdown brain microvascular endothelial cells. ( A ) The nuclear and cytoplasmic extracts were obtained in HBMEC stably transfected with Atg7 shRNA1 (Atg7 KD). Then the expression of the NF-κB p65 subunit was analyzed by western blot. β-tubulin and 38F3 were detected as marker proteins for cytoplasm and nuclear, respectively. HBMEC transfected with non-silencing shRNA were used as control. The representative images were from three independent experiments; ( B ) To quantify the results in ( A ), the band intensities of p65 in nuclear and cytoplasm fractions were measured by ImageJ software and the nuclear to cytoplasm ratios of p65 was calculated. * p < 0.05; ( C ) HBMEC stably transfected with Atg7 shRNA1 were seeded on coverslips and immunofluorescence was conducted with antibody against p65 (green). DAPI (blue) was used for counterstaining. HBMEC transfected with non-silencing shRNA were served as a control. Scale, 20 μm; ( D ) The mRNA levels of IL-6 in the HBMEC transfected with Atg7 shRNA1 were determined by real time RT-PCR, with HBMEC transfected with non-silencing shRNA as a control. When indicated, the cells were incubated with NF-κB agonist and betulinic acid (10 μg/mL) for 2 h. *** p < 0.001.

Article Snippet: Human brain microvascular endothelial cell line (HBMEC) was a kind gift from Dr. Kwang Sik Kim (Johns Hopkins University School of Medicine, Baltimore, MD, USA).

Techniques: Migration, Knockdown, Stable Transfection, Transfection, Expressing, Western Blot, Marker, shRNA, Control, Software, Immunofluorescence, Quantitative RT-PCR, Incubation

Journal: PLoS Pathogens

Article Title: Binding of Glycoprotein Srr1 of Streptococcus agalactiae to Fibrinogen Promotes Attachment to Brain Endothelium and the Development of Meningitis

doi: 10.1371/journal.ppat.1002947

Figure Lengend Snippet: (A) Fibrinogen on the surface of hBMEC pretreated with or without exogenous fibrinogen (Fg; 20 µg/ml). Nuclei were stained with DAPI (blue) and fibrinogen was detected with anti-fibrinogen IgG, followed by Alexa Fluor 488 conjugated anti-rabbit IgG (red). (B) NCTC 10/84 (WT) or PS2645 (Δ srr1 ) incubated with hBMEC, with or without fibrinogen pretreatment (20 µg/ml). Unbound bacteria were washed out and bound bacteria were counted. Values represent percent (mean ± S.D.) of total GBS inoculum bound to the monolayers. * = P<0.01.

Article Snippet: The human brain microvascular endothelial cell line (hBMEC) was developed and kindly provided by Kwang Sik Kim (Johns Hopkins University) , and cultured as previously described .

Techniques: Staining, Incubation, Bacteria

Journal: PLoS Pathogens

Article Title: Binding of Glycoprotein Srr1 of Streptococcus agalactiae to Fibrinogen Promotes Attachment to Brain Endothelium and the Development of Meningitis

doi: 10.1371/journal.ppat.1002947

Figure Lengend Snippet: (A) Binding of FLAG Srr1-BR and FLAG Srr1-BRΔlatch proteins to immobilized fibrinogen. Indicated concentration of FLAG Srr1-BR and FLAG Srr1-BRΔlatch were added to wells coated with fibrinogen or casein blocking reagent. (B) Binding of FLAG Srr1-BR and FLAG Srr1-BRΔlatch proteins to hBMEC monolayers pretreated with PBS (left panels) or fibrinogen (20 µg/ml, right panels). After washing out unbound proteins, bound proteins were detected with anti-FLAG mAb, followed by Alexa Fluor 488 conjugated anti-mouse IgG (red). Nuclei were stained with DAPI (blue). (C) Expression of Srr1-WT and Srr1Δ latch on the cell surface. Isolated cell wall proteins were probed by Western blotting with anti-Srr1 IgG. (D) GBS NCTC 10/84 WT, Δ srr1 and Δlatch variant binding to immobilized fibrinogen. Values represent percent of WT GBS binding to fibrinogen. (E) GBS NCTC 10/84 WT, Δ srr1 and Δ latch isogenic variant adherence to hBMEC monolayers. * = P<0.01.

Article Snippet: The human brain microvascular endothelial cell line (hBMEC) was developed and kindly provided by Kwang Sik Kim (Johns Hopkins University) , and cultured as previously described .

Techniques: Binding Assay, Concentration Assay, Blocking Assay, Staining, Expressing, Isolation, Western Blot, Variant Assay