Journal: Glia

Article Title: Endoplasmic Reticulum Stress Amplifies Cytokine Responses in Astrocytes via a PERK / eIF2α / JAK1 Signaling Axis

doi: 10.1002/glia.70067

Figure Lengend Snippet: JAK1 and PERK are required to augment TNF‐α induced gene expression in human glioma cells. (A) 1321N1 cells stably expressing Cas9 were transfected with non‐targeting (NT) or JAK1 guide RNAs (gRNA) to establish non‐clonal cell lines. These cells were then treated with IFN‐γ (10 ng/mL) for 30 min followed by immunoblotting. (B) NT and JAK1 gRNA (#2) cells were treated with thaps (1 μM), TNF‐α (10 ng/mL), or both for 4 h followed by qPCR. (C) 1321N1 cells stably expressing Cas9 were transfected with NT or PERK gRNA to establish non‐clonal cell lines. These cells were then treated with thaps (1 μM) for the indicated times followed by immunoblotting. (D) NT and PERK gRNA cells were treated with thaps (1 μM), TNF‐α (10 ng/mL), or both for 4 h followed by qPCR. N = 4, data are means ± standard deviation.

Article Snippet: 1321N1 human astrocytoma cell line stably expressing Cas9 nuclease was purchased from GeneCopoeia.

Techniques: Gene Expression, Stable Transfection, Expressing, Transfection, Western Blot, Standard Deviation

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

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Schematic of the proposed mechanism by which interleukin (IL)-18 amplifies macrophage (Mφ) M2 polarization and its morphological alteration, leading to excessive angiogenesis. IL-18 amplifies IL-10-induced increases in the production of osteopontin (OPN) and thrombin as soluble mediators derived from Mφ, yielding the generation of thrombin-cleaved form of OPN (Thr-OPN). Subsequently, Thr-OPN binds to integrins α4/α9 receptors on Mφ, which in turn augments M2 polarization of Mφ with higher expression of CD163 and its morphological alteration. Furthermore, CD163 may be responsible for mediating the direct cell–cell interaction between these Mφs and endothelial cells, ultimately resulting in the excessive angiogenesis.

Article Snippet: The mouse leukemic monocyte Mφ cell line, RAW264.7 cells (DS Pharma Biomedical), and the mouse endothelial cell line, b.End5 cells (DS Pharma Biomedical), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 mM l -glutamine and 10% heat-inactivated fetal bovine serum.

Techniques: Derivative Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Characteristic behavior of macrophages (Mφs) during angiogenesis. (A) Representative series of time-lapse images at 4 h intervals from 0 to 16 h extracted from Video S1 which shows live-cell imaging of Matrigel tube formation assay where endothelial cells (green) and Mφs [interleukin (IL)-10 + IL-18] (red) were cocultured. Scale bar represents 50 µm. (B) Higher magnification images of white rectangle region in panel (A) were reconstructed from 4 h 00 min to 7 h 00 min in Video S5 in Supplementary Material. The time elapsed after starting the movie is indicated in hours:minutes in the bottom left of each panel. White arrowheads highlight the characteristic behavior of Mφ (IL-10 + IL-18) as well as the cell–cell interaction with endothelium in respective image. Scale bar represents 10 µm.

Article Snippet: The mouse leukemic monocyte Mφ cell line, RAW264.7 cells (DS Pharma Biomedical), and the mouse endothelial cell line, b.End5 cells (DS Pharma Biomedical), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 mM l -glutamine and 10% heat-inactivated fetal bovine serum.

Techniques: Live Cell Imaging, Tube Formation Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Ultrastructural analysis of cell–cell interaction between macrophages (Mφs) [interleukin (IL)-10 + IL-18] and endothelia. SEM images were obtained at 4 h after coculture of b.End5 with Mφs (IL-10 + IL-18) on Matrigel. Lower images are magnified regions from red rectangles in the corresponding upper panels. Magnification and scale bars: (A) upper, ×1.0 K, 50 µm; Lower, ×5.0 K, 10 µm; (B) upper, ×1.0 K, 50 µm; lower, ×2.0 K, 20 µm; (C) upper, ×1.0 K, 50 µm; lower, ×8.0 K, 5 µm; (D) upper, ×1.0 K, 50 µm; lower, ×3.0 K, 10 µm; (E) upper, ×1.0 K, 50 µm; lower, ×5.0 K, 10 µm; (F) upper, ×1.0 K, 50 µm; lower, ×4.0 K, 10 µm; (G) upper, ×1.0 K, 50 µm; lower, ×2.5 K, 20 µm; (H) upper, ×1.0 K, 50 µm; lower, ×2.5 K, 20 µm; (I) upper, ×1.0 K, 50 µm; lower, ×3.5 K, 10 µm; respectively. Black or white arrows indicate Mφs or endothelial cells, respectively. Red arrowheads indicate pseudopodia of Mφs interacting with endothelial cells.

Article Snippet: The mouse leukemic monocyte Mφ cell line, RAW264.7 cells (DS Pharma Biomedical), and the mouse endothelial cell line, b.End5 cells (DS Pharma Biomedical), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 mM l -glutamine and 10% heat-inactivated fetal bovine serum.

Techniques: