Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

doi: 10.1177/0271678X17720816

Figure Lengend Snippet: Pericytes share glucose with astrocytes in an occludin-modulated manner. (a) Confocal microscopy of a representative live pericyte-astrocyte co-culture 30 min after plating. Pericytes were pre-loaded with 2-NBDG (green; e.g. blue thick arrows) and astrocytes with violet-BMQC (cell mask, red) but not with 2-NBDG. Colocalization of both signals (yellow-orange; e.g. white thin arrows) indicated that astrocytes had received 2-NBDG. Intensity coefficient (Ic) represents the whole 2-NBDG fluorescence intensity normalized to the surface it occupies, regardless of cell type. A larger Ic means more 2-NBDG was introduced into the system (taken up by pericytes). Transfer coefficient (Tc) represents the fraction of astrocytic surface occupied by 2-NBDG normalized against the Intensity coefficient. A larger Tc implicates more 2-NBDG was distributed across all possible astrocytes, and represents greater transferred volumes. Data correspond to the quantitation of the images shown. They are representative of three separate experiments. (b) Distribution of 2-NBDG in the same co-culture shown in (a), 24 h after plating. Pericytes are devoid of any stain (e.g. white thin arrows). 2-NBDG signal (green) colocalizing with astrocytes is seen as cyan/white (e.g. yellow thick arrows). (c) Similar co-culture as in (a) recorded 30 min post-plating; however, pericytes were treated with negative-control siRNA (SCR) before being loaded with 2-NBDG. (d) Distribution of 2-NBDG (green/cyan) in the same co-culture shown in (c), 24 h post-plating. (e) Similar co-culture as in (a) and (c), recorded 30 min post-plating; however, pericytes were treated with anti-occludin siRNA (OCC−) before being loaded with 2-NBDG. (f) Distribution of 2-NBDG (green/cyan) in the same co-culture shown in (e), 24 h post-plating. All images are representative of three separate experiments. (g) Average intensity (Ic) and H) Transfer (Tc) coefficients depicting transcellular glucose transport between pericytes and astrocytes as shown in (a) to (c). n = 3, p vs. WT.

Article Snippet: Cell culture Primary human brain capillary pericytes and astrocytes (ScienCell, Carlsbad, CA, USA) were cultured in 5% CO 2 at 37°C in pericyte or astrocyte growth medium (ScienCell), following standard cell culture procedures, and used between passages 2 and 7.

Techniques: Confocal Microscopy, Co-Culture Assay, Fluorescence, Quantitation Assay, Staining, Negative Control

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

doi: 10.1177/0271678X17720816

Figure Lengend Snippet: Pericytes share mitochondria with astrocytes in an occludin-mediated manner. (a) Live co-culture, 24 h post-plating, of astrocytes labeled with violet-BMQC (blue) and mitochondria-stained (TMRE in green) pericytes treated with anti-occludin siRNA (OCC−), negative-control siRNA (SCR), or non-treated (wild-type, WT). Thin white arrows exemplify TMRE-stained pericytes, while thick yellow arrows show pericyte mitochondria in the body of astrocytes (cyan signal). (b) Quantitation of TMRE intensities in astrocytes and pericytes in the same co-cultures shown in (a). Average ± SEM, n = 25 collected from three experiments, p vs. SCR. Only significant values are shown (c) Astrocytes treated with vehicle (Veh) or with endosulfan sulfate (ES, 4 h) to block their energetic metabolism. Middle image, surviving astrocytes exhibiting widened bodies and gross morphological alterations are exemplified by thin white arrows. Yellow arrowheads point to astrocytes that still retain their normal morphology. Right image: astrocytes treated with ES; however, isolated murine live brain capillaries (D shows a single brain capillary) were added to their growth medium 2 h post-treatment, and incubated for two additional hours. Note markedly improved astrocyte morphology. (e) Not-labeled human astrocytes cultured with murine live brain capillaries pre-labeled with TMRE (red) and 2-NBDG (green) for 2 h. TMRE and 2-NBDG transferred from microvessels to astrocytes (arrows) indicate transfer of mitochondria and glucose, respectively. (f) Similar TMRE and 2-NBDG transfer (arrows) in microvessel-rescued/ES-treated astrocytes after incubation with TMRE and 2-NBDG-labeled murine live brain capillaries.

Article Snippet: Cell culture Primary human brain capillary pericytes and astrocytes (ScienCell, Carlsbad, CA, USA) were cultured in 5% CO 2 at 37°C in pericyte or astrocyte growth medium (ScienCell), following standard cell culture procedures, and used between passages 2 and 7.

Techniques: Co-Culture Assay, Labeling, Staining, Negative Control, Quantitation Assay, Blocking Assay, Isolation, Incubation, Cell Culture

Journal: Molecular psychiatry

Article Title: A genome-wide association study of kynurenic acid in cerebrospinal fluid: implications for psychosis and cognitive impairment in bipolar disorder

doi: 10.1038/mp.2013.11

Figure Lengend Snippet: Stimulation of fetal human cortical astrocytes with interleukin-1β (IL-1β) and interferon-γ (IFN-γ). Fetal cultured human cortical astrocytes stimulated with recombinant human IL-1β (10 ng ml −1 ) and IFN-γ (200 ng ml −1 ), a prototypical indoleamine 2,3-dioxygenase 1 (IDO1) activator, as positive control. Protein levels of IDO1 and tryptophan 2,3-dioxygenase-2 (TDO2) analyzed at baseline and after exposure to IL-1β or IFN-γ for 48 h. IDO and TDO immunopositive bands are normalized to β-actin. Bar graphs here represent IDO1 and TDO2 protein levels expressed as % of vehicle-treated control cultures. All cells were analyzed for kynurenic acid (KYNA) at time points 1, 3 and 24 h. Bar graphs here represent % of vehicle ( t = 1 h) at these time points. All data are reported as mean ± s.e.m. All experiments were performed in triplicate and repeated twice. Representative western blots of IDO1 and TDO2 are shown below each bar graph (see for full size western blots). ( a ) Low levels of IDO1 protein expression were detected in unstimulated cells, but despite the marked increase of IDO1 mRNA levels following IL-1β stimulation, no changes in protein levels were observed after 48 h of IL-1β exposure (90 ± 5.13% vs 100 ± 7.85%, P = 0.41). Stimulation with IFN-γ was associated with a marked increase in protein levels of IDO1 (1084 ± 117% vs 100 ± 7.85%, P = 0.001). ( b ) Stimulation with IL-1β for 48 h increased protein levels of TDO2 compared with vehicle-treated cells (139 ± 11.1% vs 100 ± 2.70%, P = 0.006), whereas stimulation with IFN-γ did not affect protein levels of TDO2 (90 ± 11.2% vs 100 ± 8.70% P = 0.21). Immunofluorescent staining of IDO1 ( c ) protein visualized in green and TDO2 ( d ) protein visualized in red following 48 h of IL-1β stimulation confirmed low levels of IDO protein in most cells and scattered cells expressing high levels of TDO2. Images captured at × 40 magnification. Nuclear staining was performed using 4′,6-diamidino-2-phenylindole (DAPI). ( e ) At 24 h, cells stimulated with IFN-γ had more than 85 times higher KYNA concentrations (10.8 ± 0.34 nM) than vehicle-treated cells (0.14 ± 0.04 nM, P <0.0001). ( f ) At 24 h, cells stimulated with IL-1β showed 4 times higher KYNA concentrations (mean ± s.e.m.: 1.70 ± 0.11 nM) than vehicle-treated cells (0.42 ± 0.10 nM, p <0.0001). Two-sided P -values, statistical significance set to P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Human embryonic primary cortical astrocytes were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured according to the manufacturer’s recommendations.

Techniques: Cell Culture, Recombinant, Positive Control, Control, Western Blot, Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: Allopregnanolone Promotes Migration and Invasion of Human Glioblastoma Cells through the Protein Tyrosine Kinase c-Src Activation

doi: 10.3390/ijms23094996

Figure Lengend Snippet: AKR1C1-4 are differentially expressed in GB cells. The expression of AKR1C1-4 was detected in a total protein extract from normal human astrocytes (HA) and different human GB cell lines. ( a ) Representative Western blots of AKR1C1-4 and α-Tubulin, which was used as a loading control. ( b ) Densitometric analysis graph. Each column represents the mean ± SEM. n = 3; * p < 0.05 U251, T98G, and LN229 vs. HA and U87 cell lines.

Article Snippet: For Western blot determination of AKR1C1-4 (37 kDa), 20 μg of normal human primary astrocytes lysate (HA; 1806, ScienCell, Carlsbad, CA, USA), and 20 μg of protein lysate of the cell lines were mixed with Laemmli 2X buffer (100 mM Tris-base pH 6.8, 20% glycerol, 4% SDS, 10% β-mercaptoethanol, and bromophenol blue) were boiled for 5 min and separated in a 12% SDS-PAGE gels at 80 V. The separated proteins were then transferred to nitrocellulose membranes (Millipore, Burlington, MA, USA) by electrophoresis in semi-dry conditions at 30 mA per membrane for 2 h. Membranes were blocked in agitation at 37 °C with a blocking solution (TBS buffer-0.1% Tween with 5% bovine serum albumin; InVitro, MEX) for 2 h; then, membranes were incubated with a mouse monoclonal AKR1C1-4 antibody (1:1000; sc-390419, Santa Cruz, CA, USA) overnight.

Techniques: Expressing, Western Blot, Control

Journal: Cell reports

Article Title: Intrinsic antiviral immunity of barrier cells revealed by an iPSC-derived blood-brain barrier cellular model

doi: 10.1016/j.celrep.2022.110885

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human Astrocytes-midbrain , ScienCell , Cat#1850.

Techniques: Virus, Recombinant, Modification, Membrane, Knock-Out, Clinical Proteomics, Fluorescence, Plasmid Preparation, Western Blot, Mutagenesis, PCR Cloning, Cloning, Software, Cell Culture, Electroporation