Journal: Cells

Article Title: Avenanthramide-C as Alzheimer's Disease-Modifying Therapy: Early and Sustained Intervention Prevents Disease Progression in Mouse Models.

doi: 10.3390/cells14110826

Figure Lengend Snippet: Figure 2. Long-term Avn-C oral administration initiated at the early AD stage lowers Aβ1-42 production and prevents amyloid-mediated disease advancement in AD mouse models. (a) Western blot and quantitative analysis of BACE1, APP, and Aβ1-42 levels from the hippocampal lysate of WT, 5xFAD (vehicle and Avn-C group) mice (n = 4). A severe amyloid-mediated disease progression was observed from the vehicle 5xFAD group with increased APP, BACE1, and Aβ1-42 levels compared to the WT mice, and Avn-C lowers these protein expressions and halts amyloid progression in 5xFAD mice orally administered with Avn-C three months from early AD stage. (b) Fluorescent confocal z-stack image of Aβ1-42 (green), DAPI (blue) from Tg2576 vehicle and 3 months Avn-C treated mice hippocampal sections (2 sections/group) focused on CA3 region (scale bar 20 µm). (c) Quantitative analysis of Aβ42 fluorescent intensities are shown in the graph (n = 3), where Aβ1-42 levels at the CA3 hippocampal region were lowered by the Avn-C administered early at AD stage with three months of continuous treatment. In all panels, the error bar indicates S.E.M. Overall differences among groups were analyzed using the unpaired t-test two-tailed, one-way ANOVA (*** p < 0.001), followed by pairwise comparisons between groups using post hoc Tukey’s test. Statistical significance is indicated as *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Slides were mounted with ProLong Gold Antifade reagent with DAPI (Cell Signaling, Danvers, MA, USA).

Techniques: Western Blot, Biomarker Discovery, Two Tailed Test

Journal: Cells

Article Title: Avenanthramide-C as Alzheimer's Disease-Modifying Therapy: Early and Sustained Intervention Prevents Disease Progression in Mouse Models.

doi: 10.3390/cells14110826

Figure Lengend Snippet: Figure 5. Early AD-stage Avn-C administration with long-term treatment prevents chronic activation and reduces the cell body (soma) size of microglial cells in AD mouse models. (a–c) The confocal z-stack fluorescent image Iba1 (red) microglial marker and DAPI (blue) nuclei. Quantitively, cell area (soma, yellow arrow) and fluorescent intensity are measured from 5xFAD vehicle and 3 months Avn-C orally administered mice hippocampal sections (2 sections/mouse) (n = 4); for cell soma size from hippocampal sections, 6–7 cells/sections were analyzed (scale bar 20 µm). (d) Western blot and quantitative analysis of IBA1 microglial marker from WT, vehicle, and 3 months Avn-C-treated (5xFAD (n = 3) and Tg2576 (n = 4)) mice hippocampal lysate. Mean ± S.E.M.s. Overall differences among groups were analyzed using unpaired t-test two-tailed, one-way ANOVA (*** p < 0.001), and pairwise comparisons between groups were performed using post hoc Tukey’s test. Statistical significance is indicated as *** p < 0.001, ** p < 0.01, ns p > 0.05 (no significance).

Article Snippet: Slides were mounted with ProLong Gold Antifade reagent with DAPI (Cell Signaling, Danvers, MA, USA).

Techniques: Activation Assay, Marker, Western Blot, Two Tailed Test

Journal: Cells

Article Title: Avenanthramide-C as Alzheimer's Disease-Modifying Therapy: Early and Sustained Intervention Prevents Disease Progression in Mouse Models.

doi: 10.3390/cells14110826

Figure Lengend Snippet: Figure 6. Long-term oral administration of Avn-C from the early AD stage reduces large amyloid plaque deposition, strengthens the microglial cell barrier in hippocampal tissue, and protects and enhances microglial phagocytosis in BV2 cells. (a) The representative confocal z-stack fluorescent image DAPI (blue), Iba1 (green), and Aβ1-42 (red) focused on microglial recruitment and plaque deposition in the hippocampus proper and medial entorhinal cortex between vehicle and 3 months Avn-C oral administered hippocampal tissue sections (scale bar 200 µm). (b) Fluorescent confocal z-stack image from 5xFAD vehicle and three months Avn-C-administered mice hippocampal sections (50 µm, two sections per mouse) fluorescent tag with Iba1 (green) and Aβ42 (red) (scale bar 20 µm). (c) Quantification of the area covered by microglia over the plaque between vehicle and three months Avn-C orally administered mice from early AD stage from hippocampus proper. The ratio is calculated as (area of microglia)/(area of plaque) in the region of interest. Number of plaque analyzed 30/group (n = 4). (d) Confocal fluorescent image of DAPI (blue) and BV-2 microglial cells (green) pre-activated by oAβ1-42 (1.0 µM) for 30 min and continued treatment with oAβ1-42 (1.0 µM) and oAβ1-42 (1.0 µM) + Avn-C (50 µm) for different time points: 3, 6, and 12 h. Phagocytosis was observed from ingested fluorescent carboxylate microsphere (red) (scale bar 20 µm). (e) Quantification of phagocytosis from BV-2 cells treated with oAβ1-42 (1.0 µM) or oAβ1-42 (1.0 µM) + Avn-C (50 µm), analyzed based on the number of microspheres engulfed per cells from 3, 6, and 12 h time duration; 30 cells analyzed per condition. Mean ± S.E.M.s. Differences were analyzed using an unpaired t-test, two-tailed. Statistical significance is indicated as *** p < 0.001, ** p < 0.01.

Article Snippet: Slides were mounted with ProLong Gold Antifade reagent with DAPI (Cell Signaling, Danvers, MA, USA).

Techniques: Two Tailed Test

Journal: Circulation Research

Article Title: Zac1 Is an Essential Transcription Factor for Cardiac Morphogenesis

doi: 10.1161/circresaha.109.214130

Figure Lengend Snippet: Figure 6. Altered gene expression in Zac1-mutated hearts. A and B, Expression levels of Nkx2-5 and GATA4 are normal in the Zac1-mutated heart, as assessed by whole-mount in situ hybridization and quantitative RT-PCR analysis. C through E, Expression lev- els of ANF, MLC-2v, and MLC-2a are decreased in the Zac1-mutated heart, as assessed by whole-mount in situ hybridization and quantitative R T-PCR analysis. F, Representative histological sections from the wild-type and Zac1-mutated hearts at E13.5 stained in the TUNEL assay. The numbers of positive cells in 5 different hearts of each genotype are shown. G, Representative histological sec- tions from the wild-type and Zac1-mutated hearts at E13.5 stained with anti–phospho-histone H3 antibody. The numbers of positive cells in each 5 different hearts are shown.

Article Snippet: Antibodies directed against Zac1 (G-18; Santa Cruz Biotechnology, Santa Cruz, CA), actinin (EA-53; Sigma, St. Louis, MO), Lamin A/C (#2032, Cell Signaling Technology), Rho-GDI (610255, BD Biosciences), phospho-histone H3 (9071; Cell Signaling) and phalloidin (Molecular Probes, Eugene, OR) were added to the sections, followed by overnight incubation at 4°C.

Techniques: Gene Expression, Expressing, In Situ Hybridization, Quantitative RT-PCR, Staining, TUNEL Assay

Journal: Fluids and Barriers of the CNS

Article Title: A face-to-face comparison of claudin-5 transduced human brain endothelial (hCMEC/D3) cells with porcine brain endothelial cells as blood–brain barrier models for drug transport studies

doi: 10.1186/s12987-020-00212-5

Figure Lengend Snippet: Comparison of cell morphology and localization and expression of claudin-5 and Pgp in hCMEC/D3-WT, hCMEC/D3- Cldn5 -YFP and pBCECs. a Claudin-5 was indirectly stained (hCMEC/D3-WT and pBCECs) or visualized by YFP tag in the stably transduced hCMEC/D3- Cldn5 -YFP cell line (for better visualization both claudin-5 and Cldn5 -YFP are depicted in green). F-Actin is shown in red and cell nuclei are counterstained in blue by DAPI. b An xz scan of the cell layer revealed interendothelial (junctional) localization of claudin-5 (green) in the two cell lines and pBCECs. Scale bars: 10 µm. c Phase contrast micrographs of hCMEC/D3-WT, hCMEC/D3- Cldn5 -YFP and primary pBCEC cultures. d Purity of pBCEC isolation, evaluated by fluorescent staining for the endothelial cell marker CD31 (green). Cell nuclei were counterstained with DAPI (blue). e Length and width of hCMEC/D3 and pBCECs. f Representative Western blot showing claudin-5 (Cldn5) expression in hCMEC/D3-WT (7 days after seeding), hCMEC/D3- Cldn5 -YFP (7 and 21 days after seeding) and pBCEC cultures (7 days after seeding). β-actin was used as a loading control. g Quantification of Western blot bands and normalization of claudin-5 expression to β-actin. Data are represented as mean ± SEM of n = 3 independent experiments. Significant intergroup differences are indicated by asterisk (* P < 0.05). h Representative Western blot showing Pgp expression in hCMEC/D3-WT, hCMEC/D3- Cldn5 -YFP and pBCEC cultures. β-actin was used as a loading control. i Quantification of Western blot bands and normalization of Pgp expression to β-actin. Data are represented as mean ± SEM of n = 3 independent experiments. Significant intergroup differences are indicated by asterisk (* P < 0.05)

Article Snippet: The membranes were cut out of the inserts and mounted on glass slides in Prolong Gold antifade reagent with DAPI (Carl Roth, Karlsruhe, Germany).

Techniques: Comparison, Expressing, Staining, Stable Transfection, Isolation, Marker, Western Blot, Control