Journal: Virus Research

Article Title: Coronavirus nonstructural protein 1: Common and distinct functions in the regulation of host and viral gene expression

doi: 10.1016/j.virusres.2014.11.019

Figure Lengend Snippet: Analysis of the subcellular localization of TGEV nsp1 using confocal microscopy. HEK 293 cells, grown on 4-well Lab-Tek II chamber slides (Nalgene Nunc International), were transfected with a TGEV nsp1 expressing plasmid, pCAGGS-nsp1 (lower row) or an empty pCAGGS plasmid (mock; upper row). At 24 h post-transfection, the cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100. Subsequently, the cells were subjected to immunofluorescence analysis using a TGEV-nsp1 specific primary antibody, which was raised by immunizing rabbits with purified full-length C-terminally myc-tagged TGEV nsp1 protein, followed by Alexa Fluor 594-conjugated secondary antibody (Molecular Probes) and DAPI counterstaining (Cell Signaling Technology). Images were collected using a Zeiss LSM-510 META confocal laser-scanning microscope with a 100X oil immersion lens and processed with the LSM image browser (Zeiss) and ImageJ (NIH) software program. No signal was detected in the negative control (mock; upper row), confirming the specificity of the anti-TGEV nsp1 antibody.

Article Snippet: Subsequently, the cells were subjected to immunofluorescence analysis using a TGEV-nsp1 specific primary antibody, which was raised by immunizing rabbits with purified full-length C-terminally myc-tagged TGEV nsp1 protein, followed by Alexa Fluor 594-conjugated secondary antibody (Molecular Probes) and DAPI counterstaining (Cell Signaling Technology).

Techniques: Confocal Microscopy, Transfection, Expressing, Plasmid Preparation, Immunofluorescence, Purification, Laser-Scanning Microscopy, Software, Negative Control

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: Stem Cell Reports

Article Title: Long-term adherence of human brain cells in vitro is enhanced by charged amine-based plasma polymer coatings

doi: 10.1016/j.stemcr.2022.01.013

Figure Lengend Snippet: Neuronal cells detach from standard glass surfaces sooner than from tissue culture polystyrene (A) Schematic of the cell culture microenvironment and modifications tested to improve neuronal attachment. (B) Human neural progenitor cells (NPCs) were pre-matured in BrainPhys + supplements for 0–2 weeks, replated on test surfaces, and further matured for up to 27 weeks in BrainPhys + supplements before phenotypic analyses. Before replating on test surfaces, post-mitotic precursors were sorted for DAPI exclusion at 2 weeks (see Experimental procedures). (C and D) Live neuronal cultures under phase contrast at 4× and 20× magnification, respectively. Detachment was quantified from 4× magnification phase contrast images by tracing regions with no cells (yellow) and calculating their combined area as a fraction of the total field of view area for each well. (E) Detachment on standard glass across multiple coating solutions: poly- l -ornithine (PLO; n = 4), PLO-laminin (Lam) (n = 6), Matrigel (n = 2), 24 h treatment of laminin (n = 6), 4 h treatment of laminin (n = 2), uncoated (n = 6), and poly- l -lysine (PLL; n = 2); n replicates from 2 independent experiments, means ± SEMs represented. (F) Cell detachment on standard glass compared to plastic (TCPS) both with PLO-Lam. Means ± SEMs from n = 5 biologically independent experiments, 2–6 replicates per experiment. (G and H) Spontaneous multi-electrode array (MEA) recordings of hPSC-derived neuronal cultures showing network burst activity and synchrony over 15–17 weeks on TCPS plates pre-coated with PLO-Lam. Means ± SEMs data from 16 replicate wells, each well containing 16 electrodes.

Article Snippet: For most of the experiments, NPCs were differentiated into post-mitotic neuronal cultures for 2 weeks in NMM, then live cell (DAPI) sorted (BD FACSMelody) and replated at 1.58 × 10 5 –2.1 × 10 5 cells/cm on TCPS, coated glass coverslips, or standard glass coverslips, in NMM containing half-concentrations of growth factors ( , , and , detachment; , morphology; and , , and , electrophysiology).

Techniques: Cell Culture, Derivative Assay, Activity Assay

Journal: Stem Cell Reports

Article Title: Long-term adherence of human brain cells in vitro is enhanced by charged amine-based plasma polymer coatings

doi: 10.1016/j.stemcr.2022.01.013

Figure Lengend Snippet: DAP-treated glass surface supports human proliferative neuronal/glia progenitors, brain tumor cells, and post-mitotic mature neurons and astrocytes (A–D) Analysis of proliferative human brain cells on various substrates. Attachment determined by counting fixed nuclei (DAPI + ) over time. Means ± SEMs fold change in number of nuclei per well (normalized to day 0) calculated for 5–6 replicate wells/condition/time point (1 independent experiment). All of the surfaces were pre-coated with Matrigel. Proliferative human brain cells analyzed were (A) patient-derived glioblastoma tumor cells (GBMs), (B) hESC-derived glial precursor cells (GPCs), and (C) hESC-derived neural progenitor cells (NPCs). (D) Immunofluorescence staining of DNA (DAPI; blue) and cell soma (CellTracker, red) at latest time point (8 days for brain cells). See also Figure S3 . (E) Proliferation of non-neuronal human embryonic stem cells (H9, hESC) was measured as the fraction of area covered by colonies normalized to day 0 (n = 5–6 replicates from 1 independent experiment). (F) Immunofluorescence staining of DNA (DAPI; blue) and cell soma (CellTracker, red) at latest time point (5 days for stem cells). See also Figure S3 . (G and H) Cytotoxicity analysis of neuronal cultures. All of the surfaces were pre-coated with PLO-Lam. (G) Lactate dehydrogenase (LDH) relative concentrations measured in neuronal culture supernatant (n = 15 replicates from 5 independent experiments) to estimate cellular stress. (H) Without fixation, DNA marker DAPI is incorporated in the nuclei of dead or dying cells (DAPI + ), but not live cells (DAPI − ). Means ± SEMs proportions of live cells (DAPI − ). (I) Immunofluorescence staining of MAP2, GFAP, and synapsin at 10× magnification (top) and 40× magnification (bottom) in neuronal cultures after 9 weeks in maturation medium, on glass-DAP-LAM. (J) Live neuronal cultures on glass-DAP-LAM under phase contrast at 20× after 27 weeks in maturation medium. (K) FACS analysis of the proportion of neurons (Lv-synapsin-GFP) and astrocytes (Lv-GFAP-tdTomato) at 9 weeks in maturation medium on different surfaces. For (H) and (K), n = 3–5 replicates per condition from 2 independent experiments. p values calculated using Mann-Whitney tests, ns for p > 0.05.

Article Snippet: For most of the experiments, NPCs were differentiated into post-mitotic neuronal cultures for 2 weeks in NMM, then live cell (DAPI) sorted (BD FACSMelody) and replated at 1.58 × 10 5 –2.1 × 10 5 cells/cm on TCPS, coated glass coverslips, or standard glass coverslips, in NMM containing half-concentrations of growth factors ( , , and , detachment; , morphology; and , , and , electrophysiology).

Techniques: Derivative Assay, Immunofluorescence, Staining, Marker, MANN-WHITNEY