Journal: STAR Protocols

Article Title: Protocol for Isolation of Golgi Vesicles from Human and Animal Hearts by Flotation through a Discontinuous Sucrose Gradient

doi: 10.1016/j.xpro.2020.100100

Figure Lengend Snippet: Analysis of Isolated Fractions (A) Western blot of Golgi (GM130 and MYLK4), nucleus (nucleolin and MYLK4), cytoplasmic (MYLK4), and mitochondrial (NDUFB9) markers in the three fractions collected after the sucrose gradient ultracentrifuge. F1, fraction 1 (top); F2, fraction 2 (half); F3, fraction 3 (bottom). (B) Visualization of isolated Golgi vesicles using transmission electron microscopy (white arrows). Numerous smooth vesicles are seen, mostly formed by a single membrane, apparently empty with little dense content, mainly a light network of granular material. The presence of mitochondrial remains is also observed (black arrow). Bar represents 500 nm.

Article Snippet: Nucleolin primary mouse monoclonal antibody , Merk Milipore , 05-565.

Techniques: Isolation, Western Blot, Transmission Assay, Electron Microscopy, Membrane

Journal: Frontiers in Immunology

Article Title: Adenovirus vector-mediated single chain variable fragments target the nucleocapsid protein of porcine epidemic diarrhea virus and protect against viral infection in piglets

doi: 10.3389/fimmu.2023.1058327

Figure Lengend Snippet: Jejunal expression of four scFvs. After rAdV-ZW, rAdV-wild-type or PBS oral administration, jejunums were collected on days 2 ( Aa , Ba ), 10 ( Ab , Bb ), 14 ( Ac , Bc ), and 18 ( Ad , Bd ). (A) Western blotting of scFv expression in jejunum tissue showing several bands at approximately 30-35 kDa. Lanes 1–3, jejunum tissue lysate from piglets administrated rAdV-ZW (lane 1), rAdV-wild-type (lane 2), and PBS (lane 3). (B) Immunofluorescence analysis showing scFv expression in jejunum tissue. Jejunum paraffin sections were incubated with a primary mouse monoclonal anti-HA antibody to detect HA-tagged scFvs, and then stained with DAPI to observe nuclei. Red fluorescence represents scFvs and blue represents nuclei. Scale bar = 100 μm. (C) Immunohistochemical jejunum tissue staining to analyze scFv expression. Brown staining indicates scFvs and blue represents hematoxylin-stained nuclei. Scale bar = 100 μm.

Article Snippet: After blocking in 5% skim milk, membranes were incubated with a primary mouse monoclonal anti-HA antibody (CMCTAG, Milwaukee, WI, USA) overnight at 4°C, and then incubated with a horseradish peroxidase (HRP)-conjugated Affinipure goat anti-mouse IgG (H + L) antibody (Jackson ImmunoResearch, West Grove, PA, USA) at 37°C for 1 h. Protein bands were revealed using Immobilon Western Chemiluminescent HRP Substrate (Merck-Novagen, Darmstadt, Germany).

Techniques: Expressing, Western Blot, Immunofluorescence, Incubation, Staining, Fluorescence, Immunohistochemical staining

Journal: Virulence

Article Title: Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

doi: 10.1080/21505594.2021.2006960

Figure Lengend Snippet: Replication synergy in co-infection of SARS-CoV-2 variants and high antiviral potency of TG against all variants . Confluent cells were primed with 0.5 µM TG or DMSO control for 30 min, washed twice with PBS, and infected with A variant, B variant, and D variant at 0.1 MOI in single virus infections (A, B and D) and in co-infections (AB, AD, and BD) for 1.5 h, washed twice with PBS and incubated in infection media. Notably, co-infected cells received the same amount of each virus as used in single virus infection. At 24 (panel a) and 72 hpi (panel a and b), total viral RNA from media was subjected to one-step reverse transcription qPCR, using primer set (1, 2) specific to all 3 variants, to quantify viral spike glycoprotein RNA by relative Ct method. Synergy in progeny production was evident in AB and AD co-infections where total virus RNA detected was greater than the sum of corresponding single-virus infection RNA evident at 24 and 72 hpi (panel a). Indicated significance relative to corresponding A/DMSO control based on 2-way ANOVA with Tukey’s multiple comparisons. Replication of all single virus- and co-infections was effectively blocked for at least 3 days by single pre-infection priming of TG. In AD co-infection, the most prolific infection group, combined viral RNA from TG-primed cells fell by 99.6% relative to corresponding DMSO control (panel b). Indicated significance relative to corresponding DMSO control based on 2-way ANOVA with Sidak’s multiple comparisons. Data shown are representative of three independent experiments and performed in quadruplicates

Article Snippet: Briefly, Vero E6 cells infected with a fixed volume of supernatant for 18 h were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed three times with PBS, followed by peroxidase treatment for 10 min and incubation with a 1:1000 dilution of primary mouse monoclonal antibody, specific to the viral spike glycoprotein (1A9 clone, GeneTex GTX632604), at room temperature for 1 h. The cells were then washed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labeled polymer for 1 h. After further washing with TBS, the cells were incubated with DAB substrate-chromogen solution for a few min (Envision+ system-HRP kit, Dako).

Techniques: Infection, Control, Variant Assay, Virus, Incubation, Reverse Transcription

Journal: Virulence

Article Title: Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

doi: 10.1080/21505594.2021.2006960

Figure Lengend Snippet: Replication comparisons of each variant in single virus- and co-infections . Confluent Calu-3 cells were primed with 0.5 µM TG or DMSO control for 30 min, washed twice with PBS, and infected with A variant, B variant, and D variant at 0.1 MOI in single variant infections (A, B and D) and in co-infections (AB, AD, and BD) for 1.5 h, washed twice with PBS and incubated in infection media. Co-infected cells received the same amount of each virus as in single variant infection. At 24, 48 and 72 hpi, viral RNA from media was subjected to one-step reverse transcription qPCR with variant-specific primers that can discriminate between variants in co-infected samples to detect relative spike glycoprotein gene expression. All single variant- and co-infections were blocked for at least 3 days by single pre-infection priming of TG (panels a to c). Production of A variant vRNA in AB and AD co-infections at 72 hpi was clearly enhanced (relative to single-virus A variant infection) (panel a). Production of B variant vRNA in BA and BD co-infections at 72 hpi was also enhanced (panel b). However, production of D variant vRNA in DA and DB co-infections at 72 hpi was attenuated relative to single-variant D variant infection; reduction of the D variant vRNA in DA was more than 2 times that in DB co-infection (panel c). Unless otherwise indicated significance relative to corresponding DMSO control based on 2-way ANOVA with Tukey’s multiple comparisons; % reduction is relative to corresponding DMSO group. Data shown are representative of three independent experiments and performed in quadruplicates

Article Snippet: Briefly, Vero E6 cells infected with a fixed volume of supernatant for 18 h were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed three times with PBS, followed by peroxidase treatment for 10 min and incubation with a 1:1000 dilution of primary mouse monoclonal antibody, specific to the viral spike glycoprotein (1A9 clone, GeneTex GTX632604), at room temperature for 1 h. The cells were then washed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labeled polymer for 1 h. After further washing with TBS, the cells were incubated with DAB substrate-chromogen solution for a few min (Envision+ system-HRP kit, Dako).

Techniques: Variant Assay, Virus, Control, Infection, Incubation, Reverse Transcription, Gene Expression

Journal: Virulence

Article Title: Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

doi: 10.1080/21505594.2021.2006960

Figure Lengend Snippet: Comparative vRNA production of emergent SARS-CoV-2 variants . Confluent Calu-3 cells were primed with 0.5 µM TG or DMSO control for 30 min, washed twice with PBS, and separately infected with A variant, B variant, and D variant at 0.1 MOI in for 1.5 h, washed twice with PBS and incubated in infection media. At 24, 48 and 72 hpi, viral RNA from media was subjected to one-step reverse transcription qPCR with variant-specific primer sets to detect relative spike glycoprotein RNA of each variant. Increase of virus output of each variant was determined between the periods of 24 to 48 hpi, and 24 to 72 hpi. Relative rates of progeny virus production of variants are such that D > A > B (panel a). Relative increase in virus output under TG inhibition (panel b) followed the same pattern as DMSO controls but was drastically reduced. Indicated significance of D is relative to B and A variants of each corresponding interval period based on 2-way ANOVA with Tukey’s multiple comparisons. Data shown are representative of three independent experiments and performed in quadruplicates

Article Snippet: Briefly, Vero E6 cells infected with a fixed volume of supernatant for 18 h were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed three times with PBS, followed by peroxidase treatment for 10 min and incubation with a 1:1000 dilution of primary mouse monoclonal antibody, specific to the viral spike glycoprotein (1A9 clone, GeneTex GTX632604), at room temperature for 1 h. The cells were then washed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labeled polymer for 1 h. After further washing with TBS, the cells were incubated with DAB substrate-chromogen solution for a few min (Envision+ system-HRP kit, Dako).

Techniques: Control, Infection, Variant Assay, Incubation, Reverse Transcription, Virus, Inhibition

Journal: Virulence

Article Title: Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

doi: 10.1080/21505594.2021.2006960

Figure Lengend Snippet: Replication rates of A, B and D variants in single variant- and co-infections . Data points, from earlier viral supernatant results of spike glycoprotein gene expression in single virus- and co-infections at 24, 48 and 72 hpi , were used to generate indicated growth curves and equations to determine the rate of viral RNA production (gradient) at a given time point. The D variant in single-variant infection, until saturation at 72 hpi, had the highest rate of viral RNA production relative to A and B variants in DMSO control (panel a) and TG-primed (panel b) Calu-3 cells. In co-infections, the D variant boosted the production rate of A and B variants (panel c and d) but at the expense of its initial performance (panel e). Negative values in production rate are likely due to virus saturation from limited cell number and even virus breakdown in media (panel a), or low initial virus growth (panel e). Data shown are representative of three independent experiments

Article Snippet: Briefly, Vero E6 cells infected with a fixed volume of supernatant for 18 h were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed three times with PBS, followed by peroxidase treatment for 10 min and incubation with a 1:1000 dilution of primary mouse monoclonal antibody, specific to the viral spike glycoprotein (1A9 clone, GeneTex GTX632604), at room temperature for 1 h. The cells were then washed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labeled polymer for 1 h. After further washing with TBS, the cells were incubated with DAB substrate-chromogen solution for a few min (Envision+ system-HRP kit, Dako).

Techniques: Variant Assay, Gene Expression, Virus, Infection, Control

Journal: Virulence

Article Title: Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

doi: 10.1080/21505594.2021.2006960

Figure Lengend Snippet: TG reduced vRNA synthesis and progeny production of Beta variant of SARS-CoV-2 in Calu-3 cells . Confluent cells were primed with 0.5 µM TG or DMSO control for 30 min, washed twice with PBS and infected with the B variant at 0.05 MOI in infection media for 3 h followed by three washes with PBS and incubated in fresh infection media. At indicated hpi, total cellular RNA was extracted (panel a); progeny virus in media was subjected to focus forming assay (FFA) (panel b) and viral RNA extraction (panel c). Total RNA was converted to cDNA for qPCR of SARS-CoV-2, normalized to 18s rRNA (panel a). FFA was based on the quantification of virus-positive Vero E6 cells at 18 hpi by immunochemical detection of viral spike glycoprotein (error bars = SEM; ns = not significant between time points) (panel b). Viral RNA from media was subjected to one-step reverse transcription qPCR to detect relative copy number of SARS-CoV-2, based on relative Ct method (panel c). Notably, despite increasing viral RNA accumulation in media of infected cells with time (panel c), viral gene expression (panel a) and infectious progeny virus (panel b) were in relative decline by 72 hpi. Indicated significance relative to corresponding DMSO control based on 2-way ANOVA with Sidak’s multiple comparisons. Indicated % refers to reduction in viral detection relative to corresponding DMSO control

Article Snippet: Briefly, Vero E6 cells infected with a fixed volume of supernatant for 18 h were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed three times with PBS, followed by peroxidase treatment for 10 min and incubation with a 1:1000 dilution of primary mouse monoclonal antibody, specific to the viral spike glycoprotein (1A9 clone, GeneTex GTX632604), at room temperature for 1 h. The cells were then washed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labeled polymer for 1 h. After further washing with TBS, the cells were incubated with DAB substrate-chromogen solution for a few min (Envision+ system-HRP kit, Dako).

Techniques: Variant Assay, Control, Infection, Incubation, Virus, Focus Forming Assay, RNA Extraction, Reverse Transcription, Gene Expression

Journal: Virulence

Article Title: Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

doi: 10.1080/21505594.2021.2006960

Figure Lengend Snippet: TG was effective in inhibiting emergent SARS-CoV-2 variants in pre-infected Calu-3 cells . Confluent cells were separately infected with SARS-CoV-2 variants at about 0.2 MOI for 2 h followed by three washes with PBS and incubated in fresh infection media. At 24 hpi, cultured media were removed and kept for re-seeding back to the same wells. Infected cells were then primed with 0.5 µM TG or DMSO control for 30 min, washed twice with PBS and the retained media were put back to the corresponding wells. At 48 and 72 hpi (i.e. at 24 h and 48 h post-priming with TG/DMSO respectively), viral RNA was extracted from spun supernatants for one-step reverse transcription qPCR to detect relative abundance of viral RNA that codes for spike glycoprotein (using primers 1 and 2). Notably, TG was able to inhibit preexisting active infection to great effect with a single 30 min exposure dose. Indicated significance relative to corresponding DMSO control based on 2-way ANOVA with Tukey’s multiple comparisons. Indicated % refers to reduction in viral detection relative to corresponding DMSO control

Article Snippet: Briefly, Vero E6 cells infected with a fixed volume of supernatant for 18 h were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed three times with PBS, followed by peroxidase treatment for 10 min and incubation with a 1:1000 dilution of primary mouse monoclonal antibody, specific to the viral spike glycoprotein (1A9 clone, GeneTex GTX632604), at room temperature for 1 h. The cells were then washed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labeled polymer for 1 h. After further washing with TBS, the cells were incubated with DAB substrate-chromogen solution for a few min (Envision+ system-HRP kit, Dako).

Techniques: Infection, Incubation, Cell Culture, Control, Reverse Transcription

Journal: Journal of Cellular and Molecular Medicine

Article Title: Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

doi: 10.1111/j.1582-4934.2010.01131.x

Figure Lengend Snippet: Sheep MSC were characterized using FACS and RT-PCR analysis. (A) With RT-PCR analysis CD29, CD44 and CD166 expression of MSC could be proofed on mRNA level. As indicated by increased CD45 expression, ratio of hematopoietic cells was higher in directly auto-transplanted MSC as compared to expanded MSC. (B–D) FACS analysis revealed sheep MSC to express CD29, CD44 and CD166. Expanded MSC (B) were negative for the hematopoietic markers CD31 and CD45. Directly auto-transplanted cells (C) had a different expression pattern than expanded MSC. The directly auto-transplanted MSC had a weaker CD29 and CD166 but a stronger CD45 expression. Mean fluorescent indices are shown in (D).

Article Snippet: CD31 staining: After antigen retrieval with pH 6 solution (Target Retrieval Solution; Dako Cytomation) in a pressure cooker for 10 min. (Pascal; Dako Cytomation) peroxidase block (CSAII-System; Dako Cytomation) was applied for 15 min., followed by incubation with 10% goat serum (PromoCell GmbH) in PBS (PBS-Dulbecco 1×, Biochrom AG) for 30 min. and protein block with the CSA II-System for 30 min. Then sections were incubated with the primary monoclonal mouse anti-ovine antibody CD31 (Anti-CD31/PECAM-1, MorphoSys UK Ltd., Kidlington, Oxford, UK) at 1:100 diluted in 10% goat serum in PBS for 1 hr.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

doi: 10.1111/j.1582-4934.2010.01131.x

Figure Lengend Snippet: For determination of the cell type which is qualified best for bone tissue engineering purposes, different groups (expanded versus directly auto-transplanted MSC, groups 8–10) were investigated. In both groups cells were DiI labelled prior to implantation and implanted subcutaneously with or without BMP-2. (A–C) Expanded MSC (A), directly auto-transplanted MSC (B), BMP-2 in combination with directly auto-transplanted MSC (C). DiI-labelled MSC (red) could be found close to β-TCP/HA granules contributing to the newly formed bone parts. In the explants with directly auto-transplanted MSC a higher section of the DiI-labelled cells were found in the connective tissue parts of the constructs compared to the explants with expanded MSC or directly auto-transplanted MSC with BMP-2. (D–F) Sections of constructs of the groups with expanded MSC (D), directly auto-transplanted MSC (E), BMP-2 in combination with directly auto-transplanted MSC (F) were evaluated for vascularization. The constructs in all three groups are well vascularized as shown by CD31 immunohistochemistry (green). Nuclei are counterstained with DAPI (blue).

Article Snippet: CD31 staining: After antigen retrieval with pH 6 solution (Target Retrieval Solution; Dako Cytomation) in a pressure cooker for 10 min. (Pascal; Dako Cytomation) peroxidase block (CSAII-System; Dako Cytomation) was applied for 15 min., followed by incubation with 10% goat serum (PromoCell GmbH) in PBS (PBS-Dulbecco 1×, Biochrom AG) for 30 min. and protein block with the CSA II-System for 30 min. Then sections were incubated with the primary monoclonal mouse anti-ovine antibody CD31 (Anti-CD31/PECAM-1, MorphoSys UK Ltd., Kidlington, Oxford, UK) at 1:100 diluted in 10% goat serum in PBS for 1 hr.

Techniques: Construct, Immunohistochemistry

Journal: Experimental neurology

Article Title: Impaired Social Behaviors and Minimized Oxytocin Signaling of the Adult Mice Deficient in the N-Methyl-D-Aspartate Receptor GluN3A Subunit

doi: 10.1016/j.expneurol.2018.02.015

Figure Lengend Snippet: Western blot analysis was performed on brain tissues from the hippocampus and thalamus areas of WT and GluN3A KO mice. A. Western blot images and quantification of the band intensity compared to the basal level. The protein level of oxytocin receptor, serotonin receptors 5-HTR2A, 5-HTR1B, TNFR1 and CD73 showed no statistically difference between WT and KO mice. The level of CD38, however, was significantly higher in the KO hippocampus. N=6, Student t test; * p<0.05, F=1.299. B. The above assay was repeated in the thalamus. All measured protein expressions were similar between WT and KO mice, expect the 5-HTR2A level was significantly lower in this brain region. N=6; Student t test; * p<0.05, F=5.650.

Article Snippet: The primary antibodies used and the dilutions for each were mouse anti-serotonin receptor (5-HTR; BD Bioscience, San Jose, CA, USA) at 1:2,500, rabbit anti-serotonin transporter (BD Bioscience) at 1:1,000, rabbit anti-oxytocin antibody (Fitzgerald Industries International, North Acton, MA, USA) at 1:1000, goat anti-oxytocin receptor antibody (Abcam, Cambridge, MA, USA) at 1:2,000, goat anti-CD73 antibody (Santa Cruz Biotech, Inc., Dallas, TX, USA) at 1:2,000, rabbit anti-BDNF antibody (Santa Cruz Biotech) at 1:2,000, mouse anti-TNFR1 antibody (Santa Cruz Biotech) at 1:2,000, rabbit anti-GluN3A antibody (Abcam) at 1:2,000, and mouse anti-actin (Sigma-Aldrich).

Techniques: Western Blot

Journal: Experimental neurology

Article Title: Impaired Social Behaviors and Minimized Oxytocin Signaling of the Adult Mice Deficient in the N-Methyl-D-Aspartate Receptor GluN3A Subunit

doi: 10.1016/j.expneurol.2018.02.015

Figure Lengend Snippet: We investigated the possibility that the deletion of GluN3A could altered social behavior-associated genes such as serotonin receptor (5-HTR), serotonin transporter (5-HTT), BDNF, CD73, and TNFR1. A. Western blotting of serotonin receptor, serotonin transporter, BDNF, CD73, and TNFR1 in the prefrontal cortex from WT and GluN3A KO mice. B-F. Quantification of the optic density (normalized to β-actin control) of 5-HTR, 5-HTT, BDNF, CD73, and TNFR1 in the prefrontal cortex. The expression of these genes was also unchanged in GluN3A KO mice. N=6 per group.

Article Snippet: The primary antibodies used and the dilutions for each were mouse anti-serotonin receptor (5-HTR; BD Bioscience, San Jose, CA, USA) at 1:2,500, rabbit anti-serotonin transporter (BD Bioscience) at 1:1,000, rabbit anti-oxytocin antibody (Fitzgerald Industries International, North Acton, MA, USA) at 1:1000, goat anti-oxytocin receptor antibody (Abcam, Cambridge, MA, USA) at 1:2,000, goat anti-CD73 antibody (Santa Cruz Biotech, Inc., Dallas, TX, USA) at 1:2,000, rabbit anti-BDNF antibody (Santa Cruz Biotech) at 1:2,000, mouse anti-TNFR1 antibody (Santa Cruz Biotech) at 1:2,000, rabbit anti-GluN3A antibody (Abcam) at 1:2,000, and mouse anti-actin (Sigma-Aldrich).

Techniques: Western Blot, Expressing