Journal: Cell

Article Title: Nuclear Pores Promote Lethal Prostate Cancer by Increasing POM121-Driven E2F1, MYC, and AR Nuclear Import

doi: 10.1016/j.cell.2018.07.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-karyopherin a2 (B-9) , Santa Cruz Biotechnology , SC-55538; RRID: AB_831493.

Techniques: Control, Electron Microscopy, Virus, Recombinant, Extraction, shRNA, Mutagenesis, Cloning, Negative Control, Plasmid Preparation, Binding Assay, Retroviral, Luciferase, Software

Journal: Frontiers in Immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: The depletion of basophil secretory leukoprotease inhibitor (SLPI) exacerbates IgE-mediated allergic responses. (A) The experimental protocol of IgE-mediated chronic allergic inflammation in 5-fluorouracil (5-FU)-treated Fcer1g −/− mice adaptively transferred with B6 and Slpi −/− DX5 + cells containing basophils from bone marrow (BM) cells. (B) The kinetics of the ear thickness after the antigen challenge are shown. (C) Ear specimens obtained 6 days after the antigen challenge were stained with HE. Data are representative of three separate experiments and are shown as the mean ± SD. n = 4–6. * P < 0.05, ** P < 0.01.

Article Snippet: On Day 12, c-kit − DX5 + cells were isolated as basophils using the MACS system with magnetic microbead-conjugated anti-DX5 antibody (Miltenyi Biotec).

Techniques: Staining

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Expression of fibrinolytic system components in 24 h and 7 days old venous thrombi. Panels A-F show the expression (red) of (A-B) tPA, (C-D) uPA, (E-F) PAI-1, (G-H) plasminogen (Pg), (I-J) α2AP, and (K-L) their quantification in 8-µm cryosections of 24 hours and 7 days thrombi as labeled. Red color (Alexa Fluor 555) represents each component as labeled and blue color shows 4′,6-diamidino-2-phenylindole–stained nuclei. For comparison, IVC from sham mice were immunostained and quantified for each antibody under the same color range in the histogram mode of Image Pro-Plus software. The total immunostained area (arbitrary units; AU/mm2) for each protein was measured in 500 μm (original magnification ×4) images of an IVC sample. (N = 4-5 per group, mean ± SEM). *P < .05, **P < .01, ***P < .001.

Article Snippet: Proteins and reagents Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Expressing, Labeling, Staining, Software

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP deficiency on early thrombus formation. (A) Representative IVCs in different groups as labeled. (B) Vein thrombus weight milligram/gram of body weight. Each symbol represents an animal used in each group (N = 33). The bar graph shows the statistical significance between different groups by one-way ANOVA and Newman-Keuls post hoc test. ****P < .0001; ***P < .001; ns, nonsignificant. (C-F) Formalin-fixed paraffin-embedded (5 µm) sections of IVC thrombi were stained with Martius scarlet blue kit (scale bar = 500 µm). Representative image of N = 5 for each group. (C) Thrombus formation in the IVC of α2AP+/+ mice 5 hours after ligation (500 µm). (D) The composition of 5-hour-old thrombi (fibrin, red blood cell [RBC], and leukocytes; black arrows, scale bar = 100 µm). The pink/yellow color represents fibrin and nuclei are darkly stained. This panel shows a magnified image of the area in the black box highlighted in panel C. (E-F) Martius scarlet blue-stained representative images of IVC in α2AP−/− mice 5 hours after IVC ligation. The black arrows indicate areas of minimum thrombosis.

Article Snippet: Proteins and reagents Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Labeling, Formalin-fixed Paraffin-Embedded, Staining, Ligation

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP deficiency on acute thrombus formation. (A-B) Representative IVCs in α2AP+/+ and α2AP−/− mice (A) 24 hours after IVC ligation and (B) their thrombus weights. Each symbol in the bar graph represents an animal used in each group (N = 12); ****P < .0001, Student t test. (C) Martius scarlet blue (scale bar = 500 µm) staining shows 24-hour thrombi rich in fibrin (pink color). (D) Magnified image (original magnification ×20; scale bar = 100 µm) of panel C showing a fibrin-rich (pink) thrombus area. (E) Minimum thrombus formation (black arrows) in α2AP−/− mice 24 hours after IVC ligation.

Article Snippet: Proteins and reagents Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Ligation, Staining

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP deficiency on chronic thrombus formation. (A) Martius scarlet blue-stained 7-day-old thrombi (scale bar = 500 µm). (B) Magnified images (original magnification ×20) of IVC thrombus showing lamellate fibrin-rich (pink) lines of Zahn. (C) Thrombi were not formed in α2AP−/− mice after 7 days of IVC ligation. Arrow indicates the minimum thrombus formation. (D) Martius scarlet blue stained 14 days old thrombi (scale bar = 500 µm) in α2AP+/+ mice. (E) Magnified image (original magnification ×20) of panel D showing a fibrin-rich (pink), porous thrombus. (F) Martius scarlet blue-stained IVC showing minimum thrombus in α2AP−/− mice. (G) Representative images of IVC thrombus formation in different groups as labeled including sham mice (7 days). (H) Vein thrombus weight milligram/gram of body weight. Each symbol represents an animal used in each group (N = 38). One-way ANOVA; ****P < .0001, **P < .01.

Article Snippet: Proteins and reagents Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Staining, Ligation, Labeling

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP supplementation/inhibition. (A) Representative images of IVC thrombus formation in α2AP−/− mice after 5 hours’ ligation after administration of α2AP or α2AP + α2AP-inactivating antibody (α2AP-I) as labeled. (B) Vein thrombus weight in these groups in milligram/gram of body weight as labeled. Each symbol represents a single experiment, N = 14. The horizontal red and blue lines in the graph show, for reference, the mean thrombus weight in α2AP+/+ and α2AP−/− mice, respectively. ****P < .0001; one-way ANOVA (among 4 groups).

Article Snippet: Proteins and reagents Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Inhibition, Ligation, Labeling

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP on fibrinolysis. (A) Plasma D-dimer levels in α2AP+/+ and α2AP−/− mice after 5 hours of IVC ligation. The mice were supplemented with 10 mg/kg human fibrinogen. *P < .05 (Student t test). (B) Vein thrombus weight milligram/gram of body weight in α2AP+/+ and α2AP−/− mice after 5 hours’ IVC ligation used for D-dimer measurements ***P < .001 (Student t test). (C) Representative images of IVC thrombus formation in EACA-treated α2AP−/− mice 5 hours after ligation as labeled. (D) Vein thrombus weight (milligram/gram of body weight) in α2AP−/− mice with or without EACA treatment. The horizontal red line in the graph shows the mean vein thrombus weight in α2AP+/+ mice for reference. *P < .05, one-way ANOVA (among 3 groups). (E) Vein thrombus weight (milligram/gram of body weight) in Pg−/− mice in comparison with α2AP+/+ mice. (N = 21) ***P < .001, Mann-Whitney test.

Article Snippet: Proteins and reagents Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Ligation, Labeling, MANN-WHITNEY

Journal: Frontiers in Genetics

Article Title: Identification of a Functional Non-coding Variant in the GABA A Receptor α2 Subunit of the C57BL/6J Mouse Reference Genome: Major Implications for Neuroscience Research

doi: 10.3389/fgene.2019.00188

Figure Lengend Snippet: Strain variation in Gabra2 expression. (A) Low expression of Gabra2 in B6J brain relative to other inbred strains and substrains. B6J demonstrated the lowest expression of Gabra2 in hippocampus (data set GN110, probe set 1421738_at) compared to 15 inbred strains, including the D2 strain, and a closely related B6 substrain (C57BL/6ByJ). (B) Gabra2 levels measured by RNAseq are reduced in 10 mesocorticolimbic regions relative to D2, indicating a global reduction in B6J. BLA = basolateral amygdala, BST = bed nucleus stria terminalis, DLS = dorsolateral striatum, DMS = dorsomedial striatum, ILC = infralimbic cortex, NAc = nucleus accumbens core, NAs = nucleus accumbens shell, PrL = prelimbic cortex, VCX = visual cortex, VTA = ventral tegmented area. All P s < 0.001 for within region contrasts between B6J and D2. (C) Approximate time line seperating C57BL/6 substrains. (D) Average Gabra2 expression level (measured by qPCR) in the cortex, hippocampus, and striatum of D2, C57BL/6 substrains, and congenic strains relative to B6J shown at left. The B6N lineage (seperated in 1951 at generation F32 from B6J) includes C57BL/6JByJ (B6ByJ), C57BL/6NCrl (B6NCrl), and C57BL/6NJ (B6NJ) and all showed higher expression of Gabra2 relative to B6J. Even the C57BL/6EiJ (B6EiJ) substrain that diverged most recently from C57BL/6J in 1976 showed higher brain expression of Gabra2 . The only substrain with low Gabra2 expression similar to C57BL/6J is the B6(Cg)- Tyr c-2J /J (B6C2J) congenic strain. This albino mutation was detected in 1970 but the original strain harboring the spontaneous mutation was backcrossed to B6J creating a congenic line. All statistical contrasts performed relative to B6J. (E) Derivation of BXD strain cohorts. Each cohort was derived by separate crossing and inbreeding of female B6J (black) and male D2 (gray) parental inbred strains. The first cohort of BXD strains (1 through 32) was derived in the late 1970s. Another set of strains (33 through 42) was produced in the early 1990s. The last cohort of BXD strains (43 to 100) was created in the early 2000 s. (F) Hippocampal differences in Gabra2 expression between earlier and later derived BXD strains. There was little difference in the expression of Gabra2 between BXD strains with the B6 or D2 allele in the first cohort. However, inheritance of the B6 allele in the later two cohorts was associated with a dramatic reduction in Gabra2 expression. This suggests the appearance of a mutant allele in the B6J line that occurred after the late 1970’s and prior to 1990. Data from GeneNetwork data set GN110, probe set 1421738_at. (G) Protein levels of GABRA2 were reduced in B6J relative to D2 using an unbiased shotgun proteomics approach. Relative expression of GABRA2 protein shown as normalized relative intensity for GABRA2 in B6J and D2 hippocampus. Eight different peptide sequences that matched GABRA2 were used to generate normalized relative intensity counts for each sample. Each sample is 3 pooled animals (males only). (H) Hippocampal protein levels of GABRA2 are reduced in B6J ( n = 3) compared to the closely related B6N ( n = 3) substrain and D2 ( n = 2) strain (Western blot analysis). The experiment was repeated twice and both B6J sample 1 and B6N sample 1 were run each time. Tissue from Gabra2 KO mice and their wild type control strain (WT) were kindly provided by Y. Blednov at The University of Texas. These mice were originally maintained on a mixed B6J and 129/SvEv background but were backcrossed twice to B6J upon arrival at The University of Texas, thus the WT control strain has low GABRA2 protein expression similar to B6J. ANOVA for effect of strain is significant [ F (2,5) = 103.9, p < 0.001]. Post hoc tests show B6J is different from both B6N and D2 ( p < 0.001) but B6N and D2 are not significantly different from each other ( p = 0.15). Significance defined as: # p < 0.1, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For chemiluminescence western blots, blots were incubated in primary antibody for GABRA2 (1:500 rabbit polyclonal, PhosphoSolutions, Cat #822-GA2CL) overnight at 4 ° C. Blots were washed three times (5 min each) with blocking buffer and incubated with horseradish-peroxidase conjugated antibody for 1 h at room temperature.

Techniques: Expressing, Mutagenesis, Derivative Assay, Produced, Western Blot, Control

Journal: Frontiers in Genetics

Article Title: Identification of a Functional Non-coding Variant in the GABA A Receptor α2 Subunit of the C57BL/6J Mouse Reference Genome: Major Implications for Neuroscience Research

doi: 10.3389/fgene.2019.00188

Figure Lengend Snippet: Identification of a B6J private variant in Gabra2 . (A) Position of variants near the Gabra2 gene locus. An intergenic SNP (yellow arrowhead) and a single nucleotide indel (red arrowhead) in the third intron were the only confirmed variants among B6J and other inbred mouse strains. Genotyping of strains with high (red) or low (blue) expression of Gabra2 revealed that the likely causal variant is the intronic indel (single base pair deletion in B6J). (B) Evidence for disruption of mRNA processing is shown as an accumulation of intronic reads in the affected intron three (designated with an asterisk) and downstream introns (four and five) in B6J (blue) compared to D2 (red). Normalized and binned read pileup of poly-A enriched striatal RNA-seq data from 10 to 11 B6 and D2 individuals, respectively, generated by Bottomly and colleagues and hosted for viewing and analysis at the GeneNetwork mirror of the UTHSC Genome Browser ( http://ucscbrowser.genenetwork.org/ ). Gabra2 gene model is shown above (5′ to 3′ orientation with dashed line representing introns and solid vertical bars representing exons). (C) Average hippocampal expression of Gabra2 exons and introns based on inheritance of B6J (B) and D2 (D) alleles in the BXD population. All coding exons and UTRs showed significantly higher expression associated with inheritance of the D allele. In contrast, introns 4, 5, and 6 showed significantly higher expression associated with inheritance of the B allele. The variant is located in intron 3. Introns 3, 6 (distal to the fifth coding exon), 7, and 8 are not significantly different based on expression of parental alleles. There was no evidence of a splicing defect resulting in loss of exons in B6J. GeneNetwork data set GN206, UMUTAffy Hippocampus Exon (Feb09) RMA was used for the analysis and includes 45 BXD strains from BXD33 and above. 15 strains inherited the B allele and 30 inherited the D allele at the Gabra2 locus. Significance was determined by a pair-wise comparison of B and D allele expression for each intron or exon using Student’s t -test. Significance defined as: # p < 0.1, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For chemiluminescence western blots, blots were incubated in primary antibody for GABRA2 (1:500 rabbit polyclonal, PhosphoSolutions, Cat #822-GA2CL) overnight at 4 ° C. Blots were washed three times (5 min each) with blocking buffer and incubated with horseradish-peroxidase conjugated antibody for 1 h at room temperature.

Techniques: Variant Assay, Expressing, Disruption, RNA Sequencing, Generated, Comparison

Journal: Frontiers in Genetics

Article Title: Identification of a Functional Non-coding Variant in the GABA A Receptor α2 Subunit of the C57BL/6J Mouse Reference Genome: Major Implications for Neuroscience Research

doi: 10.3389/fgene.2019.00188

Figure Lengend Snippet: Repair of the private deletion in B6J is sufficient to restore GABRA2 protein and mRNA levels. (A) Site of repair of the private deletion in B6J (green), guide RNA (red) and PAM site (blue) are shown. The exon is highlighted in yellow. (B) Protein levels of GABRA2 are also restored in the hippocampus (top panel) and cortex (bottom panel) of the original Gabra2 KI/KI founder mouse ( n = 1) relative to B6J ( n = 3) and are similar to that of B6NJ ( n = 3). Western blot shown to the left and adjusted density bar graph shown to the right for each region. (C) Hippocampal and cortical Gabra2 mRNA levels were restored in the original Gabra2 KI/KI founder after insertion of a single nucleotide relative to B6J. Expression of Gabra2 in the founder mouse was similar to that of other B6 substrains. Expression was measured via qPCR and shown relative to B6J. Males and females were combined for the statistical analysis ( n = 5 to 6 per strain). Note that CRISPR founders can often be mosaic for the introduced mutation such that some cells may have the mutation, some may not, and other cells may have different mutations. We include molecular validation in the founder in order to demonstrate that our engineering approach worked, and that mosaicism is not evident in brain tissue of the Gabra2 KI/KI founder mouse. Tissue from Gabra2 knockout mice (KO, predominately B6J background mixed with 129/SvEv, see section Materials and Methods) was kindly provided by Y. Blednov from The University of Texas and used as a negative control. (D) Hippocampal, striatal, and cortical Gabra2 mRNA levels were also restored in heterozygous Gabra2 KI/- and homozygous knockin Gabra2 KI/KI mice after insertion of a single nucleotide relative to homozygous Gabra2 B6J/B6J mice. Expression measured via qPCR and data were analyzed at the ΔCt level (relative to the expression of the control gene Cyclin D). There is a significant main effect of genotype [ F (2,32) = 47.1, p = 2.9e-10] and brain region [ F (2,32) = 27.89, p = 9.7e-08]. The interaction was not significant. Pairwise significance between genotypes and within brain region were determined using Tukey’s HSD test. Significance defined as: # p < 0.1, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For chemiluminescence western blots, blots were incubated in primary antibody for GABRA2 (1:500 rabbit polyclonal, PhosphoSolutions, Cat #822-GA2CL) overnight at 4 ° C. Blots were washed three times (5 min each) with blocking buffer and incubated with horseradish-peroxidase conjugated antibody for 1 h at room temperature.

Techniques: Western Blot, Expressing, CRISPR, Mutagenesis, Biomarker Discovery, Knock-Out, Negative Control, Knock-In, Control

Journal: Frontiers in Genetics

Article Title: Identification of a Functional Non-coding Variant in the GABA A Receptor α2 Subunit of the C57BL/6J Mouse Reference Genome: Major Implications for Neuroscience Research

doi: 10.3389/fgene.2019.00188

Figure Lengend Snippet: Expression of GABA-A receptor subunit mRNA. Expression generated using the Affymetrix Clariom D Assay (microarray platform). Only subunits with significant or suggestive ( p < 0.1) differential expression between B6J and KI Gabra2 genotypes are shown. Significance defined as: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Fold change is indicated by color intensity with yellow representing increased expression in Gabra2 B6J/B6J (B6J allele) mice relative to Gabra2 KI/KI (KI allele) mice. In contrast, blue represents decreased expression in B6J allele mice relative to KI allele mice. Alterations in the mRNA levels of several alpha subunits, including the major Gabra1 subunit, are observed in the cortex and striatum of B6J allele mice which harbor a naturally occuring intronic deletion resulting in decreased Gabra2 expression. The only alpha subunit with significant and higher levels of mRNA expression (fold change >1.3) in B6J allele mice is Gabra4 (striatum). Beta and gamma subunit mRNAs are also altered at a suggestive level in cortex and hippocampus. In the hippocampus, γ1 transcript expression is significantly reduced in mice with the B6J allele. In the striatum, β1-2 and γ1-γ2 transcripts are also significantly reduced in mice with the B6J allele of Gabra2 . In contrast, striatal levels of delta subunit mRNA is increased in mice with the B6J allele relative to those with the KI allele. These results suggest brain region specific alterations in the abundance of certain classes of GABA-A receptors associated with inheritance of the Gabra2 B6J allele. For example, in the cortex, α1β2-containing receptors may be increased while α2γ1-containing subunits may be decreased. In the hippocampus, α2 and α3-containing receptors including β1 or γ1-γ2 subunits may be decreased. In the striatum there is evidence that there may be a general decrease in receptors containing α1, α2, α3 or α5; and β1 or 2; and γ1 or γ2. In contrast, there may be an increase in receptors containing α4 and δ subunits in striatum.

Article Snippet: For chemiluminescence western blots, blots were incubated in primary antibody for GABRA2 (1:500 rabbit polyclonal, PhosphoSolutions, Cat #822-GA2CL) overnight at 4 ° C. Blots were washed three times (5 min each) with blocking buffer and incubated with horseradish-peroxidase conjugated antibody for 1 h at room temperature.

Techniques: Expressing, Generated, Microarray, Quantitative Proteomics

Journal:

Article Title: Echovirus 1 Endocytosis into Caveosomes Requires Lipid Rafts, Dynamin II, and Signaling Events V⃞

doi: 10.1091/mbc.E04-01-0070

Figure Lengend Snippet: Characterization of fluorescently labeled EV1 and its uptake into the cell. (A) SDS-PAGE analysis of AF-594-labeled EV1 by silver staining (left) and by UV exposure (right) indicates that the major capsid proteins were fluorescently labeled. (B) Fluorescent virus spots, present on the CV-1 cell surface after incubation of the cells with AF-EV1 (8 × 104 EV1 particles/cell) for 1 h at 4°C (top). A detail of a picture presenting the viral particles (see arrow) is shown below. Bars, 20 and 5 μm, respectively. (C) Fluorescently labeled EV1 colocalizes with α2β1 integrin and caveolin-1. The labeled virus (red) and the AF-488-labeled antibody against α2 integrin (green) were incubated with SAOS-α2β1 cells for 2 h at 37°C, and the fixed cells were stained for caveolin-1 (purple). Colocalization is shown in the merged image. Bar, 10 μm. (D) AF-EV1 entry into CV-1 cells at 3 min p.i. The fluorescently labeled virus was bound to the cells for 1 h at 4°C followed by incubation at 37°C under a real-time microscope. The figures illustrate accumulations of viral particles on the cell surface and their entry into the cell (Supplementary Video Figure 2D_v). The time elapsed from the beginning of recording (3 min p.i.) is noted in each frame. Bar, 5 μm. (E) Association of AF-EV1 with caveolin-1–containing structures at 18 min p.i. In AF-EV1–infected (red), caveolin-1-GFP (green)–transfected CV-1 cells, virus particles (indicated by a circle) were observed to associate with caveolin-1–containing structures (Supplementary Video Figure 2E_v). In addition, some viral particles were already associated with caveolin-1–containing structures at the beginning of the observation period (indicated by an arrow). The time elapsed from the beginning of recording (18 min p.i.) is noted in each frame. Bar, 5 μm.

Article Snippet: The following primary antibodies were used in the immunofluorescent staining: rabbit anti-EV1 antibody ( Marjomäki et al., 2002 ) to detect EV1 capsid proteins, rabbit anticaveolin-1 antibody (N-20; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anticaveolin-1 antibody (2234; Transduction Laboratories, Lexington, KY), anti-CD49b mAb recognizing the I domain of the α2 integrin subunit (Immunotech, Marseille, France), AF-488-labeled (Molecular Probes) mouse anti-α2 mAb (MCA2025, Serotec, Oxford, United Kingdom), mouse anti-p230 mAb for detecting the trans-Golgi network (Transduction Laboratories), mouse mAb against PDI (ID3; from S. Fuller, University of Oxford, Oxford, United Kingdom), rabbit antitransferrin antibody (Behring Institute, Marburg, Germany), goat antibody against syntaxin 17 ( Steegmaier et al., 1998 ), rabbit antiserum against cation-independent-mannose-6-phosphate receptor (CI-MPR; Marjomäki et al., 1990 ), and mouse mAb for CD63 (Zymed, South San Francisco, CA).

Techniques: Labeling, SDS Page, Silver Staining, Incubation, Staining, Microscopy, Infection, Transfection

Journal:

Article Title: Echovirus 1 Endocytosis into Caveosomes Requires Lipid Rafts, Dynamin II, and Signaling Events V⃞

doi: 10.1091/mbc.E04-01-0070

Figure Lengend Snippet: Expression of α2β1 integrin and caveolin-1, and synthesis of the infectious virus in CV-1 cells. (A) Mock-infected CV-1 cells, immunostained for cell-surface α2 integrin (red) and for caveolin-1 (green), expressed molecules on the cell surface (first row). In EV1-infected cells, the integrin (red) showed partial intracellular colocalization with caveolin-1 (green; second row) and EV1 (green; third row) at 2 h p.i. Bars, 20 μm. (B) Infectious EV1 particles were produced in CV-1 cells after 3 h p.i., as determined using the plaque titration assay. (C) The amount of EV1 capsid proteins (VP1, VP2/VP3, indicated by arrows) increased in the cells at 4 h p.i., when analyzed using anti-EV1 antibody in immunoblot analysis. The most immunoreactive VP1 band, seen in the first three lanes (0, 2, and 3 h p.i.), represents the inoculated virus particles.

Article Snippet: The following primary antibodies were used in the immunofluorescent staining: rabbit anti-EV1 antibody ( Marjomäki et al., 2002 ) to detect EV1 capsid proteins, rabbit anticaveolin-1 antibody (N-20; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anticaveolin-1 antibody (2234; Transduction Laboratories, Lexington, KY), anti-CD49b mAb recognizing the I domain of the α2 integrin subunit (Immunotech, Marseille, France), AF-488-labeled (Molecular Probes) mouse anti-α2 mAb (MCA2025, Serotec, Oxford, United Kingdom), mouse anti-p230 mAb for detecting the trans-Golgi network (Transduction Laboratories), mouse mAb against PDI (ID3; from S. Fuller, University of Oxford, Oxford, United Kingdom), rabbit antitransferrin antibody (Behring Institute, Marburg, Germany), goat antibody against syntaxin 17 ( Steegmaier et al., 1998 ), rabbit antiserum against cation-independent-mannose-6-phosphate receptor (CI-MPR; Marjomäki et al., 1990 ), and mouse mAb for CD63 (Zymed, South San Francisco, CA).

Techniques: Expressing, Infection, Produced, Titration, Western Blot

Journal: Cancer Cell

Article Title: Genetic and pharmacological modulation of DNA mismatch repair heterogeneous tumors promotes immune surveillance

doi: 10.1016/j.ccell.2022.12.003

Figure Lengend Snippet: Immune profiling of MMR heterogeneous tumors reveals the involvement of CD8 + and γδ T cells (A) MMR heterogeneous tumors (100% Mlh1 +/+ , 80% Mlh1 +/+ 20% Mlh1 −/− , 50% Mlh1 +/+ 50% Mlh1 −/− ) were analyzed 13 days after injection in immunocompetent BALB C mice. Immune cell infiltrate was evaluated by flow cytometry. Percentages of CD8 + T, CD4 + T, γδ T (γδ-TCR + ), T reg (CD4 + FoxP3 + CD25 + ), B (CD19 + ), natural killer cells (CD49b + ), and macrophages (F4/80 + ), were calculated normalizing the absolute number of each population with the viable CD45 + fraction. The total amount of CD45 + alive cells is reported. Data are represented as average % ± standard error of the mean. The experiment was performed once. Tumors with insufficient material for immunophenotypical characterization were excluded from the analyses; n = 10 for 100% Mlh1 +/+ group, n = 9 for 80% Mlh1 +/+ 20% Mlh1 −/− group, n = 6 for 50% Mlh1 +/+ 50% Mlh1 −/− group. Statistical significance for each mixed population compared to the 100% Mlh1 +/+ control group was calculated by one-way ANOVA: ∗ p < 0.05 ∗∗ p < 0.005 ∗∗∗ p < 0.0005; (B) Ex vivo reactivity assays; CT26 100% Mlh1 +/+ and 50% Mlh1 +/+ 50% Mlh1 −/− were injected in immunocompetent BALB C mice. On day 13, mice were sacrificed and blood was taken. PBMCs were isolated and cocultured with Mlh1 +/+ or Mlh1 −/− separately (ratio PBMCs to tumor cells of 1:1). PBMCs harvested from naive mice were used as controls. IFN-γ expression was analyzed by flow cytometry as marker of activation after 5 h of coculture. The analyses of IFN-γ + CD8 + T, IFN-γ + CD4 + T, IFN-γ + γδ T cells obtained from the coculture with Mlh1 +/+ cells or (C) Mlh1 −/− cells is reported. At least three biological replicates were performed for each of the immune cell populations. Each dot represents a single biological replicate. Only conditions in which at least two biological replicates were above the threshold of 1% of IFNγ + cells (after background subtraction) were considered specifically activated. Mean ± standard error of the mean is reported (D) CT26 50% Mlh1 −/− 50% Mlh1 +/+ cells were injected in immunocompetent BALB C mice treated with depleting antibody for CD8 + T cells or γδ T cells. Twelve mice per group were used. Untreated mice served as control. Depleting CD8 + T cell antibody was given according to the following schedule: 400 μg at the day of the injection, 200 μg at the day 2, and then every 3 days after injection. Depleting γδ T cell antibody was administered according to the following schedule: 400 μg 2 days and 1 day before the injection, 400 μg at days 3 and 6 after injection followed by 400 μg every 7 days. Tumors were measured twice a week and volumes are reported as average tumor volume (mm 3 ) ± standard error of the mean; Mann-Whitney statistical analyses was performed, ∗ ∗∗∗ p < 0.0001. (E) Single mice graphs of the experiment shown in Figure 5 D are reported. (F) gDNA was extracted from four immune escaped tumors per arm (randomly selected), and ddPCR analyses was performed to determine the Mlh1 +/+ Mlh1 −/− cell content. Two or three sampling for each tumor were analyzed. Day 0 indicates the day of tumor cell injection. Data are represented as average % of cells ± standard deviation for each tumor. The experiment was performed once. See also Figure S3 .

Article Snippet: Immune staining was performed using the following antibodies purchased by BD Biosciences: PE-Cy7 Rat anti-mouse CD45 (30-F11), BB700 anti-mouse CD3e (145-2C11), BV786 Rat anti-mouse CD4 (RM4-5), APC anti-mouse CD8a (53-6.7), BB515 Rat anti-human/mouse CD11b (M1/70), APCR-700 Rat anti-mouse CD25 (PC61), BV421 Hamster anti-mouse γδ T-Cell Receptor(GL3), PE-CF594 anti mouse FOXP3 (MF23), BV480 hamster anti-mouse CD49b (HMα2), BV605 Rat anti-mouse CD19 (1D3), APC-H7 LIVE/DEAD Fixable Viability Stain 780.

Techniques: Injection, Flow Cytometry, Ex Vivo, Isolation, Expressing, Marker, Activation Assay, MANN-WHITNEY, Sampling, Standard Deviation

Journal: Cancer Cell

Article Title: Genetic and pharmacological modulation of DNA mismatch repair heterogeneous tumors promotes immune surveillance

doi: 10.1016/j.ccell.2022.12.003

Figure Lengend Snippet:

Article Snippet: Immune staining was performed using the following antibodies purchased by BD Biosciences: PE-Cy7 Rat anti-mouse CD45 (30-F11), BB700 anti-mouse CD3e (145-2C11), BV786 Rat anti-mouse CD4 (RM4-5), APC anti-mouse CD8a (53-6.7), BB515 Rat anti-human/mouse CD11b (M1/70), APCR-700 Rat anti-mouse CD25 (PC61), BV421 Hamster anti-mouse γδ T-Cell Receptor(GL3), PE-CF594 anti mouse FOXP3 (MF23), BV480 hamster anti-mouse CD49b (HMα2), BV605 Rat anti-mouse CD19 (1D3), APC-H7 LIVE/DEAD Fixable Viability Stain 780.

Techniques: In Vivo, Recombinant, Purification, Staining, Sequencing, Detection Assay, Mutagenesis, Software

Journal: International Journal of Molecular Sciences

Article Title: Human Decidual CD1a + Dendritic Cells Undergo Functional Maturation Program Mediated by Gp96

doi: 10.3390/ijms24032278

Figure Lengend Snippet: Cytokeratin expression in the decidua of normal pregnancy ( A , D , G ), blighted ovum ( B , E , H ), and missed abortion ( C , F , I ). Immunohistological labeling ( A – F ) using mouse IgG1 anti-Cytokeratin mAbs is visualized in brown color using 3,3-diaminobenzidine. Details from the lined quadrangles (( A – C ), 100×) are shown at higher magnification (( D – I ) 400×). Black arrows show the sites of trophoblast invasion ( D – F ). Graphs show the number of Cytokeratin-positive cells scattered per mm 2 and the H score for Cytokeratin in the decidual stroma ( J ) and trophoblast cells ( K ) using immunohistology (NP—normal pregnancy; BO—blighted ovum; MA—missed abortion). Ten samples were analyzed per group. Levels of significance: * p = 0.016 and ** p = 0.0078.

Article Snippet: CD91 (PE) , A2MR-α2 (mouse IgG1) , BD Biosci. San Diego, CA, USA , 20 μL/10 6 cells.

Techniques: Expressing, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Human Decidual CD1a + Dendritic Cells Undergo Functional Maturation Program Mediated by Gp96

doi: 10.3390/ijms24032278

Figure Lengend Snippet: Ki-67 and Ki-67/cytokeratin co-expression in the decidua of normal pregnancy ( A , D ), blighted ovum ( B , E ), and missed abortion ( C , F ). Immunohistology ( A – C ) shows single labeling with mouse anti-Ki-67 mAb or mouse IgG1 (inserts) using indirect immunoperoxidase staining. Ki-67 + cells are stained brown (black arrows). Double immunofluorescent labeling of Ki-67 and Cytokeratin in NP ( D ), BO ( E ), and MA ( F ). Ki-67 fluoresces red, and Cytokeratin fluoresces green. The nuclei are stained blue by DAPI. Purple fluorescence visualizes the merge of red and blue color (white arrows), while green fluorescence (white arrowheads). The inserts represent isotype-matched control. Ten samples were in each group. Magnification: 400×.

Article Snippet: CD91 (PE) , A2MR-α2 (mouse IgG1) , BD Biosci. San Diego, CA, USA , 20 μL/10 6 cells.

Techniques: Expressing, Labeling, Immunoperoxidase Staining, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Human Decidual CD1a + Dendritic Cells Undergo Functional Maturation Program Mediated by Gp96

doi: 10.3390/ijms24032278

Figure Lengend Snippet: Specific binding of gp96 for CD91 and TLR4 on CD1a + dendritic cells from the suspension of early pregnancy decidual mononuclear cells. Dot plots show the gating of decidual CD1a + cells within the region (R) 1 (back gating for CD1a + events) and R2 gate (CD1a + events) with respect to isotype mouse IgG1 ( A ). The results of binding assays are shown as the percentages of CD91 ( B ) or TLR4 ( C ) expressing CD1a + cells calculated as the difference compared to isotype-matched controls after the treatment with gp96 at the indicated concentrations. Histograms illustrate the labeling of CD1a + cells with antibodies of interest (solid histogram curves) in relation to isotype-matched control (dashed histogram curves). Levels of statistical significance: ** p = 0.001 and * p = 0.03.

Article Snippet: CD91 (PE) , A2MR-α2 (mouse IgG1) , BD Biosci. San Diego, CA, USA , 20 μL/10 6 cells.

Techniques: Binding Assay, Expressing, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Human Decidual CD1a + Dendritic Cells Undergo Functional Maturation Program Mediated by Gp96

doi: 10.3390/ijms24032278

Figure Lengend Snippet: Specification of antibodies used.

Article Snippet: CD91 (PE) , A2MR-α2 (mouse IgG1) , BD Biosci. San Diego, CA, USA , 20 μL/10 6 cells.

Techniques: Concentration Assay