Journal: Frontiers in Endocrinology

Article Title: Adipose Tissue Myeloid-Lineage Neuroimmune Cells Express Genes Important for Neural Plasticity and Regulate Adipose Innervation

doi: 10.3389/fendo.2022.864925

Figure Lengend Snippet: LysMCre +/- ::BDNF -/- (KO) mice showed no difference in body composition, had a lower energy expenditure at the basal state, and exhibited decreased neurite density in scWAT upon cold exposure. (A) The body composition of adult (14 weeks old) male LysMCre +/- :: BDNF -/- (KO) and their littermate LysMCre -/- ::BDNF fl/fl (Con) controls was measured by echoMRI; N = 7 Con, N = 4 KO. (B) The skin surface temperature above the inguinal scWAT of adult (12–14 weeks old) Con and KO mice at the basal state was measured using a thermal camera (FLIR T560); N = 4 Con, N = 4 KO. The mean temperature in the region of interest (indicated by the black circle on the thermal images) for n = 3 images per animal was qualified in FLIR Research Studio Application software, plotted in GraphPad Prism software, and analyzed by Student’s t -test (two-tailed). (C) Adult (16 weeks old) male Con ( N = 3) and KO ( N = 3) mice were assessed in metabolic cages (CLAMS) at the basal state. The KO mice displayed a lower oxygen consumption (VO 2 , top left panel); decreased energy expenditure is represented as heat calculated from the measures of VO 2 and VCO 2 (bottom left panel), and there was a higher respiratory exchange ratio (bottom right panel) compared with the littermate controls. Waveform analysis of the metabolic cage measurements taken at 15-min increments for 48 h and represented over a 24-h period. Two-way ANOVA was performed, and main effects were reported; p ≤ 0.0001. Time of day is indicated on the x -axis. The animals were maintained on a 12-h light/dark cycle (the gray box indicates the dark cycle). (D–G) Adult (19–21 weeks old) Con ( N = 4) and KO ( N = 4) male mice were cold-exposed at 5°C for 7 days. The mice were single-caged in a diurnal incubator (12-h light/dark cycle). (D) The skin surface temperature above the inguinal scWAT of adult (12–14 weeks old) Con and KO mice was measured at day 7 of cold exposure and analyzed as described in (B) . (E) Food intake was measured nearly daily throughout the cold exposure intervention. The area under the curve for cumulative food intake was calculated and compared for Con and KO animals. (F) The body mass and tissue weights were measured, and adiposity was calculated. Data were analyzed by Student’s t -test (two-tailed). (G) Whole-mount immunostaining and imaging were performed on scWAT from cold-exposed Con and KO male mice. The tissue was stained with tyrosine hydroxylase for the assessment of sympathetic innervation. Imaging was performed on a confocal microscope equipped with WLL using an objective at ×10 magnification. The images are z-maximum intensity projections (with median blur filter and 4 kernel size applied) of merged tiled z-stack imaging of the entire scWAT depot. White circles represent the SiLN region (inset to the right of each image) for which the mean intensity was quantified (far right). All error bars are SEMs. *p-value < 0.05; n. s., not significant.

Article Snippet: Mouse BDNF PicoKineTM ELISA Kit (Boster Biological Technology, Pleasanton, CA, USA; catalog #EK0309) was used as per the manufacturer’s instruction to determine the amount of BDNF present in adipose protein lysates.

Techniques: Software, Two Tailed Test, Immunostaining, Imaging, Staining, Microscopy

Journal: Frontiers in Endocrinology

Article Title: Adipose Tissue Myeloid-Lineage Neuroimmune Cells Express Genes Important for Neural Plasticity and Regulate Adipose Innervation

doi: 10.3389/fendo.2022.864925

Figure Lengend Snippet: Cx3crCreER1 +/- ::BDNF -/- (KO) mice showed no difference in body composition and had a lower energy expenditure at the basal state and decreased neurite density in scWAT upon cold exposure. (A) The body composition of adult (14–16 weeks old) male Cx3cr1CreER +/- :: BDNF -/- (KO) and their littermate Cx3cr1CrERe -/- ::BDNF fl/fl (Con) controls was measured by echoMRI; N = 6 Con, N = 4 KO. (B) The skin surface temperature above the inguinal scWAT of adult (14 weeks old) Con and KO mice at the basal state was measured using a thermal camera (FLIR T560); N = 4 Con, N = 4 KO. The mean temperature in the region of interest (indicated by the black circle on the thermal images) for n = 3 images per animal was qualified in FLIR Research Studio Application software, plotted in GraphPad Prism software, and analyzed by Student’s t -test (two-tailed). (C) Adult (25–27 weeks old) male Con ( N = 3) and KO ( N = 3) mice were assessed in metabolic cages (CLAMS) at the basal state. The KO mice displayed higher oxygen consumption (VO 2 , top left panel) and carbon dioxide respiration (VCO 2, top right panel); greater energy expenditure was represented as heat calculated from measures of VO 2 and VCO 2 (bottom left panel) and had a significantly altered respiratory exchange ratio (bottom right panel) compared with Con animals. Waveform analysis of metabolic cage measurements taken at 15-min increments for 48 h and represented over a 24-h period. Two-way ANOVA was performed, and the main effects were reported; p ≤ 0.0001. Time of day is indicated on the x-axis. The animals were maintained on a 12-h light/dark cycle (the gray box indicates the dark cycle). (D) Adult (14–16 weeks old) Con ( N = 4) and KO ( N = 4) male mice were cold-exposed at 5°C for 7 days. The skin surface temperature above the inguinal scWAT of adult (14–16 weeks old) Con and KO mice was measured at day 7 of cold exposure and analyzed as described in (B) . (E) Adult (14–16 weeks old) Con ( N = 4) and KO ( N = 4) male mice were cold-exposed at 5°C for 7 days. Food intake was measured daily throughout the cold exposure intervention. The area under the curve for cumulative food intake was calculated and compared for Con and KO animals. (F) The body mass and tissue weights were measured, and adiposity was calculated for cold-exposed Con ( N = 4) and KO ( N = 4) mice. Data were analyzed by Student’s t -test (two-tailed). All cold-exposed mice were single-caged in a diurnal incubator (12-h light/dark cycle). (G) Whole-mount immunostaining and imaging were performed on scWAT from cold-exposed Con and KO male mice. Tissue was stained with tyrosine hydroxylase for the assessment of sympathetic innervation. Imaging was performed on a confocal microscope equipped with WLL using an objective at ×10 magnification. Images are z-maximum intensity projections (with median blur filter and 4 kernel size applied) of merged tiled z-stack imaging of the entire scWAT depot. White circles represent the SiLN region (inset to the right of each image) for which the mean intensity was quantified. All error bars are SEMs. *p-value < 0.05; **p-value < 0.01, ****p-value < 0.0001; n.s., not significant.

Article Snippet: Mouse BDNF PicoKineTM ELISA Kit (Boster Biological Technology, Pleasanton, CA, USA; catalog #EK0309) was used as per the manufacturer’s instruction to determine the amount of BDNF present in adipose protein lysates.

Techniques: Software, Two Tailed Test, Immunostaining, Imaging, Staining, Microscopy

Journal: Frontiers in Endocrinology

Article Title: Adipose Tissue Myeloid-Lineage Neuroimmune Cells Express Genes Important for Neural Plasticity and Regulate Adipose Innervation

doi: 10.3389/fendo.2022.864925

Figure Lengend Snippet: Cx3crCreER1 +/- ::BDNF -/- (KO) mice exhibited increased neurite density around the SiLN, decreased expression of CD11c+ adipose SVF cells, and increased expression of CD14+ adipose SVF cells compared with littermate floxed controls (Con). (A) Regional imaging (around SiLN) of whole-mount scWAT depots described in Figure 5G , where neurite sprouting was most pronounced. Images are representative of N = 3 per group. (B) Adult (15–17 weeks old) male Cx3cr1CrERe +/- :BDNF -/- (KO) and their littermate Cx3cr1CrERe -/- ::BDNF fl/fl (Con) mice were cold-exposed (5°C) for 7 days. Stromal vascular fraction from bilateral inguinal scWAT depots was isolated, and flow cytometry was performed. Percentage change of CD11b+ CD11c+ cells and CD11b+ CD14+ cells in Con and KO mice. Data were analyzed by two-tailed Student’s t -test; N = 3 per group. All error bars are SEMs. * p < 0.05, ** p < 0.01.

Article Snippet: Mouse BDNF PicoKineTM ELISA Kit (Boster Biological Technology, Pleasanton, CA, USA; catalog #EK0309) was used as per the manufacturer’s instruction to determine the amount of BDNF present in adipose protein lysates.

Techniques: Expressing, Imaging, Isolation, Flow Cytometry, Two Tailed Test

Journal: Cells

Article Title: Early Dietary Exposures Epigenetically Program Mammary Cancer Susceptibility through Igf1-Mediated Expansion of the Mammary Stem Cell Compartment

doi: 10.3390/cells11162558

Figure Lengend Snippet: The HT-A and HT-B dietary regimens increased mammary Igf1 levels and decreased Igfbp5 levels. ( A ) Mammary tissue Igf1 mRNA levels of 5-week-old ( n = 8/group) and 10-week-old ( n = 5/group) animals. Relative Igf1 mRNA levels are expressed as fold change over group LT-C; ( B ) Igf1 protein levels in 5-week-old and 10-week-old ( n = 6/group) mammary tissues. Top panel, representative Western blot, bottom panel, densitometric quantification; ( C ) Igfbp5 mRNA levels in 5-week-old and 10-week-old ( n = 4/group) mammary tissues. Relative mRNA levels are expressed as fold change over group HT-A; ( D ) Igfbp5 protein levels in 5-week-old and 10-week-old ( n = 6/group) mammary tissues. Top panel, representative Western blot, bottom panel, densitometric quantification. Circles in the bar graphs represent individual data points collected from independent experiments. Mean ± SEM is shown. One-way ANOVA was used for statistical analysis, and pairwise comparisons were performed using Tukey’s posttest. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Protein concentrations of Igf1 in CM were measured using the Mouse Igf1 ELISA Kit PicoKineTM (Boster Bio, cat #EK0378, Pleasanton, CA, USA), following the manufacture’s protocol.

Techniques: Western Blot

Journal: Cells

Article Title: Early Dietary Exposures Epigenetically Program Mammary Cancer Susceptibility through Igf1-Mediated Expansion of the Mammary Stem Cell Compartment

doi: 10.3390/cells11162558

Figure Lengend Snippet: The HT-A and HT-B dietary regimens increased Igf1 production in mammary stromal cells and promoted MaSC self-renewal. ( A ) Igf1 mRNA levels in FACS-sorted mammary stromal cells from 5-week-old and 10-week-old tissues from the HT and LT groups ( n = 4/group). Relative Igf1 mRNA levels are expressed as fold change over group LT-C; ( B ) Experimental procedure of the mammosphere assay using stromal cell-conditioned medium (CM); ( C ) Representative images and ( D ) bar graph showing numbers of mammospheres formed in CM produced by HT and LT groups. Unconditioned DMEM/Ham’s F-12 medium was used as a control; ( E ) Representative images and ( F ) bar graph showing numbers of mammospheres formed in LT-C-CM and LT-D-CM, supplemented with DMSO vehicle or 7.5 nM recombinant Igf1; ( G ) Representative images; ( H ) Bar graph showing numbers of mammospheres formed in HT-A-CM and HT-B-CM, supplemented with DMSO vehicle or 10 μM picropodophyllin (PPP). Scale bar of images = 100 µm. Four independent experiments ( n = 4) were performed, and circles in the bar graphs represent individual data points collected from independent experiments. Mean ± SEM is shown. One-way ANOVA was used for statistical analysis, and pairwise comparisons were performed using Tukey’s posttest. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Protein concentrations of Igf1 in CM were measured using the Mouse Igf1 ELISA Kit PicoKineTM (Boster Bio, cat #EK0378, Pleasanton, CA, USA), following the manufacture’s protocol.

Techniques: Produced, Control, Recombinant

Journal: Cells

Article Title: Early Dietary Exposures Epigenetically Program Mammary Cancer Susceptibility through Igf1-Mediated Expansion of the Mammary Stem Cell Compartment

doi: 10.3390/cells11162558

Figure Lengend Snippet: The HT-A and HT-B dietary regimens decreased DNA methylation of the Igf1 Pr1 promoter in mammary stromal cells, and Dnmt3b levels were also decreased. ( A ) Graphical representation of the mouse Igf1 promoters 1 (Pr1) and 2 (Pr2). Transcription start sites (TSSs, +1) are shown as arrows, CG sites are shown as vertical bars; ( B ) mRNA levels of Pr1 and Pr2 transcripts in mammary stromal cells from 5-week-old animals ( n = 4/group). Relative mRNA levels are expressed as fold change over group LT-C; ( C ) Representative Pr1 DNA methylation patterns from each group. Each circle represents a CG site; closed circles are methylated. Each row indicates sequencing results of a single clone; ( D ) Quantification of DNA methylation percentages for each CG site of the Pr1 promoter in mammary stromal cells from 5-week-old animals ( n = 4/group); ( E ) Scatter plot of Pr1 mRNA levels and combined Pr1 methylation percentages (Pearson’s correlation test, R 2 = 0.77, p < 0.0001, one-tailed); ( F ) mRNA levels of DNA methyltransferases in sorted mammary stromal cells from 5-week-old animals ( n = 4/group). Relative mRNA levels are expressed as fold change over group HT-A; ( G ) Dnmt3b protein levels in stromal cells. Top panel, representative Western blot, bottom panel, densitometric quantification ( n = 5/group). Circles in the bar graphs represent individual data points collected from independent experiments. Mean ± SEM is shown. One-way ANOVA was used for statistical analysis and pairwise comparisons were performed using Tukey’s posttest. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Protein concentrations of Igf1 in CM were measured using the Mouse Igf1 ELISA Kit PicoKineTM (Boster Bio, cat #EK0378, Pleasanton, CA, USA), following the manufacture’s protocol.

Techniques: DNA Methylation Assay, Methylation, Sequencing, One-tailed Test, Western Blot

Journal: Cells

Article Title: Early Dietary Exposures Epigenetically Program Mammary Cancer Susceptibility through Igf1-Mediated Expansion of the Mammary Stem Cell Compartment

doi: 10.3390/cells11162558

Figure Lengend Snippet: Dnmt3b knockdown decreased Igf1 Pr1 DNA methylation and increased Igf1 mRNA expression. ( A ) mRNA levels of Dnmts in 3T3 cells transfected with non-targeting siRNA or Dnmt3b -specific siRNA. Relative mRNA levels are expressed as fold change over non−targeting control. Four independent experiments ( n = 4) were performed; ( B ) Dnmt3b protein levels in non-targeting siRNA- and Dnmt3b siRNA−treated groups. Top panel, representative Western blot, bottom panel, densitometric quantification of four independent experiments; ( C ) Representative Igf1 Pr1 DNA methylation patterns (top panel) and quantification of methylated CG sites in four independent experiments (bottom panel); ( D ) Igf1 mRNA levels in transfected 3T3 cells. Four independent experiments ( n = 4) were performed, and circles in the bar graphs represent individual data points collected from independent experiments. Mean ± SEM is shown. Pairwise comparisons were performed using Student’s t -test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Protein concentrations of Igf1 in CM were measured using the Mouse Igf1 ELISA Kit PicoKineTM (Boster Bio, cat #EK0378, Pleasanton, CA, USA), following the manufacture’s protocol.

Techniques: Knockdown, DNA Methylation Assay, Expressing, Transfection, Control, Western Blot, Methylation

Journal: bioRxiv

Article Title: A Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO.IL-2Rγc KO. NOD) for COVID-19

doi: 10.1101/2020.08.19.251249

Figure Lengend Snippet: a. Positive control (+) = human lung mRNA. Negative control (−) = primers alone. upper row , PCR amplicons of hACE2 from the lungs of 4 HIS-DRAGA mice (2 females and 2 males) and 2 non-HIS-reconstituted DRAGA mice (1 female and 1 male), amplified using hACE2-specific primers. Lower row, PCR amplicons of the same lung samples in the upper row, amplified using mACE2-specific primers. b. hACE2 protein expression on alveolar epithelia as indicated by binding of the S1(RBD)-mFc□2a protein + goat anti-mouse IgG-FITC in lung sections from a representative HIS-DRAGA female mouse and two HIS-DRAGA male mice. Upper panels, merged images of S1(RBD)-mFc□2a binding (green) and nuclei (blue, DAPI), and an enlargement of the hACE2 + alveolar epithelia. Lower panels , binding of S1(RBD)-mFc□2a protein + goat anti-mouse IgG-FITC and an enlargement of the hACE2 + alveolar epithelia. c. Same staining protocol as in a for lung sections from two non-HIS reconstituted DRAGA mice showing very weak binding (female mouse) and no detectable binding (male mouse) of S1(RBD)-mFc◻2a protein, indicating siginificantly weaker avidity of the S1 viral protein for the murine than the human ACE2 receptor. d. lung section from a representative HIS-DRAGA female mouse showing hTMPRSS2 co-localization with hACE2 as revealed by S1(RBD)-mFc◻2a protein + goat anti-mouse IgG-FITC (green) and anti-human TMPRSS2-PE (red) on the epithelial wall of a bronchiole ( b ) and the endothelial wall of a pulmonary arteriole ( a ). A section of the bronchiole image is also shown enlarged 200X.

Article Snippet: The amounts of hACE2 protein in these immunoprecipitates were then quantified using the highly sensitive human ACE2 ELISA kit PicoKineTM (Boster Biological Technology, Pleasanton, CA) per the manufacturer’s protocol.

Techniques: Positive Control, Negative Control, Amplification, Expressing, Binding Assay, Staining

Journal: bioRxiv

Article Title: A Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO.IL-2Rγc KO. NOD) for COVID-19

doi: 10.1101/2020.08.19.251249

Figure Lengend Snippet: Human ACE2 levels in immunoprecipitates obtained from non-infected HIS-DRAGA and human lung lysates using S1(RBD)-mFc◻2a protein + rat anti-mouse IgG2a-magnetic beads were quantified by ELISA. Of note, the OD450nm values for protein immunoprecipitated from a pool of 10 non-infected, non-HIS-humanized DRAGA mouse lung lysates (negative control) fell below the limit of detection (OD450nm <0.05). Insert shows Western blot detection of hACE2 protein in the concentrated immunoprecipitates probed with a mouse monoclonal anti-human ACE2 IgG followed by goat anti-mouse IgG-HRP with ECL detection. Lane 1 , human lung immunoprecipitate; lane 2 , HIS-DRAGA mouse lungs immunoprecipitate; lane 3 , DRAGA mouse lungs immunoprecipitate (note this sample did not contain detectable hACE2). Lower panel shows the experimental conditions for immunoprecipitation of hACE2, quantification by ELISA, and the ratio of hACE2 in human versus HIS-DRAGA mouse lung samples.

Article Snippet: The amounts of hACE2 protein in these immunoprecipitates were then quantified using the highly sensitive human ACE2 ELISA kit PicoKineTM (Boster Biological Technology, Pleasanton, CA) per the manufacturer’s protocol.

Techniques: Infection, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Negative Control, Western Blot

Journal: bioRxiv

Article Title: A Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO.IL-2Rγc KO. NOD) for COVID-19

doi: 10.1101/2020.08.19.251249

Figure Lengend Snippet: a,b. Co-localization of hACE2 with alveolar hCD326 + ECs (orange) revealed by co-staining of S1(RBD)-mFc◻2a protein + goat anti-mouse IgG-FITC (green) and anti-hCD326-PE (red) in representative HIS-DRAGA female and male mice. c. Lack of hCD326 + ECs and negligible binding of S1(RBD)-mFc◻2a protein to a representative lung section from a non-HIS-reconstituted DRAGA female mouse.

Article Snippet: The amounts of hACE2 protein in these immunoprecipitates were then quantified using the highly sensitive human ACE2 ELISA kit PicoKineTM (Boster Biological Technology, Pleasanton, CA) per the manufacturer’s protocol.

Techniques: Staining, Binding Assay

Journal: bioRxiv

Article Title: A Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO.IL-2Rγc KO. NOD) for COVID-19

doi: 10.1101/2020.08.19.251249

Figure Lengend Snippet: a. Sections of renal cortex from HIS-DRAGA survivors #F1 (upper panels) and #F2 (lower panels) of SARS-CoV-2 infection with 2.8×10 4 pfu and 2.8×10 3 pfu, respectively, at the experimental endpoint (14 dpi). Sections were co-stained with DAPI (blue) and S1(RBD)-mFc◻2a protein + goat anti-mouse IgG-FITC (green). Shown is the S1(RBD) protein bound to the epithelium layer of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) surounding the glomeruli (G). Enlargement of a peripheral glomerular area shows nuclei (blue) of podocytes (p) and the endothelia (green) of glomerular microcapilaries (mc) in close proximity to the podocytes (green). b. Expression of hACE2 revealed by S1(RBD)-mFc◻2a binding (green) on kidney epithelial cells labeled with an anti-hCD31-AF594 antibody (red).

Article Snippet: The amounts of hACE2 protein in these immunoprecipitates were then quantified using the highly sensitive human ACE2 ELISA kit PicoKineTM (Boster Biological Technology, Pleasanton, CA) per the manufacturer’s protocol.

Techniques: Infection, Staining, Expressing, Binding Assay, Labeling

Journal: bioRxiv

Article Title: A Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO.IL-2Rγc KO. NOD) for COVID-19

doi: 10.1101/2020.08.19.251249

Figure Lengend Snippet: a. Merged images of tissue sections from a representative non-infected HIS-DRAGA mouse stained with mouse anti-hACE2 followed by goat anti-mouse IgG-FITC (green) and DAPI (blue). b. Minimal background binding of the secondary antibody (goat anti-mouse IgG-FITC) (left) overlapped with DAPI staining (right) of tissue sections adjacent to those shown in panel a .

Article Snippet: The amounts of hACE2 protein in these immunoprecipitates were then quantified using the highly sensitive human ACE2 ELISA kit PicoKineTM (Boster Biological Technology, Pleasanton, CA) per the manufacturer’s protocol.

Techniques: Infection, Staining, Binding Assay

Journal: bioRxiv

Article Title: A Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO.IL-2Rγc KO. NOD) for COVID-19

doi: 10.1101/2020.08.19.251249

Figure Lengend Snippet: Titers of hIgM and hIgG antibodies to S1(RBD) protein ( a ) S-trimer protein ( b ) and N protein ( c) in sera (diluted 1:20) from 8 HIS-DRAGA mice infected with SARS-CoV-2 (10 3 pfu), as measured by ELISA at 25 dpi. An anti-S1(RBD) antibody provided in the kit (Bethyl Laboratories) was included as a positive control in panels a and b (C+). The antibody titers against the N protein in serum from a non-infected mouse served as a negative control in panel c. OD450nm values were corrected by subtracting values (ranging from 0.045–0.067) of serum samples from the same mice prior to infection. Standard deviations (+/−SD) for each serum sample in duplicate wells were determined at 99% interval of confidence by SigmaPlot v.14 software.

Article Snippet: The amounts of hACE2 protein in these immunoprecipitates were then quantified using the highly sensitive human ACE2 ELISA kit PicoKineTM (Boster Biological Technology, Pleasanton, CA) per the manufacturer’s protocol.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control, Software

Journal: Chemico-biological interactions

Article Title: Signature of paraoxonases in the altered redox homeostasis in Alzheimer's disease.

doi: 10.1016/j.cbi.2023.110839

Figure Lengend Snippet: Fig. 4. PON1 (panel A) and PON2 (panel B) protein levels in transgenic (3xTg-AD) and non-transgenic (Non-Tg; i.e. controls) mice brain homogenate at different post-natal days (PD). Data are reported as mean ± S.E.M.; (n = 4/group). Statistical analysis was performed by 2-way ANOVA and subsequent Bonferroni post-hoc test for multiple comparisons. *p < 0.05, **p < 0.01.

Article Snippet: PON1 and PON2 proteins concentration in homogenized brains were determined by PicokineTM Mouse PON1 ELISA kit (Boster Biochem, Cambridge, MA, USA) and mouse PON2 ELISA kit (Abcam, Cambridge, UK) following the manufactured procedures.

Techniques: Transgenic Assay

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: XCL1 plasma levels rise after running and XCL1 treatment increases the number of neurospheres. ( a ) XCL1 plasma levels measured by an ELISA in standard-housed mice (STD, n = 5 mice) and mice housed for 4 days with a running wheel (RUN, n = 6 mice). * p < 0.05, Student’s t -test. ( b ) qPCR gene expression analysis of lymphotactin receptors reveals that neural precursor cells express Itga9 but not Xcr1 (left), although both Itga9 and Xcr1 are detected in splenic control tissue (right). Uncropped gels are presented in Supplementary Fig. . ( c ) Representative images of a SVZ neurosphere (top) and a DG neurosphere (bottom). Scale bars: 100 μm. ( d ) Neurosphere assays with DG-derived primary cells cultured with XCL1. n = 9 to 10 independent experiments, * p < 0.05, one-way ANOVA with Dunnett test. ( e ) Neurosphere assays with SVZ-derived primary cells cultured with XCL1. n = 6 to 9 independent experiments, ** p < 0.01, one-way ANOVA with Dunnett test. ( f ) Neurosphere assays with XCL1-neutralizing antibodies. n = 3 to 6 independent experiments, * p < 0.05, one-way ANOVA with Dunnett test. ( g ) Size distribution of DG-derived neurospheres cultured with XCL1. n = 7 to 8 independent experiments. Dashed lines represent control cultures normalized to 100%. All data represent the mean ± SEM.

Article Snippet: Protein levels were also measured using the mouse XCL1 PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Gene Expression, Control, Derivative Assay, Cell Culture

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: XCL1 promotes neuronal differentiation in adherent monolayer and neurosphere cultures. ( a ) Viability assay in adherent NPC cultures with XCL1. n = 5 to 6 independent experiments. ( b ) CFSE proliferation assay in adherent NPC cultures with XCL1. n = 3 independent experiments, ** p < 0.01, one-way ANOVA with Dunnett test. ( c ) Motility of adherent monolayer-cultured NPCs determined by semi-automated tracking. Data are plotted as the 5 th /95 th percentile with outliers represented as circles. XCL1: n = 92 cells, Control: n = 104 cells, *** p < 0.001, Student’s t -test. ( d ) Quantifica t ion of β-tubulin + cells in proliferating NPC cultures two days after the addition of XCL1. n = 4 independent experiments, * p < 0.05, one-way ANOVA with Dunnett test. ( e ) Representative image of differentiated NPCs in adherent monolayer cultures showing GFAP + astrocytes in green and β-tubulin + neurons in red. Scale bar: 50 μm. ( f ) Quantification of GFAP + and β-tubulin + cells in differentiated adherent monolayer cultures treated with XCL1. n = 4 to 5 independent experiments, * p < 0.05, *** p < 0.001, one-way ANOVA with Dunnett test. ( g ) Representative image of differentiated neurospheres showing GFAP + astrocytes in green and β-tubulin + neurons in red. Scale bar: 50 μm. ( h ) Quantification of GFAP + and β-tubulin + cells in differentiated neurosphere cultures treated with XCL1. n = 5 independent experiments, *** p < 0.001, one-way ANOVA with Dunnett test. Dashed lines represent control cultures normalized to 100%. All data represent the mean ± SEM.

Article Snippet: Protein levels were also measured using the mouse XCL1 PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Viability Assay, Proliferation Assay, Cell Culture, Control

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: XCL1 influences the cell cycle progression of NPCs in vitro . ( a ) Representative images of a dividing NPC followed by time-lapse microscopy. Images are 5 min apart. Yellow arrows mark the process of cell division. Scale bar: 10 μm. ( b ) Example of a generation tree of a re-dividing cell obtained from semi-automated cell tracking of NPCs to calculate the mean generation time. ( c ) Generation time of NPCs cultured with and without XCL1 determined by semi-automated cell tracking. Data are plotted as the 5 th /95 th percentile with outliers represented as circles. Control: n = 23 cells, XCL1: n = 26 cells. ( d ) Representative flow cytometry plots of the click-iT EdU proliferation assay. Viable cells were first defined using forward scatter and side scatter (left). Doublets were then excluded from single cell signals by plotting Hoechst-width against Hoechst-area (middle). Finally, to determine the cell cycle phase, the DNA content (Hoechst label) was plotted against the EdU signal (right). ( e ) Percentage of NPCs in S phase. n = 4 independent experiments, ** p < 0.01, one-way ANOVA with Dunnett test. ( f ) Percentage of NPCs in G2/M phases. n = 4 independent experiments. ( g ) Percentage of NPCs in G1/G0 phases. n = 4 independent experiments. All data represent the mean ± SEM.

Article Snippet: Protein levels were also measured using the mouse XCL1 PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: In Vitro, Time-lapse Microscopy, Cell Tracking Assay, Cell Culture, Control, Flow Cytometry, Proliferation Assay

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: Neurogenesis in XCL1 KO mice is reduced ex vivo . ( a ) Neurosphere assays with primary DG cells from XCL1 KO mice (−/−) and WT littermates (+/+). n = 6 mice per group, * p < 0.05, paired Student’s t -test. ( b ) Representative image of differentiated neurospheres showing GFAP + astrocytes in green and β-tubulin + neurons in red. Scale bar: 50 μm. ( c ) Quantification of GFAP + and β-tubulin + cells in differentiated neurosphere cultures from XCL1 −/− and +/+ mice. n = 4 mice per group, * p < 0.05, Student’s t -test. ( d ) and ( e ) Neurosphere assays with primary DG cells from XCL1 −/− and +/+ mice in the presence of ( d ) potassium chloride (n = 4 to 5 independent experiments) and ( e ) norepinephrine (n = 6 independent experiments). *** p < 0.001, **** p < 0.0001, Student’s t -test.

Article Snippet: Protein levels were also measured using the mouse XCL1 PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Ex Vivo