Journal: Arthritis Research & Therapy

Article Title: Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium

doi: 10.1186/s13075-022-02817-7

Figure Lengend Snippet: Nrp2 expression in activated macrophages during joint inflammation. A Nrp2 expression in immune cells in the CIA synovium. The hind paws of CIA-induced mice were digested and subjected to flow-cytometric analysis. The representative histograms of Nrp2 staining and the cumulative data are shown. Data are expressed as the means ± SD. N = 3 from three independent experiments. B The characteristics of Nrp2-high macrophages. The representative histograms of CD86 and MHC class II expression on joint-infiltrating macrophages (left) and their cumulative data (right) are shown. N = 6 from two independent experiments. C Nrp2 expression on BMMs. BMMs were cultured under the indicated conditions, and Nrp2 expression was determined by flow cytometry. The representative histograms and the cumulative data are shown. N = 3 from three independent experiments. D NRP2 expression in synovium-infiltrating cells in RA. The deposited single-cell RNA-seq data were reanalyzed, and several cell types were defined by UMAP. NRP2 expression is denoted by purple dots. The statistical analyses were performed using an unpaired t-test

Article Snippet: Anti-neuropilin2 (Nrp2) polyclonal antibodies conjugated with FITC were purchased from Alomone Labs (#ANR-062).

Techniques: Expressing, Staining, Cell Culture, Flow Cytometry, RNA Sequencing

Journal: Scientific Reports

Article Title: Cell release during perfusion reflects cold ischemic injury in rat livers

doi: 10.1038/s41598-020-57589-4

Figure Lengend Snippet: Release of structural liver cells into the perfusate after cold ischemia. ( a ) Percentages of presumed hepatocytes (left/purple), sinusoidal endothelial cells (middle/green), and stellate cells (right/blue) in the perfusate, relative to the total number of nucleated cells (TNCs) in the perfusate. The perfusate recirculated during 3 hours of subnormothermic machine perfusion. Stars denote statistical significance (two-way ANOVA, followed by Tukey’s post-hoc test): *0.01 < p < 0.05; **0.001 < p < 0.01; ***0.0001 < p < 0.001; ****p < 0.0001. Error bars: SEM. ( b ) Imaging flow cytometry for quantification of presumed hepatocytes (CD45−/SE1−/ASGR1+/OX62−), liver sinusoidal endothelial cells (LSECs) (CD45−/SE1+/ASGR1−/OX62−), and stellate cells (CD45−/CD105+/SE1−/CD14+). Left: fresh livers. Right: 24-h-CI livers. ( c ) Representative images of surface marker expression of hepatocytes (top), LSEC (middle), and stellate cells (below). Scale bars: 5 µm.

Article Snippet: The aliquot for panel 2 was additionally stained for the detection of stellate p and Kupffer p cells with Alexa 405-conjugated CD105 (1:87; Novus Biologicals; Cat# NB500-452AF405), FITC-conjugated SE1 (1:100; Novus Biologicals; Cat# NB110-68095F), and Cy3-conjugated CD14 (2:100; Bioss, Woburn, MA, USA; Cay# bs-1192R-Cy3) antibodies.

Techniques: Imaging, Flow Cytometry, Marker, Expressing

Journal: Scientific Reports

Article Title: Cell release during perfusion reflects cold ischemic injury in rat livers

doi: 10.1038/s41598-020-57589-4

Figure Lengend Snippet: Alterations in liver-resident immune cell release into the perfusate after cold ischemia. ( a ) Percentage of presumed Kupffer cells (left/orange), liver-resident natural killer cells (also known as pit cells) (middle/red), and dendritic cells (right/yellow) in the perfusate, relative to the total number of nucleated cells (TNCs) that are released into the perfusate from fresh (n = 4), 24-h-cold ischemic (CI) (n = 5), and 72-h-CI (n = 4) livers. The perfusate recirculated during 3 hours of subnormothermic machine perfusion. Stars denote statistical significance (two-way ANOVA, followed by Tukey’s post-hoc test): *0.01 < p < 0.05; **0.001 < p < 0.01; ***0.0001 < p < 0.001; ****p < 0.0001. Error bars: SEM. ( b ) Imaging flow cytometry for the quantification of presumed Kupffer cells (CD45+/CD105−/SE1−/CD14+) and pit cells (CD45+/NKRP1A+/CD3−/OX62−). Dendritic cells were manually selected from a CD45+/NKRP1A−/CD3−/OX62+ population (not shown). Left: fresh livers. Right: 24-h-CI livers. ( c ) Representative images of surface marker expression images of Kupffer cells (top), pit cells (middle), and dendritic cells (below). Scale bars: 5 µm.

Article Snippet: The aliquot for panel 2 was additionally stained for the detection of stellate p and Kupffer p cells with Alexa 405-conjugated CD105 (1:87; Novus Biologicals; Cat# NB500-452AF405), FITC-conjugated SE1 (1:100; Novus Biologicals; Cat# NB110-68095F), and Cy3-conjugated CD14 (2:100; Bioss, Woburn, MA, USA; Cay# bs-1192R-Cy3) antibodies.

Techniques: Imaging, Flow Cytometry, Marker, Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Coronary and Aortic Endothelial Function Affected by Feedback Between Adiponectin and Tumor Necrosis Factor α in Type 2 Diabetic Mice

doi: 10.1161/atvbaha.110.214700

Figure Lengend Snippet: Figure 4. Colocalization and regulation of adiponectin expres- sion in coronary microvessels. Dual fluorescence combining adi- ponectin with markers for endothelial cells (von Willebrand fac- tor [vWF], vascular smooth muscle [-actin], and fibroblast marker) with the use of specific primary antibodies followed by fluorescence-labeled secondary antibodies. A through C, Dual labeling of adiponectin (red) and vWF (green) in control mouse heart tissue. D through F, Dual labeling of adiponectin (red) and vWF (green) in Leprdb mouse heart tissue. G through I, Dual labeling of adiponectin (red) and vWF (green) in dbTNF/dbTNF

Article Snippet: Primary antibodies for adiponectin (goat polyclonal, R&D, AF1119), and endothelial cell marker, von Willebrand factor (rabbit polyclonal, Abcam), or smooth muscle -actin (rabbit polyclonal, Abcam) or fibroblast (rat monoclonal, Novus Biologicals) were used for sequential double immunofluorescence staining.

Techniques: Marker, Labeling, Control

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Detection of Pdcd1 expression and autophagy in Pdcd1-overexpressing H9c2 (H9c2/Pdcd1) cells and negative control (H9c2/mock) cells. Pdcd1-overexpressing cells were established by transfecting H9c2 cells with Pdcd1-encoding plasmid DNA. After 24 h of transfection, the Pdcd1 protein expression (red fluorescence) was determined through immunofluorescence analysis in H9c2/mock ( a ) or H9c2/Pdcd1 cells ( b ). Autophagy was detected through staining with DAL Green, an autolysosome detection reagent (green fluorescence), and by the expression of LC3B, an autophagy marker, using immunofluorescence analysis in H9c2/mock ( c , e ) and H9c2/Pdcd1 cells ( d , f ). Magnification = × 400. Scale bar = 100 μm

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Expressing, Negative Control, Plasmid Preparation, Transfection, Fluorescence, Immunofluorescence, Staining, Marker

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Effects of Pdcd1 overexpression on doxorubicin (DOX)-induced apoptosis and caspase-3/7 activation in H9c2 cells. a After the cells were incubated with 1 μM DOX for various time periods (1–24 h), apoptosis was evaluated through a luciferase assay indicating the increase in Annexin V-positive cells. b Activity of caspase-3/7, an executor enzyme of apoptosis, was assessed using a luciferase assay in cells incubated with 1 μM DOX for 8 h; the values were expressed as the percentage of DOX-untreated H9c2/mock control cells. c Apoptosis levels in response to various concentrations of DOX (0.03–1 μM). The apoptosis was determined after a 24 h incubation with DOX, using the Annexin V luciferase assay. Each value was expressed as a percentage of that in H9c2/mock control cells. d Nuclear morphological observations and quantification of the apoptotic cells stained with H33342 after a 24 h incubation with various concentrations of DOX (0.1–1 μM). The yellow arrows indicate a typical feature of apoptotic cells. Magnification = × 200. Scale bar = 50 μm. Data are presented as the mean ± SEM of three samples. * p < 0.05, ** p < 0.01 vs. the DOX-untreated mock (control) cells. ## p < 0.01 vs. the DOX-untreated Pdcd1 over-expressed cells. † p < 0.05, †† p < 0.01 vs. the corresponding DOX-treated mock cells

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Over Expression, Activation Assay, Incubation, Luciferase, Activity Assay, Control, Staining

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Expression of phosphorylated mTOR (p-mTOR) and mTOR proteins in H9c2/mock and H9c2/Pdcd1 cells under DOX-untreated basal condition. a The expression of p-mTOR and mTOR in both cell types were evaluated through western blotting. b Quantitative levels of p-mTOR/mTOR protein expression. Data are presented as the mean ± SEM of three samples. * p < 0.05 vs. the mock cells. c Immunofluorescence detection of p-mTOR expression in H9c2/mock and H9c2/Pdcd1 cells. Magnification = × 200. Scale bar = 50 μm

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Expression of Beclin-1, Atg5, and Atg3 proteins in H9c2/mock and H9c2/Pdcd1 cells under DOX-untreated basal condition. a The expression of Beclin-1, Atg5, and Atg3 in both cell types were evaluated using western blotting, with β-actin as the loading control. b – d Quantitative levels of protein expression. Data are presented as the mean ± SEM of three samples. * p < 0.05 vs. the mock cells

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Expressing, Western Blot, Control

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Expression levels of caspase-3, Bad, and Bax proteins in H9c2/mock and H9c2/Pdcd1 cells under DOX-untreated basal condition. a The expression of the caspase-3, Bad, and Bax proteins in both cell types were evaluated using western blotting, with β-actin as the loading control. b – d Quantitative levels of protein expression. Data are presented as the mean ± SEM of three samples

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Expressing, Western Blot, Control

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Changes in the basal levels of autophagy and apoptosis after incubation for 6 h with Rap, Baf, or their combination in H9c2/mock and H9c2/Pdcd1 cells under DOX-untreated basal condition. a Autophagy was evaluated using the autophagy LC3 HiBiT reporter assay. b Apoptosis was determined using a luciferase assay indicating increase in Annexin V-positive cells. Each value is expressed as a percentage relative to that in H9c2/mock control cells. Data are presented as the mean ± SEM of three samples. * p < 0.05, ** p < 0.01 vs. the control group in H9c2/mock cells; ## p < 0.01 vs. the control group in H9c2/Pdcd1 cells; † p < 0.05, †† p < 0.01 vs. the corresponding Rap-treated group

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Incubation, Reporter Assay, Luciferase, Control

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Effects of Pdcd1 overexpression on doxorubicin (DOX)-induced apoptosis in human cancer cell lines, K562 (human erythroleukemia cells) and MCF-7 (human breast cancer cells). a Caspase-3/7 activity in the control (mock) and Pdcd1-overexpressing (Pdcd1) K562 cells was measured using a luciferase assay after incubation with 1 μM DOX for 8 h. The values were expressed as a percentage relative to that in DOX-untreated mock cells. b , c The apoptosis was assessed after a 24 h incubation with various concentrations of DOX (0.03–1 μM) using the Annexin V luciferase assay in K562 ( b ) and MCF-7 cells ( c ). Each value was expressed as a percentage relative to that in DOX-untreated control (mock) cells. d Quantification of apoptotic cells after a 24 h incubation with various concentrations of DOX (0.1–1 μM) in the control (mock) and Pdcd1-overexpressing MCF-7 cells. Nuclear morphological observations were performed after staining with H33342. e Control (mock) and Pdcd1-overexpressing (Pdcd1) MCF-7 cells stained with H33342 after a 24 h incubation with 1 μM DOX. Yellow arrows indicate a typical feature of apoptotic cells. Magnification = × 200. Scale bar = 50 μm. Data are presented as the mean ± SEM of three samples. * p < 0.05, ** p < 0.01 vs. the DOX-untreated mock (control) cells; ## p < 0.01 vs. the DOX-untreated Pdcd1-overexpressing cells; † p < 0.05, †† p < 0.01 vs. the corresponding DOX-treated mock cells

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Over Expression, Activity Assay, Control, Luciferase, Incubation, Staining

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Schematic representation of the molecular mechanisms by which Pdcd1 overexpression plays a protective role against DOX-induced apoptosis and cell viability reduction in the cardiomyocyte H9c2 cells. Pdcd1-mediated signaling inhibits the expression of mTOR; and therefore, increases the expression of its downstream proteins, Atg3, Atg5, and Beclin-1, leading to the activation of autophagy, which inhibits the DOX-induced caspase activation and subsequent apoptosis. Therefore, Pdcd1 could be an effective molecule for cardioprotection from DOX-induced toxicity

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Over Expression, Expressing, Activation Assay