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: Cysteine redox state regulates human β2-adrenergic receptor binding and function

doi: 10.1038/s41598-020-59983-4

Figure Lengend Snippet: Agonism of β2AR on human lung airway epithelial cells stimulates ROS generation. ( A ) CALU3 cells were preloaded with the intracellular ROS-sensitive fluorescent probe CM-H 2 DCFDA (10 µM) and stimulated with 10 μM ISO for 5 min or as otherwise shown, prior to fixation and confocal imaging. ISO stimulation elicited robust fluorescence, indicative of ROS generation, at the cell membrane, and this effect was blocked by pretreatment with alprenolol (10 µM). The overall degree of fluorescence was equivalent to that seen upon exogenous treatment with 1 mM H 2 O 2 . The upper panel is 40X image and the lower panel depicts further zoom on a single cell from that image. ( B ) Fluorescent microscopy of cells that were pretreated with the NADPH oxidase inhibitors apocynin (APO; 100 µM) and diphenyleneiodonium chloride (DPI; 10 µM) reveals marked decreases in the fluorescent intensity ( B ) and total number of fluorescent cells ( C ) in APO and DPI treated states. Images in ( B ) represent a 20X objective image capture with the top image representing FITC fluorescence, the middle image representing the respective transilluminated image, and the lower image representing the overlay. ( D , E ) Agonism of CALU3 cells with 1 μM ISO for 5 or 15 minutes oxidized cysteine residues on the β2AR enabling selective dimedone alkylation of these sulfenic acid residues with 5 mM dimedone (15 minutes) as can be seen by either β2AR immunoprecipitation followed by anti-Cys-dimedone immunoblotting ( D ) or Cys-dimedone immunoprecipitation followed by β2AR immunoblotting ( E ). Full length immunoblots are shown in Supplementary Fig. .

Article Snippet: FITC conjugated anti-mouse antibody was from Rockland Immunochemicals (Limerick, PA).

Techniques: Imaging, Fluorescence, Membrane, Microscopy, Immunoprecipitation, Western Blot

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