Journal: Journal of neuroscience research

Article Title: Polyomavirus JC Infection Inhibits Differentiation of Oligodendrocyte Progenitor Cells

doi: 10.1002/jnr.23135

Figure Lengend Snippet: Preparation of human neural progenitor cells (hNPC) and their differentiation into human oligodendrocyte progenitor cells (hOPC) and human oligodendrocytes (hOL). A: Schematic representation of the preparation of hNPC from human fetal brain tissue (hfB, embryonic age 16 weeks) based on protocols of Espinosa-Jeffrey at al. (2009), with slight modifications. After dissociation of brain tissue, cells were plated onto nontissue-culture-grade Petri dishes coated with anti-PSA-NCAM antibody. Unattached cells were cultured in neural stem cell medium (NSCM) either as two- or as three-dimensional hNPC cultures. The hNPC were then gradually switched mixed with glial defined medium plus growth factors (GDM+) to differentiate them into hOPC and then to glial defined medium minus growth factors (GDM) to differentiate them into hOL. B: The hNPC stage of cultures was ascertained by immunolabeling of hNPC plated on slides with antibodies specific to nestin, A2B5, βIII-tubulin, and GFAP. Nuclei were stained with DAPI. C: Immunolabeling of hOPC for expression of nestin, A2B5, βIII-tubulin, and GFAP. Nuclei were stained with DAPI. D: Immunolabeling of hOL cultures for expression of GalC, A2B5, βIII-tubulin, and GFAP. Nuclei were stained with DAPI. Average percentages of cells expressing specific markers were quantified in at least 15 fields of view (~700–800 cells total). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: After removal of meninges and dissociation of brain tissue in the presence of Tryple Express (Gibco, Grand Island, NY; catalog No. 12605) and DNase I (10 U/ml; Sigma, St. Louis, MO), cells were triturated through a fire-polished Pasteur pipette and plated onto nontissue-culture-grade Petri dishes coated with anti-PSA-NCAM antibody (Developmental Studies Hybridoma Bank, University of Iowa [DSHB]; 5A5) in neural stem cell medium (NSCM: Neurobasal medium; Invitrogen, Carlsbad, CA; catalog No. 10888-022), N2 supplement (Invitrogen; catalog No. 17502048), B27 supplement without vitamin A (Invitrogen; catalog No. 12587-010), GlutaMAX (Invitrogen), 10 ng/ml basic fibroblast growth factor (bFGF; Invitrogen), 10 ng/ml recombinant human epidermal growth factor (EGF; Invitrogen), penicillin/streptomycin, and fungizone.

Techniques: Cell Culture, Immunolabeling, Staining, Expressing

Journal: Frontiers in immunology

Article Title: Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

doi: 10.3389/fimmu.2023.1252384

Figure Lengend Snippet: FIGURE 2 PCSK9-Il1a-/-, PCSK9-Nlrp3-/-, and PCSK9-Il1b-/- mice show reduced levels of circulating cytokines. (A) Volcano plot of proteins regulated in Olink mouse exploratory panel comparing serum protein levels of mice fed a NC vs. PCSK9-WT mice. Red data points indicate the difference of the NPX mean between PCSK9-WT and NC for each protein. The dashed line intersecting the y-axis indicates the significance of p<0.05. (B) Shiny GO (19) (Version 0.77) enrichment analysis annotating the significantly regulated proteins to the Gene Ontology (GO) Biological Process (false detection rate (FDR) cutoff: 0.1). The top 10 regulated pathways are presented as a barplot with the colors indicating the -log10 FDR, with red as the highest and blue as the lowest. (C) Heatmap depicting the dynamics of proteins significantly regulated in Il1a-/- compared to PCSK9-WT. Data is presented as fold change of PCSK9-WT, PCSK9-Il1a-/-, PCSK9-Nlrp3-/-, and PCSK9-Il1b-/- to NC. (D) Venn Diagram representing serum proteins specifically regulated in only NC or PCSK9-Il1a-/- animals compared to PCSK9-WT. Shared regulated proteins are displayed at the intersection of both areas. (E) Bargraphs of serum IL-1a levels measured in the Olink 96 mouse exploratory panel. The dashed line indicates the limit of detection of the assay. Data is presented with the Olink NPX value as mean ± SD, *p<0.05. PCSK9-WT (n=5), PCSK9-Il1a-/- (n=6), PCSK9-Nlrp3-/- (n=5), and PCSK9-Il1b-/- (n=4).

Article Snippet: The primary antibodies hIL-1a (1:50, sc-271618, clone G10, Santa Cruz, USA) and IL1R1 Polyclonal Antibody (1:100, PA5-117479, Invitrogen, USA) were diluted in Duolink antibody dilution and stained overnight.

Techniques:

Journal: Frontiers in immunology

Article Title: Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

doi: 10.3389/fimmu.2023.1252384

Figure Lengend Snippet: FIGURE 3 Cell surface translocation of IL-1a is not influenced by NLRP3 and IL-1b in murine BMDC. (A) Schematic overview of the experimental setup. Bone marrow-derived dendritic cells (BMDCs) were replated after 7 days of differentiation and stimulated with 100 ng/ml ultrapure Lipopolysaccharide (upLPS) overnight. Afterward, cells were harvested, and fractions were isolated using a detergent-based method. (B) Representative immunoblot of the membrane and cytoplasmic fraction of BMDCs with or without upLPS stimulation. Protein levels of IL-1a, GAPDH (glycerinaldehyde-3- phosphate-dehydrogenase, cytoplasmatic marker), and NaK ATPase (sodium–potassium ATPase, membrane marker) are presented. (C) Densiometric quantification of IL-1a in cytoplasmic fraction normalized to GAPDH and IL-1a in membrane fraction normalized to NaK ATPase. (D) Representative flow cytometry scatter plot of staining for IL-1a on stimulated BMDCs after gating for viable, non-fixated (7AAD-) cells. Total IL-1a was measured on fixed cells. (E) Barplot depicting the percentage of total IL-1a positive monocytes (7AAD+) with or without upLPS stimulation. (F) Barplot depicting the percentage of csIL-1a positive monocytes (7AAD-) with or without upLPS stimulation. (G) Barplot representing the percentage of non-permeable monocytes with or without upLPS stimulation. (H) Representative immunoblot of membrane and cytosolic fraction from upLPS-stimulated BMDCs of PCSK9-WT, PCSK9-Il1a-/-, PCSK9-Nlrp3-/-, and PCSK9-Il1b-/- animals. Protein levels of IL-1a, GAPDH (cytoplasmatic marker), and NaK ATPase (membrane marker) are presented with corresponding densiometric quantification (I) of log-transformed IL-1a expression. All data are presented as mean ± SEM, *p<0.05.

Article Snippet: The primary antibodies hIL-1a (1:50, sc-271618, clone G10, Santa Cruz, USA) and IL1R1 Polyclonal Antibody (1:100, PA5-117479, Invitrogen, USA) were diluted in Duolink antibody dilution and stained overnight.

Techniques: Translocation Assay, Derivative Assay, Isolation, Western Blot, Membrane, Marker, Cytometry, Staining, Transformation Assay, Expressing

Journal: Frontiers in immunology

Article Title: Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

doi: 10.3389/fimmu.2023.1252384

Figure Lengend Snippet: FIGURE 5 Myristoylation regulates csIL-1a in murine bone marrow cells and human monocytes. (A) Barplot depicting the percentage of murine bone marrow cells with myristoylated proteins. Bone marrow cells were cultured and myristoylated proteins were labeled overnight. Data are presented as mean ± SEM of NC (n=3) and PCSK9-WT (n=3), *p<0.05. (B) Mean fluorescence intensity of human primary monocytes under culture conditions (con) or with overnight incubation of N-myristoyltransferase inhibitor IMP-1088 [1 µM]. Data are presented as mean ± SEM of three independent experiments. One-sided paired t-test was performed, *p<0.05. (C) Percentage of csIL-1a presenting monocytes stimulated with 100 ng/ml upLPS and 1 µM IMP-1088 as indicated. Data are presented as mean ± SEM of four independent experiments, *p<0.05.

Article Snippet: The primary antibodies hIL-1a (1:50, sc-271618, clone G10, Santa Cruz, USA) and IL1R1 Polyclonal Antibody (1:100, PA5-117479, Invitrogen, USA) were diluted in Duolink antibody dilution and stained overnight.

Techniques: Cell Culture, Labeling, Incubation

Journal: Frontiers in immunology

Article Title: Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

doi: 10.3389/fimmu.2023.1252384

Figure Lengend Snippet: FIGURE 4 IL-1a surface expression on human monocytes induces VCAM1 expression and leads to increased adhesion on endothelial cells. (A) Schematic principle of proximity ligation assay (PLA) and representative picture of PLA. Direct binding of csIL-1a to the Interleukin-1 receptor 1 (IL1R1) leads to a fluorescence signal detectable at 594 nm. Human umbilical vein endothelial cells (HUVECs) were treated with monocytes for 6h, stimulated with and without upLPS (100 ng/ml). Cells were imaged at a 40× magnification (scale bar 50µm). HUVECs and monocytes are presented in blue, IL-1a/IL1R1 PLA signal is visible as red dots. (B) Representative flow cytometry histogram of vascular cell adhesion molecule–1 (VCAM1) stained HUVECs after 4h treatment with upLPS-stimulated monocytes. 10 µg/ml neutralizing IL1R1 (nIL1R1) antibody was added 1h before HUVEC-monocyte co-incubation. (C) Bar graph depicting the percentage of VCAM1- positive HUVECs after treatment with unstimulated and upLPS-stimulated monocytes. HUVECs were incubated with and without nIL1R1 antibody (10 µg/ml) for 1h before co-incubation. Data are presented as mean ± SEM of seven independent experiments; *p< 0.05. (D): Schematic experimental setup of monocyte adhesion assay. Primary monocytes were treated as indicated, labeled with Calcein and 4x washings. HUVECs were treated with and without 1 µg/ml neutralizing IL-1a antibody (nIL-1a) 1h before co-incubation. Then, HUVECs were treated with labeled monocytes for 4h. The initial fluorescence of adhering monocytes was measured as well as after two washes. Cells were imaged (4× magnification), scalebar 100 µM. (E) Quantification of adhering monocytes to HUVECs presented as mean ± SEM of four independent experiments. Repeated measure ANOVA was performed, followed by Sidak’s multiple comparison test (*p< 0.05).

Article Snippet: The primary antibodies hIL-1a (1:50, sc-271618, clone G10, Santa Cruz, USA) and IL1R1 Polyclonal Antibody (1:100, PA5-117479, Invitrogen, USA) were diluted in Duolink antibody dilution and stained overnight.

Techniques: Expressing, Proximity Ligation Assay, Binding Assay, Cytometry, Staining, Incubation, Cell Adhesion Assay, Labeling, Comparison

Journal: Acta Neuropathologica

Article Title: Xenografted human iPSC-derived neurons with the familial Alzheimer’s disease APP V717I mutation reveal dysregulated transcriptome signatures linked to synaptic function and implicate LINGO2 as a disease signaling mediator

doi: 10.1007/s00401-024-02755-5

Figure Lengend Snippet: Histology of APP V717I and Ctrl grafts 2 months after cell injection into the cortex and striatum of adult mice. a Schematic illustration of NPC transplantation into the brain of an immunocompromised adult mouse. Post-transplantation analysis, including histology and snRNA-seq, was conducted after 8 weeks of grafting. b H&E staining and volume quantification of mouse brain slices with human grafts. Human cells appear lighter compared to mouse cells. Graft sizes were comparable between APP V717I and Ctrl. c and d Quantification of neurons ( c ) and astrocytes ( d ) in APP V717I and Ctrl grafts. Around 90% human cells differentiated into neurons and 10% of cells differentiated into astrocytes in both groups. e Immunostainings of Ctrl and APP V717I grafts for neurons using antibodies for NeuN and human nuclear antigen (HNA) with DAPI nuclear staining. f DAB stainings for NeuN highlighting neurons within Ctrl and APP V717I grafts. g and h DAB stainings for the neural cell adhesion molecule NCAM using a human-specific antibody (hNCAM) highlighting neurons in the center ( g ) and periphery ( h ) of Ctrl and APP V717I grafts. i Immunostainings of Ctrl and APP V717I grafts for astrocytes using an antibody for human glial fibrillary acidic protein (hGFAP) with DAPI nuclear staining.

Article Snippet: Primary antibodies used in this study include: HNA (human nuclear antigen; MilliporeSigma; MAB1281; 1:200), NeuN (Abcam; ab104225; 1:500); LINGO2 (Thermo Fisher; PA5-99869; 1:200); hNCAM (human-specific NCAM; ERIC1; Santa Cruz; sc-106; 1:200); hGFAP (human-specific GFAP; Takara Bio; STEM123; 1:500), GFAP (MilliporeSigma; MAB360; 1:500), IBA1 (Wako; 1919741; 1:500), and SPP1 (R&D; AF808SP; 1:50).

Techniques: Injection, Transplantation Assay, Staining

Journal: Acta Neuropathologica

Article Title: Xenografted human iPSC-derived neurons with the familial Alzheimer’s disease APP V717I mutation reveal dysregulated transcriptome signatures linked to synaptic function and implicate LINGO2 as a disease signaling mediator

doi: 10.1007/s00401-024-02755-5

Figure Lengend Snippet: Comparative single nucleus RNA-seq analysis of grafted and human postmortem APP V717I neurons captures overlapping transcriptome signatures. a Venn diagram of upregulated DEGs derived from snRNA-seq of human postmortem AD versus Ctrl neurons, AD GWAS hits (GWAS Catalog; EMBL-EBI), snRNA-seq of transplanted human APP V717I versus Ctrl neurons, and cultured APP V717I versus Ctrl neurons. Red circle indicates DEGs overlapping between grafted APP V717I neurons and human AD brains/GWAS. Blue circle indicates DEGs overlapping between in vitro, graft and human AD brains/GWAS. b Pathway analysis of DEGs overlapping between all 4 groups (red circle in a ). Grafted APP V717I neurons show preservation of dysfunctional pathways of AD brains that are related to synaptic function. The x -axis shows the gene ratio. The adjusted p value and number of genes are also denoted by color and by size, respectively. c Western blot images with quantification after micro-dissection of APP V717I and Ctrl grafts showing reduced expression of PSD95 and hNCAM in APP V717I grafts. d Violin plots showing expression levels of indicated genes in grafted APP V717I and Ctrl neurons. e RTqPCR validation of the expression of selected DEGs in cultured APP V717I versus Ctrl neurons. f and g Immunostainings ( f ) with quantification ( g ) for the validation of increased expression of LINGO2 and RBFOX1 in grafted human APP V717I cells. Student t -test was used in c , e and g , * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Primary antibodies used in this study include: HNA (human nuclear antigen; MilliporeSigma; MAB1281; 1:200), NeuN (Abcam; ab104225; 1:500); LINGO2 (Thermo Fisher; PA5-99869; 1:200); hNCAM (human-specific NCAM; ERIC1; Santa Cruz; sc-106; 1:200); hGFAP (human-specific GFAP; Takara Bio; STEM123; 1:500), GFAP (MilliporeSigma; MAB360; 1:500), IBA1 (Wako; 1919741; 1:500), and SPP1 (R&D; AF808SP; 1:50).

Techniques: RNA Sequencing, Derivative Assay, Cell Culture, In Vitro, Preserving, Western Blot, Dissection, Expressing, Biomarker Discovery

Journal: BMC Genomics

Article Title: Comparative proteomic analysis of malformed umbilical cords from somatic cell nuclear transfer-derived piglets: implications for early postnatal death

doi: 10.1186/1471-2164-10-511

Figure Lengend Snippet: Immunohistochemical analysis of ECs derived from control (a), scNT-N (b), and scNT-MUC (c) . Cells were cultured in vitro and then stained using an anti-CD31 (PECAM-1, red) antibody, followed by counterstaining of nuclear DNA with DAPI (blue). Insets show individual cells.

Article Snippet: The purity of each PUVEC population was confirmed by immunostaining with an anti-PECAM-1 (CD31) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and counterstaining with 4',6-diamidino-2-phenylindole (DAPI; 1:15,000, Sigma, St. Louis, MO).

Techniques: Immunohistochemical staining, Derivative Assay, Control, Cell Culture, In Vitro, Staining

Journal: PLoS ONE

Article Title: Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation

doi: 10.1371/journal.pone.0115325

Figure Lengend Snippet: (A) Adhesion assay to analyze osteoblast adhesion to various concentrations of recombinant CTGF, used to coat the wells. The number of cells adhered to CTGF is compared to cell adhesion to 1% BSA (negative control). (B) Adhesion assay comparing osteoblast adhesion to 2 μg/ml CTGF, 2 μg/ml fibronectin, or 1% BSA (negative control) coated wells. (C) Adhesion assay comparing osteoblast adhesion to 2 μg/ml of full-length CTGF, third domain of CTGF, fourth domain of CTGF, or 1% BSA (negative control) coated wells. (D) Adhesion assay analyzing effect of osteoblast treatment with fourth domain of CTGF prior to culture on full-length CTGF coated wells. (E) Adhesion assay analyzing effect of divalent cation chelation by EDTA on osteoblast adhesion to full-length CTGF, and reversing this effect by adding excessive amount of divalent cations to the culture. (F) Adhesion assay studying the effect of blocking heparin binding site of CTGF molecule via adding heparin to osteoblast cell suspension prior to culture on CTGF coated wells. n = 6, *p<0.05; **p<0.01; ***p<0.001. Adhesion assays were repeated three times with similar results.

Article Snippet: For Western blot analysis, goat polyclonal anti-CTGF antibody (Santa Cruz Biotechnology) or rabbit polyclonal anti-β 1 integrin (Cell Signaling) diluted 1:200 in blocking buffer, and then a horseradish peroxidase conjugated donkey anti- goat or donkey anti-rabbit secondary antibody (Jackson Immunoresearch) diluted 1:5000 in blocking buffer were applied, as described previously.

Techniques: Cell Adhesion Assay, Recombinant, Negative Control, Blocking Assay, Binding Assay, Suspension

Journal: PLoS ONE

Article Title: Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation

doi: 10.1371/journal.pone.0115325

Figure Lengend Snippet: (A) Adhesion assay of osteoblasts treated with different blocking integrin antibodies prior to culture on CTGF or BSA (negative control) coated wells. The number of adhered cells in each treatment group was compared to the adhesion level of untreated cells. Adhesion to CTGF matrix after treatment with β 3 or β 5 integrin antibodies was not significantly different from untreated cells. (B) Western blot analyzing CTGF levels following immunoprecipitation of different integrin heterodimers from osteoblast cell lysates and control blot analyzing β 1 integrin levels to confirm that comparable amounts of integrin heterodimers were pulled down during the immunoprecipitation using their respective antibodies. IgG was used as negative control for integrin antibodies. (C) Immunofluorescence staining of osteoblasts cultured on CTGF coated slides for 24 hours at 37°C. Cells stained for α v β 1 (green), F-actin (red in left panel), or vinculin (red in right panel). Lower panel: Scale bar = 50 μm. Upper panel: Scale bar = 10 μm. n = 6, *p<0.05; **p<0.01; ***p<0.001. Experiments were repeated three times with similar results.

Article Snippet: For Western blot analysis, goat polyclonal anti-CTGF antibody (Santa Cruz Biotechnology) or rabbit polyclonal anti-β 1 integrin (Cell Signaling) diluted 1:200 in blocking buffer, and then a horseradish peroxidase conjugated donkey anti- goat or donkey anti-rabbit secondary antibody (Jackson Immunoresearch) diluted 1:5000 in blocking buffer were applied, as described previously.

Techniques: Cell Adhesion Assay, Blocking Assay, Negative Control, Western Blot, Immunoprecipitation, Control, Immunofluorescence, Staining, Cell Culture

Journal: PLoS ONE

Article Title: Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation

doi: 10.1371/journal.pone.0115325

Figure Lengend Snippet: (A) Immunofluorescence staining of F-actin in osteoblasts cultured on 1% BSA, 2 μg/ml of CTGF or fibronectin coated slides for 8 hours at 37°C. Scale bar = 50 μm. (B) Cell spreading area of osteoblasts cultured on BSA, CTGF or fibronectin for 8 hours and stained for actin were measured by ImageJ; n = 50. **p<0.01; ***p<0.001. (C) Western blot analysis of active Rac1, total Rac1 and actin to study Rac1 activation levels. Osteoblasts were cultured for 2 hours on uncoated plates or plates coated with BSA, CTGF or fibronectin. Abbreviations: fibronectin (FN) and negative control (Cont). Experiments were repeated three times with similar results.

Article Snippet: For Western blot analysis, goat polyclonal anti-CTGF antibody (Santa Cruz Biotechnology) or rabbit polyclonal anti-β 1 integrin (Cell Signaling) diluted 1:200 in blocking buffer, and then a horseradish peroxidase conjugated donkey anti- goat or donkey anti-rabbit secondary antibody (Jackson Immunoresearch) diluted 1:5000 in blocking buffer were applied, as described previously.

Techniques: Immunofluorescence, Staining, Cell Culture, Western Blot, Activation Assay, Negative Control

Journal: PLoS ONE

Article Title: Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation

doi: 10.1371/journal.pone.0115325

Figure Lengend Snippet: (A) Alkaline Phosphatase (ALP) staining of osteoblasts cultured on 2 μg/ml CTGF or 1% BSA coated plates for 14 days. Scale bar = 2 mm. (B) ALP activity quantified at day 14 of culture and normalized to total protein content; n = 9 wells. (C) Alizarin red staining of osteoblasts cultured on 2 μg/ml CTGF or 1% BSA coated plates for 35 days. Same magnification as in A. (D) Number of nodules formed after 35 days of culture; n = 9 wells. (E) Area of nodules measured by ImageJ software. **p<0.01; ***p<0.001. Experiments were repeated three times with similar results.

Article Snippet: For Western blot analysis, goat polyclonal anti-CTGF antibody (Santa Cruz Biotechnology) or rabbit polyclonal anti-β 1 integrin (Cell Signaling) diluted 1:200 in blocking buffer, and then a horseradish peroxidase conjugated donkey anti- goat or donkey anti-rabbit secondary antibody (Jackson Immunoresearch) diluted 1:5000 in blocking buffer were applied, as described previously.

Techniques: Staining, Cell Culture, Activity Assay, Software

Journal: PLoS ONE

Article Title: Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation

doi: 10.1371/journal.pone.0115325

Figure Lengend Snippet: (A) Western blot analysis of p-FAK, total FAK and actin protein levels at day 7 of osteoblast culture on 2 μg/ml CTGF or 1% BSA coated plates while treated with osteogenic medium. (B) P-FAK levels were normalized to actin. (C) Total FAK levels were normalized to actin. (D) Western blot analysis of p-ERK, total ERK and actin protein levels at day 7 of osteoblast culture on 2 μg/ml CTGF or 1% BSA coated plates. (E) P-ERK levels were normalized to actin. (F) Total ERK levels were normalized to actin. All Western blots were repeated a minimum of three times with similar results.

Article Snippet: For Western blot analysis, goat polyclonal anti-CTGF antibody (Santa Cruz Biotechnology) or rabbit polyclonal anti-β 1 integrin (Cell Signaling) diluted 1:200 in blocking buffer, and then a horseradish peroxidase conjugated donkey anti- goat or donkey anti-rabbit secondary antibody (Jackson Immunoresearch) diluted 1:5000 in blocking buffer were applied, as described previously.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation

doi: 10.1371/journal.pone.0115325

Figure Lengend Snippet: Alkaline Phosphatase (ALP) staining of osteoblasts cultured on 1% BSA (A) or 2 μg/ml CTGF coated plates in the absence of ERK inhibitor (B) and in the presence of ERK inhibitor (C) for 14 days. Scale bar = 2 mm. (D) ALP activity of osteoblasts cultured on BSA or CTGF coated plates, quantified at day 14 of culture and normalized to total protein content; n = 9 wells.

Article Snippet: For Western blot analysis, goat polyclonal anti-CTGF antibody (Santa Cruz Biotechnology) or rabbit polyclonal anti-β 1 integrin (Cell Signaling) diluted 1:200 in blocking buffer, and then a horseradish peroxidase conjugated donkey anti- goat or donkey anti-rabbit secondary antibody (Jackson Immunoresearch) diluted 1:5000 in blocking buffer were applied, as described previously.

Techniques: Staining, Cell Culture, Activity Assay

Journal: PLoS ONE

Article Title: Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation

doi: 10.1371/journal.pone.0115325

Figure Lengend Snippet: (A) ChIP assay performed on osteoblasts cultured on CTGF coated, BSA coated or uncoated plates for 7 days while treated with osteogenic medium. Runx2 antibody or acetyl-Histone H4 antibody (positive control) used for chromatin immunoprecipitation. Quantitative PCR was performed using osteocalcin gene promoter primers. n = 3, ***p<0.001. ChIP assay repeated three times with similar results. (B) Quantitative PCR performed on osteoblasts cultured on CTGF or BSA coated plates for 7 days while treated with osteogenic media. OCN = osteocalcin, ALP = alkaline phosphatase. n = 3, **p<0.01; ***p<0.001. Experiments repeated three times with similar results.

Article Snippet: For Western blot analysis, goat polyclonal anti-CTGF antibody (Santa Cruz Biotechnology) or rabbit polyclonal anti-β 1 integrin (Cell Signaling) diluted 1:200 in blocking buffer, and then a horseradish peroxidase conjugated donkey anti- goat or donkey anti-rabbit secondary antibody (Jackson Immunoresearch) diluted 1:5000 in blocking buffer were applied, as described previously.

Techniques: Cell Culture, Positive Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed in BM-DCs and LECs and supports DC transmigration. ( A ) Flow cytometry analysis of immature (−LPS) and LPS-matured (+LPS) BM-DCs (gated on live/single cells). ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression of 11 independent experiments. ( C – F ) FACS analysis of CD112 expression in ( C ) LPS-matured BM-DCs and ( E ) primary LN-LECs, derived from WT and CD112 KO mice. ( D , F ) Summary of the ∆MFI values of CD112 expression of 4–6 independent experiments. Data points of the same experiment in ( B , D , F ) are connected by a line, and the mean ΔMFI values are indicated by horizontal lines. ( G ) Set up of the transmigration experiments to investigate the transmigration of BM-DCs (WT or KO) across an LEC monolayer (WT or KO). ( H ) Impact of ICAM-1 blockade on transmigration of WT BM-DCs. ( I,J ) Impact of loss of CD112 in either ( I ) LECs or ( J ) BM-DCs on transmigration. ( K ) Impact of simultaneous loss of CD112 in LECs and BM-DCs on transmigration. For each condition in ( H – K ), one representative experiment with n = 3 technical replicates is shown on the left, and a summary of the averages of 4 independent experiments (biological replicates, each experiment in a different color) is shown on the right. Data points of the same experiment are connected by a line. ( L ) Adhesion assay of WT and KO BM-DCs to WT or KO lymphatic endothelium. The pool of two independent experiments with three replicates per condition is shown (each dot represents a sample). # BM-DCs: number of BM-DCs. Data in all graphs show mean ± standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant.

Article Snippet: In the case of human DC transmigration, LECs were treated with 20 μg/mL mouse anti-human CD54 (clone: BBIG-II, R&D Systems), 20 μg/mL mouse anti-human CD112 (R2.525, Santa Cruz Biotechnology, Dallas, TX, USA) or 20 μg/mL normal mouse IgG1 (Santa Cruz) isotype control.

Techniques: Transmigration Assay, Flow Cytometry, Staining, Expressing, Derivative Assay, Cell Adhesion Assay

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 expression is high in LECs but low in DCs present in murine skin. ( A , B ) FACS analysis was performed to detect CD112 expression in dermal LECs and BECs. ( A ) Depiction of the gating strategy in one representative experiment. ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression observed in 5 independent experiments. ( C – G ) Impact of TPA-induced skin inflammation on the expression of CD112 in LECs. ( C ) Schematic depiction of the experiment: Inflammation was induced in the murine ear skin by topical application of TPA and the ear skin and draining auricular LNs analyzed 24 h later. ( D – G ) FACS analyses were performed to quantify CD112 expression levels in LECs present in control or inflamed tissues. ( D , E ) Analysis of murine ear skin and ( F , G ) auricular LN single-cell suspensions. ( E , G ) The summary of ∆ MFI values was recorded in 5–6 different experiments performed in one control (CTL) and one TPA-inflamed (TPA) ear skin. ( H , I ) FACS gating and quantification of CD112 expression in DCs present in CTL and TPA-inflamed ear skin. ( H ) Gating strategy and ( I ) summary of ∆MFI values recorded in 3 different experiments. ( J – P ) Crawl-out experiments. ( J ) Schematic depiction of the experiment performed to evaluate CD112 expression in ( K – M ) DCs that had emigrated from murine ear skin into the culture medium or in ( N – P ) DCs that had remained in the cultured ear skin at the end of the experiment. Representative ( K , N ) FACS dot plots (gating on single/live cells), identifying DCs as MHCII + CD11c + cells. ( L , O ) Representative histogram plots showing CD112 expression in WT and KO DCs as well as the corresponding fluorescence minus one (FMO) control. ( M , P ) Summary of ∆MFI values (defined as specific staining—FMO) recorded in 4 different experiments performed with one WT and one KO mouse each. Data points in ( B , E , G , I , M , P ) of the same experiment are connected by a line.

Article Snippet: In the case of human DC transmigration, LECs were treated with 20 μg/mL mouse anti-human CD54 (clone: BBIG-II, R&D Systems), 20 μg/mL mouse anti-human CD112 (R2.525, Santa Cruz Biotechnology, Dallas, TX, USA) or 20 μg/mL normal mouse IgG1 (Santa Cruz) isotype control.

Techniques: Expressing, Staining, Control, Cell Culture, Fluorescence

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Loss of CD112 does not impact the in vivo migration of adoptively transferred or endogenous DCs to dLNs. ( A – D ) Adoptive transfer experiment. ( A ) Scheme of the experiment. ( B ) Gating strategy to identify fluorescently labeled adoptively transferred BM-DCs in popliteal LNs. ( C ) The ratio of KO–WT DCs recovered from popliteal LNs draining control (CTL) or CHS-inflamed (CHS) footpads of WT or KO mice. ( D – J ) FITC painting experiment. ( D ) Scheme of the experiment. ( E ) ΔEar thickness, defined as the difference between the ear thickness measured at the start and at the end of the experiment. ( F ) Cellularity and ( G ) weight of the ear-draining auricular LN at the end of the experiment. ( H ) Gating strategy to identify and quantify the number (#) of ( I ) all CD11c + MHCII hi migratory DCs (mDCs) and ( J ) FITC + mDCs. Summaries of three ( A – D ) and two ( D – J ) independent experiments, each with 2–7 mice per condition, are shown. Each dot represents one mouse. Mann–Whitney t -test was used. Red bars in all graphs show the mean. ns: not significant.

Article Snippet: In the case of human DC transmigration, LECs were treated with 20 μg/mL mouse anti-human CD54 (clone: BBIG-II, R&D Systems), 20 μg/mL mouse anti-human CD112 (R2.525, Santa Cruz Biotechnology, Dallas, TX, USA) or 20 μg/mL normal mouse IgG1 (Santa Cruz) isotype control.

Techniques: In Vivo, Migration, Adoptive Transfer Assay, Labeling, Control, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Blockade of CD112 decreases in vitro transmigration of human moDCs across human dermal LEC monolayers. ( A – C ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in in vitro-differentiated ( A ) immature (−LPS) and ( B ) LPS-matured (+LPS) human moDCs. LPS was added 24 h prior to FACS analysis. Representative FACS plots are shown in ( A , B ). ( C ) Summary of the delta mean fluorescent intensity (∆MFI; defined as specific-isotype staining) values recorded for each corresponding marker in 3–6 independent experiments (biological replicates). Data points of the same experiment are connected by a line, and the means of the ΔMFI values are indicated by horizontal red lines. ( D , E ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in primary human dermal LECs. ( D ) Representative FACS histograms recorded upon gating on CD31 + podoplanin + cells, and ( E ) summary of the MFI values recorded for all markers and corresponding isotype controls in 4–5 independent experiments performed on LECs from two different donors. Data points of the same experiment are connected by a line, and the means of the MFI values are indicated by horizontal red lines. ( F – I ) Transmigration experiments involving human moDCs and human dermal LECs, performed in the presence/absence of ( F , G ) αICAM-1 or of ( H , I ) αCD112 or the corresponding isotype controls; ( F – I ) The number of transmigrated DCs (# DCs) was assessed. ( F , H ) show representative results from one representative experiment with n = 6 technical replicates per condition. ( G , I ) show the summaries of four independent experiments (i.e., different biological replicates, shown with different colors) with 3–6 replicates per condition. The averages from each experiment are connected by a line. The standard error of the mean (SEM) is shown; the Mann–Whitney t -test was used. * p < 0.05; ** p < 0.01.

Article Snippet: In the case of human DC transmigration, LECs were treated with 20 μg/mL mouse anti-human CD54 (clone: BBIG-II, R&D Systems), 20 μg/mL mouse anti-human CD112 (R2.525, Santa Cruz Biotechnology, Dallas, TX, USA) or 20 μg/mL normal mouse IgG1 (Santa Cruz) isotype control.

Techniques: In Vitro, Transmigration Assay, Expressing, Staining, Marker, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed by DCs and LECs in human skin. ( A – D ) FACS-based analysis of CD112 expression in endothelial cells and DCs present in human skin. ( A , C ) Gating strategy used to detect CD112 expression in ( A ) BECs and LECs and ( C ) DCs. ( B , D ) Summary of mean fluorescent intensity (MFI) values of CD112 expression in ( B ) LEC and BECs or ( D ) HLA-DR + CD86 + DCs in 2 independent experiments (i.e., different biological replicates) was analyzed. Data points of the same experiment are connected by a line. ( E , F ) Confocal images of human skin sections depicting ( E ) CD112 expression (white) by dendritic cells (examples indicated by white arrows), identified as HLA-DR + (green) and CD11c + (red). Scale bar = 100 μm ( F ) CD112 expression (white) by lymphatic vessels, LYVE-1 (green) and PLVAP (red). Scale bar = 100 μm. ( G ) Top: Gating strategy and Bottom: representative histogram plot showing CD112 expression on DCs that had emigrated from a human breast skin punch biopsy. ( H ) Crawl-out experiments from punch biopsies derived from either breast or abdominal skin were performed in the presence of a CD112-blocking antibody or media/isotype control (CTL) in the culture medium. Top: Representative FACS gating plot from abdominal skin. Bottom: Quantification of emigrated HLA-DR+CD86 + DCs. Pooled data from 5 independent experiments with 4–10 punches per condition are shown. ( I ) Crawl-out experiment from abdominal skin punch biopsies to verify the expression of CD112-binding partners DNAM-1, TIGIT and CD113 on human DCs, identified as live, HLA-DR + cells. Representative stainings from one out of three independent experiments are shown. The mean and standard deviation (SD) are shown in (H). Mann–Whitney t -test was used. ** p < 0.01.

Article Snippet: In the case of human DC transmigration, LECs were treated with 20 μg/mL mouse anti-human CD54 (clone: BBIG-II, R&D Systems), 20 μg/mL mouse anti-human CD112 (R2.525, Santa Cruz Biotechnology, Dallas, TX, USA) or 20 μg/mL normal mouse IgG1 (Santa Cruz) isotype control.

Techniques: Expressing, Derivative Assay, Blocking Assay, Control, Binding Assay, Standard Deviation, MANN-WHITNEY

Journal: Cellular and Molecular Immunology

Article Title: Anti-inflammatory mechanisms of the novel cytokine interleukin-38 in allergic asthma

doi: 10.1038/s41423-019-0300-7

Figure Lengend Snippet: Effect of IL-38 on cytokine/chemokine release in cocultures of human primary bronchial epithelial cells (HBE) and eosinophils (EOS) upon stimulation by poly (I:C)/LyoVec or TNF-α. Primary bronchial epithelial cells (1 × 105) and purified eosinophils (3 × 105) were cocultured with or without human IL-38 pretreatment for 10 min, followed by poly (I:C)/LyoVec (2 μg/ml) or TNF-α (20 ng/ml) treatment for an additional 20 h. Release of a IL-6, b CCL5, c CXCL10, d IL-1β, and e IFN-β into the supernatant of the poly (I:C)/LyoVec-treated coculture was determined. f Gating strategies for bronchial epithelial cells and eosinophils are shown. Bronchial epithelial cells and eosinophils in the cocultures were gated based on the FSC and SSC parameters. The cell surface expression of ICAM-1 on g HBE/EOS single-cultured cells and h cocultured cells was analyzed by flow cytometry. The levels of i IL-6, j CCL5, and k CXCL10 in the supernatant of the TNF-α-treated coculture were measured. The expression of ICAM-1 on l HBE/EOS single-cultured cells and m cocultured cells after stimulation with TNF-α was determined by flow cytometry. A negative control of 56 °C heat-inactivated human IL-38 (100 ng/ml) and a positive control of dexamethasone (1 μM) were included. Abbreviations: HBE, human primary bronchial epithelial cells; PLV, poly (I:C)/LyoVec; EOS, eosinophils; HBE-EOS, coculture of human bronchial epithelial cells and eosinophils; Co-HBE, human primary bronchial epithelial cells in coculture; and Co-EOS, eosinophils in coculture. The results are shown as the mean ± SEM of triplicate independent experiments with a total of three donors. *P < 0.05, **P < 0.01, and ***P < 0.001 when compared between the denoted groups

Article Snippet: Primary bronchial epithelial cells (1 × 10 5 ) and purified eosinophils (3 × 10 5 ) were cocultured with or without human IL-38 (100 ng/ml) pretreatment for 10 min, followed by poly (I:C)/LyoVec or human TNF-α (R&D Systems, Minneapolis, MN, USA) stimulation for an additional 20 h. To analyze the expression of ICAM-1 using a mouse anti-human ICAM-1 antibody (BioLegend), mixed bronchial epithelial cells and eosinophils were differentially analyzed by using a FACSCalibur flow cytometer (BD Biosciences) and distinguished based on the forward-scatter and side-scatter parameters.

Techniques: Purification, Expressing, Cell Culture, Flow Cytometry, Negative Control, Positive Control

Journal: Cellular and Molecular Immunology

Article Title: Anti-inflammatory mechanisms of the novel cytokine interleukin-38 in allergic asthma

doi: 10.1038/s41423-019-0300-7

Figure Lengend Snippet: Graphical summary of the anti-inflammatory activities of IL-38 in allergic airway inflammation. In murine allergic asthma, IL-38 can inhibit the accumulation of eosinophils, ILC2s, and Th2 cells, and the release of CCL11, ECP, and the Th2-related cytokines IL-4, IL-5, and IL-13. Moreover, IL-38 can promote Tregs, which are regulated by IL-10 to maintain immune homeostasis. In our in vitro study, we focused on the interaction between eosinophils and bronchial epithelial cells. The activation of cocultured human primary epithelial cells and eosinophils by the viral mimic dsRNA RLR ligand poly (I:C)/LyoVec or proinflammatory TNF-α could be suppressed by IL-38, and this suppression was mediated by downregulation of the p38, STAT1, STAT3, ERK1/2, and NF-κB pathways, as well as upregulation of the expression of the airway host defense gene POU2AF1 and anti-allergic response gene RGS13 in bronchial epithelial cells, leading to significantly reduced expression of ICAM-1 and proinflammatory cytokines and chemokines. In addition, IL-38 was capable of decreasing the phosphorylation of p38, STAT1, STAT3, ERK1/2, and IκBα in eosinophils in the coculture system, thereby ameliorating allergic airway inflammation

Article Snippet: Primary bronchial epithelial cells (1 × 10 5 ) and purified eosinophils (3 × 10 5 ) were cocultured with or without human IL-38 (100 ng/ml) pretreatment for 10 min, followed by poly (I:C)/LyoVec or human TNF-α (R&D Systems, Minneapolis, MN, USA) stimulation for an additional 20 h. To analyze the expression of ICAM-1 using a mouse anti-human ICAM-1 antibody (BioLegend), mixed bronchial epithelial cells and eosinophils were differentially analyzed by using a FACSCalibur flow cytometer (BD Biosciences) and distinguished based on the forward-scatter and side-scatter parameters.

Techniques: In Vitro, Activation Assay, Expressing

Journal: Journal of Tissue Engineering

Article Title: Triple co-culture and perfusion bioreactor for studying the interaction between Neisseria gonorrhoeae and neutrophils: A novel 3D tissue model for bacterial infection and immunity

doi: 10.1177/2041731420988802

Figure Lengend Snippet: Histological and immunofluorescence analysis of the triple co-culture model: (a) H&E staining of the triple co-culture 3D tissue model, showing three layers of cells including epithelial (T84, top layer), fibroblasts (HDFib, middle layer), and endothelial (HUVEC, basal layer) cells and (b–f) the tissue model was stained with anti-E-cadherin (red), anti-fibroblast (cyan), anti-MUC1 (magenta), anti-CD31 (green), anti-VE-cadherin (yellow) and DAPI (blue). The images show the formation of an epithelial (stained by E-cadherin) and endothelial (stained by VE-cadherin and CD31) monolayer and the presence of a connective tissue (stained by anti-fibroblast) and mucin layer (stained by anti MUC1). The scale bars are 50 µm.

Article Snippet: The cells were stained with anti E-Cadherin (rabbit, 1:100, Proteintech, Illinois, USA), anti ZO-1 (rabbit, 1:100, Proteintech, Illinois, USA), anti-fibroblast (mouse, 1:100, Antikörper, Aachen, Germany), anti-neutrophil cytosolic factor 2 (rabbit, 1:100, Antikörper, Aachen, Germany), anti-VE cadherin (mouse, 1:100, Gibco/Thermo Fisher scientific, Massachusetts, USA), anti-CD31 (mouse, 1:100, Abcam, Cambridge, United Kingdom), and anti-MUC1 (rabbit, 1:100, Abcam, Cambridge, United Kingdom) antibodies.

Techniques: Immunofluorescence, Co-Culture Assay, Staining

Journal: Journal of Tissue Engineering

Article Title: Triple co-culture and perfusion bioreactor for studying the interaction between Neisseria gonorrhoeae and neutrophils: A novel 3D tissue model for bacterial infection and immunity

doi: 10.1177/2041731420988802

Figure Lengend Snippet: Immunofluorescence analysis using confocal laser microscopy of triple co-culture model. Immunofluorescence analysis of the triple co-culture model (T84/HDFib/ HUVECs) after maturation: (a) XY and YZ view of the apical side of the tissue model (epithelial, T84), showing E-cadherin junctions. Z-stacks were acquired using confocal fluorescence microscopy from the top of the epithelial layer and were reconstructed using FIJI and (b) XY and YZ view of the basal side of the tissue model (endothelial, HUVECs), depicting endothelial cell adhesion, which was stained by CD31. Z- stacks were made from the top of the endothelial layer to the beginning of the SIS scaffold. (C) XZ view of the triple co-culture model, showing the apical and basolateral side together. The middle layer is SEM image of the SIS scaffold, showing the fibrous structure, with fibroblasts represented by cartoons. E-Cadherin (red), CD31 (green), and DAPI (blue).

Article Snippet: The cells were stained with anti E-Cadherin (rabbit, 1:100, Proteintech, Illinois, USA), anti ZO-1 (rabbit, 1:100, Proteintech, Illinois, USA), anti-fibroblast (mouse, 1:100, Antikörper, Aachen, Germany), anti-neutrophil cytosolic factor 2 (rabbit, 1:100, Antikörper, Aachen, Germany), anti-VE cadherin (mouse, 1:100, Gibco/Thermo Fisher scientific, Massachusetts, USA), anti-CD31 (mouse, 1:100, Abcam, Cambridge, United Kingdom), and anti-MUC1 (rabbit, 1:100, Abcam, Cambridge, United Kingdom) antibodies.

Techniques: Immunofluorescence, Microscopy, Co-Culture Assay, Fluorescence, Staining

Journal: Journal of Inflammation Research

Article Title: Tellimagrandin II Stimulates Inflammasomes by Causing an Accumulation of 3-Aminopropanal, Which Promotes Apoptosis of Endometriotic Cells While Inhibiting Invasion

doi: 10.2147/JIR.S558146

Figure Lengend Snippet: Western blot analysis of EECs treated with Tellimagrandin II: ( a ) protein immunoblot; ( b ) ALDH7A1; ( c ) SMOX; ( d ) NLRP3; ( e ) Caspase-1; ( f ) β-catenin; ( g ) cleaved Caspase-3; ( h ) BAX; ( i ) MMP2; ( j ) TIMP1. Each experiment was repeated three times. *:P <0.05; **:P <0.01; ***:P <0.001; ****P <0.0001.

Article Snippet: Tellimagrandin II (Yongjian: B02179 ), Spermine Oxidase (SMOX) Polyclonal antibody (proteintech: 15052-1-AP), Aldehyde Dehydrogenase 7A1 (ALDH7A1) Polyclonal antibody (proteintech: 10368-1-AP), Anti-NLR Family, Pyrin Domain Containing Protein 3 (NLRP3) Rabbit pAb (Servicebio: GB114320-50), Recombinant Anti-beta Catenin (β-Catenin) antibody (Servicebio: GB150016-100), Anti-Matrix Metallopeptidase 2 (MMP2) Rabbit pAb (Servicebio: GB11130-100), Anti- Tissue Inhibitors of Metalloproteinase 1 (TIMP1) Rabbit pAb (proteintech: 16644-1-AP), Anti-Caspase-3 Rabbit pAb (Servicebio: GB115600-100), Anti-Bcl-2-associated X(Bax) Rabbit pAb (Servicebio: GB11690-100), Anti-Caspase-1 Rabbit pAb (Servicebio: GB11383-100), DMEM/F12 (Glutamine and HEPES)(M&C GENE: CM10092), Fetal Bovine Serum Gold (YEASEN: 40130ES76), penicillin streptomycin mixture (Servicebio: P1400), 1×PBS buffer (Solarbio: P1020), trypsin-EDTA digestion solution (Phenol Red)(Solarbio: T1320), CCK-8 kit (Sola rbio: CA1210), Annexin V-FITC Apoptosis detection kit (Beyotime: C1062S), 96-well cell culture plate (Servicebio:) CCP-96H, SWE matrix adhesive (Solarbio: G4131-5ML), crystal violet staining solution (Solarbio: G1014-50ML), paraformaldehyde fixative (Solarbio: G1101-500ML), Transwell chamber (Corning: WG3422) Cell culture flasks (Corning: 430639), 12Z EECs line (Bioharbor: LH-H337), DMSO (Servicebio: GC203006-10mL), Propidium Iodide (proteintech: CM18819), Flow cytometer (BD: FACSAria II), Gel imaging system (Alpha: 2200) Electrophoresis apparatus (Thermo: EC250-90), Thermo Corporation, USA; Pendulum type decolorizing shaker (model: DS-2S100), Nanodrop micro spectrophotometer (Illumina: Nanodrop2000), sequencing platform machine (Illumina: NovaSeq X Plus).

Techniques: Western Blot

Journal: Journal of Inflammation Research

Article Title: Tellimagrandin II Stimulates Inflammasomes by Causing an Accumulation of 3-Aminopropanal, Which Promotes Apoptosis of Endometriotic Cells While Inhibiting Invasion

doi: 10.2147/JIR.S558146

Figure Lengend Snippet: Molecular docking diagram: ( a ) 3D representation of the docking between ALDH7A1 and Tellimagrandin II; ( b ) 2D representation of the docking between ALDH7A1 and Tellimagrandin II; ( c ) 3D representation of the docking between SMOX and Tellimagrandin II; ( d ) 2D representation of the docking between SMOX and Tellimagrandin II.

Article Snippet: Tellimagrandin II (Yongjian: B02179 ), Spermine Oxidase (SMOX) Polyclonal antibody (proteintech: 15052-1-AP), Aldehyde Dehydrogenase 7A1 (ALDH7A1) Polyclonal antibody (proteintech: 10368-1-AP), Anti-NLR Family, Pyrin Domain Containing Protein 3 (NLRP3) Rabbit pAb (Servicebio: GB114320-50), Recombinant Anti-beta Catenin (β-Catenin) antibody (Servicebio: GB150016-100), Anti-Matrix Metallopeptidase 2 (MMP2) Rabbit pAb (Servicebio: GB11130-100), Anti- Tissue Inhibitors of Metalloproteinase 1 (TIMP1) Rabbit pAb (proteintech: 16644-1-AP), Anti-Caspase-3 Rabbit pAb (Servicebio: GB115600-100), Anti-Bcl-2-associated X(Bax) Rabbit pAb (Servicebio: GB11690-100), Anti-Caspase-1 Rabbit pAb (Servicebio: GB11383-100), DMEM/F12 (Glutamine and HEPES)(M&C GENE: CM10092), Fetal Bovine Serum Gold (YEASEN: 40130ES76), penicillin streptomycin mixture (Servicebio: P1400), 1×PBS buffer (Solarbio: P1020), trypsin-EDTA digestion solution (Phenol Red)(Solarbio: T1320), CCK-8 kit (Sola rbio: CA1210), Annexin V-FITC Apoptosis detection kit (Beyotime: C1062S), 96-well cell culture plate (Servicebio:) CCP-96H, SWE matrix adhesive (Solarbio: G4131-5ML), crystal violet staining solution (Solarbio: G1014-50ML), paraformaldehyde fixative (Solarbio: G1101-500ML), Transwell chamber (Corning: WG3422) Cell culture flasks (Corning: 430639), 12Z EECs line (Bioharbor: LH-H337), DMSO (Servicebio: GC203006-10mL), Propidium Iodide (proteintech: CM18819), Flow cytometer (BD: FACSAria II), Gel imaging system (Alpha: 2200) Electrophoresis apparatus (Thermo: EC250-90), Thermo Corporation, USA; Pendulum type decolorizing shaker (model: DS-2S100), Nanodrop micro spectrophotometer (Illumina: Nanodrop2000), sequencing platform machine (Illumina: NovaSeq X Plus).

Techniques:

Journal: Theranostics

Article Title: Sulforaphane Inhibits the Acquisition of Tobacco Smoke-Induced Lung Cancer Stem Cell-Like Properties via the IL-6/ΔNp63α/Notch Axis

doi: 10.7150/thno.33812

Figure Lengend Snippet: Long-term TS exposure-induced acquisition of the CSC-like phenotype. HBE cells were exposed to 2 % CSE (cigarette smoke extract) for 55 passages. Images of cell colonies (A) were taken and the number of colonies (B) was counted. (C) ZO-1, E-cadherin, Vimentin, and N-cadherin levels in TS-treated HBE cells were determined using A549 cells as positive controls. (D) Densitometric analyses of western blots of ZO-1, E-cadherin, Vimentin, and N-cadherin were performed following β-actin normalization. (E) 1 × 10 6 non-CSE exposed control HBE cells, TS-exposed HBE cells, and A549 cells were subcutaneously injected in the front dorsum of nude mice and tumor incidence was analyzed 2 weeks later. (F) CHBE and TS-treated HBE cells were cultured in serum-free medium (SFM) for 7 days. Western blot analysis of lung CSC markers was then performed. (G) Densitometric analyses of western blots of CD133, ALDH1A1, Oct4, and Nanog were measured after β-actin normalization. Three independent experiments were performed. Data are expressed as the mean ± SD. Significance was assessed by one-way ANOVA or unpaired two-tailed Student's t tests. * P < 0.05, ** P < 0.01 compared to CHBE cells. THBE: HBE cells exposed to CSE for 55 passages; CHBE: HBE cells cultured under the same conditions for 55 passages, but not exposed to CSE. A549 cells were used as positive controls.

Article Snippet: The membranes were blocked with 5 % nonfat dry milk at 25 °C for 1 h on a rotary shaker and incubated overnight at 4 °C with the following primary antibodies: ΔNp63α (TA327976; OriGene Technologies, Rockville, MD, USA); CD133 (18470-1-AP), ALDH1A1 (15910-1-AP), Nanog (14295-1-AP), Oct4 (60242-1-Ig), Sox2 (11064-1-AP), ZO-1 (21773-1-AP), E-cadherin (20874-1-AP), Vimentin (10366-1-AP), N-cadherin (22018-1-AP), IL-6 (21865-1-AP), and β-tubulin (10094-1-AP; Proteintech, Rosemont, IL, USA); NICD (ab83232) and Hes1 (ab119776; Abcam, Cambridge, Massachusetts, US); β-actin (AP0060; Biogot Technology, Nanjing, China).

Techniques: Western Blot, Control, Injection, Cell Culture, Two Tailed Test