Journal:

Article Title: Enhanced Expression of Keratinocyte Growth Factor and Its Receptor Correlates with Venous Invasion in Pancreatic Cancer

doi: 10.2353/ajpath.2007.060935

Figure Lengend Snippet: Effect of KGF on VEGF-A expression in pancreatic cancer cells. A: Time course of VEGF-A induction. Q-PCR analysis showed significant increases in VEGF-A mRNA levels at 3 and 6 hours after the addition of 10 ng/ml rhKGF to MIA PaCa-2 cells. Each experiment was performed twice, and gene expression measurements were performed in triplicate. Bars represent the mean ± SE (*P = 0.038, **P = 0.041). B: Effects of exogenous KGF on VEGF-A protein levels. After the addition of rhKGF (0 to 100 ng/ml) to MIA PaCa-2 cells, VEGF-A levels in the culture supernatant were measured by ELISA and were found to be increased significantly in a dose-dependent manner. Results shown represent the mean ± SE of two separate experiments, each conducted in triplicate (*P = 0.034, **P = 0.018). C and D: Effects of engineered expression of KGF on VEGF-A expression. PANC-1-KGF and PANC-1-Mock cells were incubated in serum-free medium for 72 hours. KGF and VEGF-A mRNA levels were then determined by Q-PCR analysis. KGF (*P < 0.0001; C) and VEGF-A (*P = 0.004; D) mRNA levels in PANC-1-KGF were significantly higher than in PANC-1-Mock cells. Each experiment was performed twice, and each measurement was performed in triplicate. Bars represent the mean ± SE. E and F: Effects of sh-KGFR on VEGF-A expression. Western blot analysis showed that sh-KGFR inhibited the expression of KGFR protein levels in MIA PaCa-2 cells compared with sh-control (*P = 0.0006; E). Cells were treated with rhKGF (10 ng/ml) after being transfected with sh-KGFR or sh-control. sh-KGFR transfection was associated with significantly reduced VEGF-A protein levels in the conditioned medium after the addition of rhKGF (10 ng/ml) compared with sh-control (*P = 0.0016; F). Each measurement was performed in triplicate. Bars represent the mean ± SE. G and H: Effects of sh-KGF on VEGF-A expression. sh-KGF and sh-control were transiently transfected into KLM-1 cells. Q-PCR revealed that sh-KGF significantly reduced KGF and VEGF-A mRNA levels in KLM-1 cells compared with sh-control (*P = 0.0002, **P = 0.0046; G). Furthermore, sh-KGF significantly reduced VEGF-A protein levels in conditioned medium compared with sh-control (*P = 0.0016; H). Each experiment was performed twice, and each measurement was performed in triplicate. Bars represent the mean ± SE.

Article Snippet: The chemicals and reagents were purchased as follows: Isogen from Nippon Gene (Tokyo, Japan); a Takara RNA PCR kit (AMV) version 3.0 and pBAsi-hU6 Neo DNA vector from Takara Biotech (Tokyo, Japan); RNeasy mini kit from Qiagen GmbH (Hilden, Germany); Transcriptor First Strand cDNA Synthesis kit and LightCycler FastStart DNA Master SYBR Green I, FuGENE 6, and FuGENE HD transfection reagent from Roche Diagnostics GmbH (Mannheim, Germany); human VEGF Quantikine Colorimetric Sandwich enzyme-linked immunosorbent assay (ELISA) kit, goat polyclonal anti-FGF-7 antibodies, and recombinant human KGF (rhKGF) from R&D Systems Inc. (Westerville, OH); Immobilon P transfer membrane from Millipore (Yonezawa, Japan); M-PER Mammalian Protein Extraction reagent and Super Signal West Pico chemiluminescent substrates from Pierce (Rockford, IL); SERVA Blau G from Serva Electrophoresis GmbH (Heidelberg, Germany); Histofine Simple Stain Max PO (G) or (R) kit from Nichirei Biosciences, Inc. (Tokyo, Japan); anti-rabbit IgG-horseradish peroxidase secondary antibody and rabbit polyclonal anti-VEGF-A antibodies (A-20) from Santa Cruz Biotechnology (Santa Cruz, CA); Human Tissue Microarray 1 and Human Digestive Tissue Sets from Novagen (Darmstadt, Germany); fluorescein 5-isothiocyanate-conjugated anti-rabbit IgG and Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole dihydrochloride from Vector Laboratories, Inc. (Burlingame, CA); silane-coated slides and a malinol mounting medium from Muto Pure Chemicals Co., Ltd. (Tokyo, Japan); and pIRES2-EGFP vector from Clontech (Palo Alto, CA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Transfection

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 1 | Identification of CD177+ and CD275+ ADE subpopulations. a, Schematic representation of hESC differentiation toward DE. b,c, Representative FACS plots of apparently homogeneous FOXA2+/SOX17+ DE (b) showing a heterogenous population marked by CXCR4+/CD117+ cells (c) (n = 3 (b), n = 6 (c) biologically independent experiments). d–g, Gene expression profiles of CXCR4+/CD117−, CXCR4high/CD117high, CXCR4mid/CD117mid and CXCR4low/CD117low cells for FOXA2 (d), SOX17 (e), CER1 (f) and HHEX (g) (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h, Summary of the antibody screen identifying and isolating CD177 and CD275 as markers of ADE subpopulations. CXCR4 and FOXA2 are used as controls to identify the whole DE. i, hPSCs and hPSC-derived DE stained for CXCR4, CD177 and CD275 as shown by live-cell FACS (n = 10 biologically independent experiments). AA, activin A; D, day.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Gene Expression, Derivative Assay, Staining

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 2 | Molecular profiling of CD177+, CD275+ and CXCR4+ DE subpopulations reveals distinct signatures. a, Summary of differentiation protocol toward DE/ADE followed by MACS to enrich for CD177, CD275 and CXCR4 populations. b, Principal component analysis showing that mRNA-derived transcriptome profiles are characteristic of different DE/ADE subpopulations (n = 3 biologically independent experiments). c–e, Bar graphs of selected and significantly enriched gene ontology terms in CD275+ versus CXCR4+ (c), CD177+ versus CD275+ (d) and CD177+ versus CXCR4+ (e) DE populations (n = 3 biologically independent experiments). Enrichment P values are calculated by HOMER findGO.pl based on the cumulative hypergeometric distribution. f,g, Validation of the microarray analysis by qPCR for noncanonical WNT/PCP components and ligands (f) and canonical WNT components and ligands (g). Data were normalized to 18S (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h,i, Western blot analysis (h) and quantification (i) of WNT/PCP components such as p-JNK and DVL2 in ADE subpopulations (n = 3 biologically independent experiments). GAPDH is used as a loading control. Data are represented as mean ± s.e.m. j, Immunofluorescence analysis validated the exclusive localization of β-catenin in the membrane in CD177+ ADE cells and in the cytoplasm and nucleus in CD275+ ADE and CXCR4+ DE cells (n = 3 biologically independent experiments). FOXA2 is used as a nuclear marker. Scale bars, 20 µm and 10 µm in inset. PC1/2, principal component 1/2.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Derivative Assay, Biomarker Discovery, Microarray, Western Blot, Control, Immunofluorescence, Membrane, Marker

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Immunohistochemistry staining with antibody against phosphorylated Smad2 (pSmad2) and TGFβ receptor II (TβRII) showed increased nuclear signal for pSmad2 in the invasive ECdnT organotypic cultures. Scale bar 50 micron. (b) Analysis of immunohistochemistry staining for TβRII and pSmad2 in 83 ESCC cases in a tissue microarray shows no significant correlation. Fisher’s exact test, two tailed p= 0.3182. (c) Five paired normal adjacent and ESCC tissues (GSE17531) were analyzed for INHBA mRNA expression, which identified upregulation of INHBA in four ESCC samples. (d) Waterfall plot of a publically available dataset (GSE23400) represented upregulation of INHBA in the ESCC (grey bars) samples vs. normal (black bars).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 µM A83-01 (Tocris, Bristol, UK) and 1 µM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Staining, Microarray, Two Tailed Test, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures with either fetal esophageal fibroblasts (FEF) or cancer-associated fibroblasts (CAF) embedded in the underlying matrix. Immunofluorescence staining with antibody against αSMA (green) and podoplanin (red) showed similar expression pattern in the cultures. Scale bar is 50 micron. (b) Activin A concentration in conditioned media from organotypic cultures is higher in invasive cultures as measured using indirect ELISA. * p=0.003, ** p= 0.005, *** p=0.03 (c) Stimulation of epithelial cells with Act A in monolayer plastic culture demonstrated phosphorylation of Smad. Neutralizing antibody against Activin (nAb) prevented the induction of pSmad2 by Act A. Following stimulation with Act A or with conditioned media from organotypic culture increased expression of vimentin was detected after 48 hours by Western Blot. The increase was reversed in the presence of neutralizing antibody (nAb). (d) Inhibition with the Act A antagonist, Follistatin, or a pan-TGFβ inhibitor A83-01 suppressed MMP-9 secretion in E, EC and ECdnT cells as measured by gelatin zymography. Upper bands reflect pro-MMP, lower bands activated, cleaved MMP (arrow).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 µM A83-01 (Tocris, Bristol, UK) and 1 µM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Immunofluorescence, Staining, Concentration Assay, Indirect ELISA, Western Blot, Inhibition, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures in the presence of recombinant Activin A (Act A), its antagonist Follistatin or a neutralizing antibody against Activin A (nAb); H&E staining. Stimulation with Act A inhibited invasion of E and EC cells, but failed to suppress ECdnT cell invasion. Follistatin increased cell invasion in all cell types, while the neutralizing antibody prevented invasion of E and EC cells, without an effect on ECdnT cells. (b) Immunohistochemistry staining with ki67-antibody showed no differences in cell proliferation. Scale bars are 50 micron. (c) Indirect ELISA with antibody against Act A measured increased levels after addition of recombinant Act A in fibroblasts (FEF) and ECdnT. Untreated ECdnT cells (Control) secreted higher baseline levels of Act A than FEF, which were reduced by Follistatin. (d) TGFβ1 concentration was increased in response to stimulation with Act A and overall baseline secretion was higher in control ECdnT cells than fibroblasts as measured by indirect ELISA. Follistatin inhibited TGFβ1 secretion.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 µM A83-01 (Tocris, Bristol, UK) and 1 µM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Recombinant, Staining, Immunohistochemistry, Indirect ELISA, Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Separating the cellular matrix and epithelium of the organotypic cultures growing ECdnT cells through a collagen I layer, dashed lines, prevented cell invasion in the absence (control) and presence of Act A (+Act A). When the cellular matrix of the organotypic culture was treated with puromycin to kill the embedded fibroblasts before the ECdnT cells were seeded, epithelial formation occurred but invasion was inhibited with and without Act A stimulation. (b) Treatment of ECdnT organotypic cultures with a pan-MMP inhibitor, GM6001, suppressed cell invasion, which was not restored in the presence of Act A. Untreated (no tx) control ECdnT cells in organotypic culture invaded into the underlying matrix. TGFβ1 treatment inhibited epithelial cell invasion. Scale bars are 50 micron. (c) Immunohistochemistry showed nuclear localization of phosphorylated Smad (pSmad2, red) in control and Act A stimulated conditions. Collagen IV, red, was disrupted in invasive cultures after Act A treatment. Loss of the fibroblasts (FEF), labeled green with antibody against vimentin (no staining in the lower panels), had no effect on the nuclear localization of pSmad2. The collagen IV layer was not disrupted in non-invasive cultures in the absence of FEFs.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 µM A83-01 (Tocris, Bristol, UK) and 1 µM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Labeling, Staining

Journal: Journal of Korean Medical Science

Article Title: Prediction of Microbial Infection of Cultured Cells Using DNA Microarray Gene-Expression Profiles of Host Responses

doi: 10.3346/jkms.2012.27.10.1129

Figure Lengend Snippet: The microarray experimental design in three dimensional spaces according to source of infection (x axis), cell type (y axis) and day of culture (z axis).

Article Snippet: Experiments were performed using the microarray system (Oligo-Human 10K, Macrogen Inc., Seoul, Korea) according to the manufacturer's protocol.

Techniques: Microarray, Infection

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Hyperlipidemia stimuli induce IL-17 and its receptor subunit expression. A, the plasma IL-17 concentration was determined by ELISA. WT and ApoE−/− mice were fed with normal chow or high fat diet (HFD) for 8 weeks starting at 8 weeks of age. Plasma from mice was collected for ELISA analysis. B, IL-17RA mRNA expression in MAECs was determined by qRT-PCR. MAECs were treated with ox-LDL (50 μg/ml) for 24 h, and the IL-17RA mRNA expression was quantified. IL-17RA expression was significantly increased with ox-LDL treatment relative to the control cells. Data were normalized to β-actin. C and D, the protein expressions of IL-17RA and IL-17RC in HAECs were quantified by flow cytometry. HAECs were treated with LDL (50 μg/ml) or ox-LDL (50 μg/ml) for 24 h. C, IL-17RA expression was increased with ox-LDL treatment in HAECs. D, ox-LDL treatment significantly increased IL-17RC expression in HAECs. Data are presented as means ± S.E. (error bars) (n = 3). *, p < 0.05; **, p < 0.01 versus untreated control cells.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Treatment of HAECs with cytokine IL-17 increases the non-treated monocyte adhesion to HAECs. A, IL-17-treated HAECs have increased THP-1 cell adhesion. HAECs were treated with IL-17 (100 ng/ml) for the indicated time, and untreated labeled monocytes were allowed to adhere onto the endothelial monolayer. IL-17 treatment increased THP-1 monocytic cell adhesion onto HAECs in a time-dependent manner. The adhesion is shown as a percentage of the control group. B, untreated primary human monocyte adhesion to HAECs treated with IL-17 gradually increased up to 24 h. C, fluorescence microscopic images of labeled primary human monocyte adhered to HAECs. Data are presented as means ± S.E. (error bars) (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Labeling, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Depletion of IL-17 gene in ApoE−/− mice significantly decreases in vivo leukocyte rolling and adhesion to endothelium. ApoE−/− mice and ApoE−/−/IL-17A−/− mice were fed with a high fat diet for 3 weeks before intravital microscopy analysis was performed. A, intravital microscopy was used to determine the endothelial activation by determining leukocyte rolling and adhesion to endothelium in vivo. CCD, charge-coupled device. B, mean vessel diameters of venules were not significantly different between ApoE−/− and ApoE−/−/IL-17A−/− mice used for the intravital microscopy study. C, number of cells that rolled past an imaginary line that was perpendicular to the vessels during a 1-min period were not significant in both animal groups used in the study. D, the number of cells adhered to 100 μm of endothelium along the vessel for 1 min was significantly reduced in IL-17-null mice relative to the controls. Data are presented as means ± S.E. (error bars) (n = 7–8; five vessels per mouse were recorded). *, p < 0.05 (significant); n.s., not significant.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: In Vivo, Intravital Microscopy, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: IL-17 induces the gene expression of four proinflammatory cytokines and chemokines, namely IL-6, GM-CSF, CXCL1, and CXCL2, in mouse and human aortic endothelial cells. The expressions of 84 genes related to EC biology were profiled by using the Mouse Endothelial Cell Biology RT2 Profiler PCR Array. MAECs were treated with IL-17 for 24 h and used in the Mouse Endothelial Cell Biology RT2 Profiler PCR Array. A, 84 genes assessed by the array. B, scatter plot of the changes of gene expression in IL-17-treated MAECs. Expressions of four genes were dramatically induced by IL-17 treatment; blue lines indicate a -fold change of 4. C, IL-17-mediated increases in the expressions of CXCL1, CXCL2, IL-6, and CSF2 (GM-CSF) were further confirmed in MAECs by quantitative PCR. Data are presented as means ± S.E. (n = 5). *, p < 0.05; **, p < 0.01; ***p < 0.001 versus control. D, human IL-17 treatment significantly enhanced the expressions of CXCL1, CXCL2, IL-6, and CSF2 in HAECs treated with IL-17 (100 ng/ml) for 12 h as detected by qRT-PCR. Data were normalized to β-actin expression and are presented as means ± S.E. (n = 3). *, p < 0.05 versus control. E, the blockage of proinflammatory cytokines IL-6 and GM-CSF and chemokines CXCL1 and CXCL2 with specific antibodies decreased non-treated monocyte adhesion to IL-17-stimulated endothelial cells. Endothelial cells were treated with IL-17, and the blocking antibodies against CXCL1/2, IL-6, and/or GM-CSF were added 30 min prior to the adhesion assay. Antibodies against CXCL1/2, IL-6, and GM-CSF reversed the effect of IL-17 on increasing monocyte adhesion to endothelial cells relative to the IgG control. F, fluorescence microscopic image of labeled non-treated THP-1 cells adhered to endothelial cells. Data are presented as means ± S.E. (error bars) (n = 3). *, p < 0.05 versus control; #, p < 0.05 versus IL-17-treated group.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Blocking Assay, Cell Adhesion Assay, Fluorescence, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: IL-17 and IL-6 synergistically induce ICAM-1 up-regulation in HAECs. HAECs were treated with IL-6 (20 ng/ml), IL-17 (50 ng/ml), or IL-6 (20 ng/ml) + IL-17 (50 ng/ml) for 6 h. After the treatment, cells were collected, and ICAM-1 expression was quantified by a flow cytometry method with fluorescence-conjugated anti-ICAM-1 antibody. A, histograms showing an increase of ICAM-1 expression after IL-17 (50 ng/ml) treatment that was further increased when the cells were treated with IL-6 (20 ng/ml) plus IL-17 (50 ng/ml). B, bar graph showing quantification of ICAM-1 expression in HAECs. Data are presented as mean ± S.E. (error bars) (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Mouse aortic microarray results indicate that IL-17 up-regulates the expressions of many genes in mouse aortas including IL-17RA, endothelial cell adhesion molecules, and MAPK pathway components. A, mouse aortic RNAs were isolated from ApoE−/−mice and IL-17−/−/ApoE−/− mice fed with a high fat diet for 3 weeks and subjected to Affymetrix microarray analysis. B, volcano plot (scatter plot) of -fold change versus p value for all gene transcripts determined by the Affymetrix GeneChip Mouse Gene 2.0ST Arrays in mouse aortas from ApoE−/−mice and IL-17−/−/ApoE−/− mice fed with a high fat diet for 3 weeks. The volcano plot shows that there are numerous genes that were modified with statistical significance in IL-17−/−/ApoE−/− mouse aortas relative to ApoE−/− mouse aortas. C, the heat map and -fold changes of IL-17 receptor signaling genes. D, heat map and -fold changes of endothelial adhesion molecule genes. E, heat map and -fold changes of p38 MAPK (MAPK14) pathway genes. The values with p < 0.05 are highlighted in red.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Microarray, Isolation, Modification

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Depletion of IL-17 in ApoE−/− mice decreases the phosphorylation of p38 MAPK but not the phosphorylation of JNK MAPK nor the phosphorylation of eNOS in mouse aortas, and IL-17 also induces the phosphorylation of p38 MAPK in vitro. A, mouse aortic proteins were isolated from ApoE−/−mice and IL-17−/−/ApoE−/− mice fed with a high fat diet for 3 weeks. Representative Western blotting analyses of phosphorylated and total p38, phosphorylated and total JNK, and phosphorylated and total eNOS, and β-actin expressions are shown. B, quantification of total p38. C, quantification of total JNK. D, quantification of the phosphorylated p38. E, quantification of phosphorylated JNK. F, schematic representation of how IL-17 transcriptionally and post-translationally activates the p38 MAPK pathway but not JNK MAPK pathway. G, quantitation of phosphorylated eNOS (Ser-1177). H, quantification of phosphorylated eNOS (Thr-495). I, IL-17 treatment induced the phosphorylation of p38 MAPK and its upstream kinase Map2k3 in HAECs. Representative Western blotting analyses of phosphorylated p38 MAPK, total p38 MAPK, phosphorylated Map2k3, and total Map2k3 are shown. J, IL-17 treatment did not induce JNK and its upstream kinase Map2k4 in HAECs. Western blotting analyses of phosphorylated JNK, total JNK, phosphorylated Map2k4, and total Map2k4 are represented. Data are presented as means ± S.E. (error bars). *, p < 0.05 (significant); n.s., not significant.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: In Vitro, Isolation, Western Blot, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Inhibition of IL-17-mediated phosphorylation of p38 MAPK attenuates the inflammation and endothelial activation in human aortic endothelial cells in vitro. A, p38 MAPK deficiency decreased IL-6 and ICAM-1 gene expressions. Microarray database mining analysis was carried out by analyzing the National Institutes of Health NCBI GEO data set GSE7342, which studied the role of p38α in regulating the gene expression in mouse embryonic development. B, inhibition of p38 MAPK decreased the expression of ICAM-1 in IL-17-treated HAECs. HAECs were pretreated for 1 h with p38 inhibitor (SB203580; 10 μm) followed by IL-17 (100 ng/ml) treatment for 6 h. The cells were stained with fluorescent dye allophycocyanin-conjugated ICAM-1 antibody for 30 min and analyzed by flow cytometry with IgG isotype control. C and D, the HAECs were pretreated with SB203580 (10 μm) for 1 h followed by IL-17 treatment (100 ng/ml) for 24 h. The CXCL1/2 gene expression was measured by quantitative PCR. C, p38 MAPK inhibitor significantly ameliorated IL-17-induced CXCL1 gene expression. D, p38 MAPK inhibitor significantly ameliorated CXCL2 gene expression mediated by IL-17. E, p38 MAPK inhibitor significantly attenuated the IL-17-induced non-treated monocyte adhesion to HAECs. The HAECs were pretreated with SB203580 (10 μm) for 1 h prior to incubation with IL-17 (100 ng/ml) for 24 h. Data are presented as means ± S.E. (error bars). ***, p < 0.001; **, p < 0.01; *, p < 0.05.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Inhibition, Activation Assay, In Vitro, Microarray, Expressing, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Deficiency of IL-17A in ApoE−/− mice modulates the lipid profile but does not significantly affect atherosclerotic lesion formation. A, mouse experimental design. Male ApoE−/− mice and IL-17−/−/ApoE−/− mice were put on either a high fat diet or regular chow diet at 8 weeks of age for 3 weeks. B, body weight, heart weight, and spleen weight of ApoE−/− mice and IL-17−/−/ApoE−/− mice that were on either a normal chow diet or a high fat diet for 3 weeks. Data are presented as means ± S.E. (n = 7–10). C, the plasma lipid profiles of mice that were fed a high fat (HF) diet or normal chow diet for 3 weeks. FFA, free fatty acids. Data are presented as means ± S.E. (n = 7–10). *, p < 0.05. D, atherosclerotic plaque formation in the whole aorta was assessed with Sudan IV staining. The upper panel represents the images of 3-week-diet mouse group aortas stained with Sudan IV. The lower panel indicates the quantification of atherosclerotic area percentage in the whole aorta of the 3-week-diet mouse group. Data are presented as means ± S.E. (n = 4–11). E, atherosclerotic plaque in aortic sinus was determined by Oil Red O staining of aortic cross-sections. The upper panel represents the images of aortic sinus cross-section stained with Oil Red O in 3-week-diet mouse groups. The lower panel shows the quantification of atherosclerotic area percentage in the aortic sinus of 3-week-diet mouse groups. Data are presented as means ± S.E. (error bars) (n = 5).

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: The proposed novel working model. IL-17 promotes endothelial cell activation by inducing p38 MAPK, which leads to up-regulation of proinflammatory cytokines, chemokines, and endothelial cell adhesion molecules.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Activation Assay

Journal: Advanced Science

Article Title: Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet‐Induced Adiposity

doi: 10.1002/advs.202207152

Figure Lengend Snippet: Rare and low‐frequency RSPO1 nonsynonymous variants identified in young Chinese with obesity and controls

Article Snippet: SVF cells from wild‐type and Lgr4 m/m mice were treated with 100 ng mL −1 recombinant human RSPO1 protein (hRSPO1) (R&D, 4645‐RS), 100 ng mL −1 Wnt3a or their combination.

Techniques:

Journal: Advanced Science

Article Title: Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet‐Induced Adiposity

doi: 10.1002/advs.202207152

Figure Lengend Snippet: RSPO1 is enriched in visceral fat and increases with adiposity. A) TOP‐Flash luciferase reporter assay of HEK293T cells transfected with plasmids of wild‐type RSPO1 and 18 rare/low‐frequency RSPO1 nonsynonymous variants described in Table , respectively. β ‐catenin plasmids were applied as positive control ( n = 5 per group). Statistical significances were calculated between wild‐type and each variant using unpaired Student's t ‐test. B) Heat map representing the expression of mouse Rspo1/2/3/4 in diverse tissues and cell lines, as analyzed using a publicly available microarray data set (GSE10246). C) Quantitative PCR validation of Rspo1 expression in multiple tissues of 8‐week‐old male C57BL/6J mice ( n = 3–6 per group). D) Rspo1 mRNA expression in the stromal vascular fractions (SVF) and mature adipocytes (AD) of eWAT, iWAT, and BAT, respectively ( n = 3 per group). E,F) Rspo1 mRNA expression in iWAT and eWAT of mice fed normal chow diet (NCD) and high‐fat diet (HFD) ( n = 13–16 per group) (E), or in that of wild‐type (WT) and ob / ob mice ( n = 10 per group) (F). G) RSPO1 mRNA expression in the subcutaneous adipose tissue (SAT) and visceral adipose tissues (VAT) of obese subjects versus normal weight controls ( n = 10–17 per group). eWAT, epididymal white adipose tissue; pWAT, pararenal white adipose tissue; iWAT, inguinal white adipose tissue; BAT, brown adipose tissue. SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue. Data are shown as the mean ± sem. p values were calculated using unpaired Student's t ‐test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: SVF cells from wild‐type and Lgr4 m/m mice were treated with 100 ng mL −1 recombinant human RSPO1 protein (hRSPO1) (R&D, 4645‐RS), 100 ng mL −1 Wnt3a or their combination.

Techniques: Luciferase, Reporter Assay, Transfection, Positive Control, Variant Assay, Expressing, Microarray, Real-time Polymerase Chain Reaction

Journal: Advanced Science

Article Title: Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet‐Induced Adiposity

doi: 10.1002/advs.202207152

Figure Lengend Snippet: Human RSPO1 overexpression promotes diet‐induced obesity by suppressing adipose thermogenesis. A–C) A representative global image (A), body weight curve (B), and body composition (C) of human RSPO1 transgenic (h RSPO1 Tg ) and WT littermate mice fed HFD for 14 weeks ( n = 8 per group). D,E) ANCOVA analysis of 24‐hour O 2 consumption (D) and CO 2 production (E) with body weight as a covariate in HFD‐fed WT and h RSPO1 Tg mice ( n = 8 per group). F) Representative images of H&E staining and UCP1 immunohistochemical staining of BAT in HFD‐fed WT and h RSPO1 Tg mice ( n = 4 per group). Scale bar, 100 µm. G) The mitochondrial DNA (mtDNA) content of BAT in HFD‐fed WT and h RSPO1 Tg mice ( n = 6–7 for per group). H,I) Quantitative PCR analysis (H) and Western blotting (I) of mitochondrial respiratory complexes and thermogenic genes in BAT of HFD‐fed WT and h RSPO1 Tg mice ( n = 6–7 per group). J) Representative images of H&E staining and UCP1 immunohistochemical staining in iWAT of WT and h RSPO1 Tg mice exposed to chronic cold stimulation (4 °C) for 10 days ( n = 4 per group). Scale bar, 100 µm. K) The volcano plot of genes differentially expressed in iWAT of h RSPO1 Tg versus WT mice under chronic cold stimulation ( n = 3 per group). Biomarkers associated with mitochondrial functions were indicated. L) The top‐downregulated (FDR < 0.05) pathways revealed by GSEA based on GO:BP database in iWAT of h RSPO1 Tg versus WT mice under chronic cold stimulation. (M) and (N) GSEA results of mitochondrial gene expression (M) and adaptive thermogenesis (N) in iWAT of h RSPO1 Tg versus WT mice under chronic cold stimulation. O–Q) Relative mtDNA content (O), quantitative PCR analysis (P), and Western blotting analysis (Q) of mitochondrial respiratory complexes and thermogenic genes in iWAT of h RSPO1 Tg and WT mice under chronic cold stimulation ( n = 7–8 per group). Data are shown as the mean ± sem, and statistical differences between genotypes were assessed by unpaired Student's t ‐test (B,C,G,H,O,P); FDR below 0.05 was considered as the criteria for evaluating differentially expressed genes between genotypes (K); FDR below 0.1 was considered as statistical significance in GSEA (L–N). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: SVF cells from wild‐type and Lgr4 m/m mice were treated with 100 ng mL −1 recombinant human RSPO1 protein (hRSPO1) (R&D, 4645‐RS), 100 ng mL −1 Wnt3a or their combination.

Techniques: Over Expression, Transgenic Assay, Staining, Immunohistochemical staining, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: Advanced Science

Article Title: Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet‐Induced Adiposity

doi: 10.1002/advs.202207152

Figure Lengend Snippet: Rspo1 deficiency promotes adipose thermogenesis and resists HFD‐induced obesity. A–C) A representative global image (A), body weight curve ( n = 15–22 per group) (B), and body composition ( n = 7–8 per group) (C) of Rspo1 −/− and WT mice fed HFD for 13 weeks. D) Hourly (left) and total (right) energy expenditure over 24 h between Rspo1 −/− and WT mice fed with 1‐week HFD ( n = 8 per group). The hourly measurements were assessed by two‐way ANOVA model to evaluate the interaction between genotype and time, and pairwised t ‐test with Benjamini–Hochberg correction was used as post‐hoc test for evaluating the differences between genotypes in each hour. The total energy expenditure over 24 h was compared with unpaired Student's t ‐test. E) Representative images of H&E staining and UCP1 immunohistochemical staining in BAT of WT and Rspo1 −/− mice fed HFD ( n = 4 per group). Scale bar, 100 µm. F) The mtDNA content of BAT in Rspo1 −/− and WT mice fed HFD ( n = 7–8 per group). G) Representative images of H&E staining and UCP1 immunohistochemical staining in eWAT of WT and Rspo1 −/− mice exposed to chronic cold stimulation (4 °C) for 10 days ( n = 4 per group). Scale bar, 100 µm. H) The volcano plot of genes differentially expressed in eWAT of Rspo1 −/− versus WT mice under chronic cold stimulation ( n = 3 per group). Biomarkers associated with mitochondrial functions were indicated. I) The top‐upregulated pathways (FDR < 0.05) revealed by GSEA analysis based on GO:BP database in eWAT of Rspo1 −/− versus WT mice under chronic cold stimulation. J,K) GSEA results of mitochondrial gene expression (J) and adaptive thermogenesis (K) in eWAT of Rspo1 −/− versus WT mice under chronic cold stimulation. L,N) Relative mtDNA content (L), quantitative PCR analysis (M), and Western blotting (N) analysis of mitochondrial respiratory complexes and thermogenic genes in eWAT of Rspo1 −/− and WT mice under chronic cold stimulation ( n = 7–8 per group). Data are shown as the mean ± sem, and statistical differences between genotypes were assessed by unpaired Student's t ‐test (B,C,F,L,M); FDR below 0.05 was considered as the criteria for evaluating differential expressed genes between genotypes (H); FDR below 0.1 was considered as statistical significance in GSEA (I–K). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: SVF cells from wild‐type and Lgr4 m/m mice were treated with 100 ng mL −1 recombinant human RSPO1 protein (hRSPO1) (R&D, 4645‐RS), 100 ng mL −1 Wnt3a or their combination.

Techniques: Staining, Immunohistochemical staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Advanced Science

Article Title: Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet‐Induced Adiposity

doi: 10.1002/advs.202207152

Figure Lengend Snippet: Human RSPO1 protein suppresses beige adipocyte thermogenesis in an Lgr4/ β ‐catenin dependent manner. A) Quantitative PCR analysis of Rspo1 mRNA levels in eWAT (upper panels) and iWAT (bottom panels) in response to either chronic cold exposure (right panels) ( n = 7–8 per group) or β 3‐AR agonist (CL316243) injection (right panels) ( n = 6 per group). B,C) Changes of mitochondrial respiratory complexes and thermogenic proteins (UCP1 and PGC‐1 α ) (B) and OCR (C) in fully differentiated beige adipocytes with human RSPO1 (hRSPO1) recombinant protein or PBS treatment ( n = 5 per group). D,E) Changes of mitochondrial respiratory complexes and thermogenic proteins (D) and OCR (E) in fully differentiated beige adipocytes treated with Rspo1‐shRNA (shRspo1) or vector‐shRNA (LV) ( n = 4 per group). F,G) Quantitative PCR analysis of thermogenic genes and mitochondrial respiratory complexes (F) and protein quantification of UCP1 and PGC‐1 α (G) in fully differentiated beige adipocytes derived from WT and Lgr4 m/m mice in response to hRSPO1 or PBS treatment, respectively ( n = 4–6 per group). H) Alterations of cytoplasmic and nuclear β ‐catenin protein in the SVFs derived from WT and Lgr4 m/m mice in response to hRSPO1, Wnt3a, and their combinations. I,J) Quantitative PCR analysis of thermogenic genes and mitochondrial respiratory complexes (I) and protein quantification of UCP1 and PGC‐1 α (J) in fully differentiated beige adipocytes treated with hRSPO1 or PBS in the presence or absence of IWR‐endo1, respectively ( n = 3 per group). WT, wild‐type mice; Lgr4 m/m , Lgr4 mutant mice. Data are shown as the mean ± sem. Statistical differences between groups were assessed by unpaired Student's t ‐test (A,C,E,F,I). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: SVF cells from wild‐type and Lgr4 m/m mice were treated with 100 ng mL −1 recombinant human RSPO1 protein (hRSPO1) (R&D, 4645‐RS), 100 ng mL −1 Wnt3a or their combination.

Techniques: Real-time Polymerase Chain Reaction, Injection, Recombinant, shRNA, Plasmid Preparation, Derivative Assay, Mutagenesis

Journal: Advanced Science

Article Title: Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet‐Induced Adiposity

doi: 10.1002/advs.202207152

Figure Lengend Snippet: RSPO1 p.R219W/Q mutations disrupt their electrostatic interaction with the ECM and activate Wnt pathway. A) Schematic representation of the full‐length human RSPO1 protein and the location of p.R219W/Q mutations in the C‐terminal (CT) region with a cluster of positively charged amino acid residues (Figure , Supporting Information). SP, N‐terminal signal peptide; FR, furin‐like domains; TSR, thrombospondin protein 1 domain. 12 obese cases and 3 lean controls harboring the mutations were identified in GOCY cohort (Figure , Supporting Information). B) The consensus sequence of the conserved Arginine 219 (R219) residue in RSPO1 protein across different species. C) Protein secretion of wild‐type RSPO1 (WT) and the two mutants (M1, p.R219W; M2, p.R219Q) in conditioned medium (CM) and by extracellular matrix (ECM) and cell lysates in the absence or presence of 50 µg mL −1 heparin, respectively. EV, empty vector. D) RSPO1 protein levels in the conditioned media of the indicated groups (Figure ) measured by ELISA ( n = 3 per group). E) Heparan sulfate (HS) pull‐down assay (Figure , Supporting Information) of RSPO1–HS complex obtained from co‐incubation of HS‐beads and conditioned media (CM) derived from HEK293T cells transfected with wild‐type or mutant RSPO1 plasmids, respectively. Both CM (as Input) and HS‐beads put‐down (as Pull‐down) fractions were immunoblotted with the anti‐Flag antibody. F) A TOP‐Flash luciferase reporter assay was performed in HEK293T cells. The wild‐type and the two mutant RSPO1 plasmids were used for the transfection at the indicated dosages (5, 30, and 40 ng of expression plasmids), and pRL‐SV40 (expressing Renilla luciferase) was used as a normalized control ( n = 3 per group). A representative result of three independent experiments is shown. G) The β ‐catenin translocation examination was performed in HEK293T cells transfected with wild‐type or the two mutant RSPO1 plasmids, and treated with PBS or 100 ng mL −1 Wnt3a, respectively. H) A TOP‐Flash luciferase reporter assay was performed in HEK293T cells treated with conditioned media, collected from HEK293T cells transfected with empty vector, wild‐type and two mutant RSPO1 plasmids, in the presence or absence of 2 µg mL −1 RSPO1 neutralizing antibody ( n = 3–4 per group). Data are shown as the mean ± sem. Statistical differences between groups were assessed by unpaired Student's t ‐test (D,F,H) * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: SVF cells from wild‐type and Lgr4 m/m mice were treated with 100 ng mL −1 recombinant human RSPO1 protein (hRSPO1) (R&D, 4645‐RS), 100 ng mL −1 Wnt3a or their combination.

Techniques: Sequencing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Pull Down Assay, Incubation, Derivative Assay, Transfection, Mutagenesis, Luciferase, Reporter Assay, Expressing, Translocation Assay

Journal: Advanced Science

Article Title: Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet‐Induced Adiposity

doi: 10.1002/advs.202207152

Figure Lengend Snippet: Homologous Rspo1 p.R219W mutation suppresses fat thermogenesis and leads to obesity in vivo. A–D) A representative global image (A), body weight curve ( n = 9–11 per group) (B), body composition (C), and the weight of three fat tissues (D) of female homozygous Rspo1 R219W and WT littermate mice fed HFD for 10 weeks ( n = 8 per group). E–G) O 2 consumption, CO 2 production, and energy expenditure of WT and Rspo1 R219W mice fed chow diet shifting from room temperature (22 °C, RT) to cold conditions (4 °C, cold) ( n = 12 per group). The hourly measurements were assessed by two‐way ANOVA model to evaluate the interaction between genotype and time, and pairwised t ‐test with Benjamini–Hochberg correction was used as post‐hoc test to evaluate the differences between genotypes in each hour. H–K) Representative images of H&E staining (H), UCP1 immunofluorescence staining (I), protein expression changes of mitochondrial respiratory complexes and thermogenic genes (J), and electron microscopic images of mitochondria (K) in iWAT of WT and Rspo1 R219W mice under chronic cold stimulation (4 °C) for 10 days ( n = 3 per group). UCP1 (red) and perilipin protein (green) were used to indicate beige adipocytes and lipid droplets, respectively. Scale bars are indicated in the panels. L) Quantitative PCR analysis of thermogenic genes in fully differentiated beige adipocytes derived from the iWAT of WT and Rspo1 R219W mice in the presence or absence of IWR‐endo1 treatment ( n = 4 per group). Data are shown as the mean ± sem, and statistical significances between groups were assessed by unpaired Student's t ‐test (B–D,L). * p < 0.05; ** p < 0.01; *** p <0.001.

Article Snippet: SVF cells from wild‐type and Lgr4 m/m mice were treated with 100 ng mL −1 recombinant human RSPO1 protein (hRSPO1) (R&D, 4645‐RS), 100 ng mL −1 Wnt3a or their combination.

Techniques: Mutagenesis, In Vivo, Staining, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Systematic comparison of barcoding strategies used in the category of molecular barcodes.

Article Snippet: Primer-associated approach , Sequence-based barcodes , Two unique barcodes embedded in primers at the stage of RT , + (the left and the right barcodes) , + , S , n/a , Commercial pooled human saliva from healthy individuals with spiked-in synthetic viral RNA , INSIGHT [isothermal NASBA (nucleic acid sequence–based amplification) sequencing–based high- throughput test]; Illumina MiSeq (PE 150bp) , FASTX_trimmer , Multiplex samples , Diagnostics (48 samples) , .

Techniques: Software, Sequencing, Multiplex Assay, CRISPR, Plasmid Preparation, Microarray, Binding Assay, Amplification, Ligation, DNA Sequencing, Multiplexing, Generated, Staining, Flow Cytometry, High Throughput Screening Assay, Inhibition, Blocking Assay, Conjugation Assay, RNA Sequencing Assay, Transmission Assay, Incubation, Diagnostic Assay, Next-Generation Sequencing, Infection

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: Upregulation of CXCL1 in DCs isolated from mice and patients with CRC. Increased CXCL1, CXCL2 and CXCL3 ( A ) in SW620-conditioned TADCs, as determined by microarray. Elevated CXCL1 in SW620-conditioned TADCs at mRNA ( B ) and protein ( C ) levels. TADCs were generated by culturing CD14 + monocytes with RPMI, L-15 medium (50%), and SW620-CM (50%) and presenting in GM-CSF (10 ng/mL) and IL-4 (10 ng/mL) for five days. The expressions of mRNA and protein were assessed by microarray, qRT-PCR and Luminex assays. ( D ) The level of CXCL1 in the DCs isolated from patients with CRC. CD11c + cells were isolated from healthy donors and patients with CRC, and the levels of CXCL1 were measured by Luminex assay. ( E ) The levels of CXCL1 in DCs isolated from colon cancer-bearing mice. Mouse colon cancer CT26 cells were injected into mice via intraperitoneal injection. After 14 days, peritoneal lymph nodes were harvested. CD11c + DCs were isolated from the lymph nodes, and the culture medium collected after 24 h incubation. CXCL1 levels were determined by ELISA. Results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Isolation, Microarray, Generated, Quantitative RT-PCR, Luminex, Injection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: CXCL1 increased cancer stem cell (CSC) properties in SW620 colon cancer cells. Anchorage-independent growth ( A ) and tumor spheroid formation ( B ) of CXCL1-treated SW620 cells. The expression of CD44, CD326 and CD133 ( C ) and aldehyde dehydrogenase (ALDH) activity ( D ) in tumor spheroids. SW620 cells were cultured with CXCL1 protein in soft agar (0.4%) or ultra-low attachment wells for 40 and 10 days, respectively. The tumor spheroids were counted by microscope. ALDH activity and surface markers of tumor spheroids were determined by ALDEFLUOR assay and flow cytometry. ( E ) Level of CSC-related transcription factors in tumor spheroids. Results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Activity Assay, Cell Culture, Microscopy, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: CXCL1 enhanced cancer progression in SW620 cells. CXCL1 increased cell migration, as determined by wound healing ( A ) and transwell ( B ) analysis. The migration ability of SW620 cancer cells was assessed by wound healing assay and transwell assay. CXCL1 acted as a chemoattractant for cancer migration in the transwell system for 24 h. The effect of CXCL1 on the expression of MMP-7 ( C ), EMMPRIN ( D ) and epithelial-to-mesenchymal transition (EMT) ( E ) in SW620 cells. SW620 cells were treated with CXCL1 for 24 h, and the levels of various MMPs and EMT markers were determined by Luminex assay and Western blot. Results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Migration, Wound Healing Assay, Transwell Assay, Expressing, Luminex, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: Gene Profile of CXCL1-Treated SW620 Cells.

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: The correlation of CXCL1-regulated genes with CRC patient outcome. The correlation of overall survival ( A ), relapse-free ( B ) and metastasis-free rates ( C ) with CXCL-1 regulated genes. ( D ) The effect of CXCL1 in the expression of PTHLH and TCF4 at mRNA levels. CXCL1 increased the expression of PTHrP ( E ) in SW620 cells. SW620 cells were treated with CXCL1 for 24 h, and the expressions of PTHLH and TCF4 were determined by qRT-PCR and ELISA, respectively. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: The effect of CXCL1 on the expression of microRNAs (miRNAs) in colon cancer. ( A ) The regulatory effect of CXCL1 on the expressions of miRNAs in colon cancer. Prognostic significance of miR-105 and miR-597 in overall survival ( B ), relapse-free ( C ) and metastasis-free rates ( D ) in colon cancer patients. The effect of CXCL1 on the levels of miR-105 ( E ) and miR-597 ( F ) in colon cancer. SW620 cells were treated with CXCL1 for 24 h, and the expression of miR-105 and miR-597 determined by qRT-PCR. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*). Hsa, Homo sapiens; TCGA, The Cancer Genome Atlas; HR, hazard ratio; PVAL, p -value.

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: Proposed model of TADCs in colon cancer. Soluble factors in colon cancer cause abnormal phenotypes of dendritic cells, which in turn increase cancer stem cell properties, cell mobility, and EMT of colon cancer by producing the inflammatory chemokine CXCL1. Transcriptome analysis indicates that CXCL1 increases potential oncogene expression in colon cancer, including PTHLH , TYRP1 , FOXO1 , TCF4 , ZNF880 and the onco-miR miR-105. Our study indicates a new mechanism by which the colon cancer milieu exploits DC plasticity to support cancer progression.

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing

Journal: Immunity

Article Title: Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche

doi: 10.1016/j.immuni.2019.08.017

Figure Lengend Snippet:

Article Snippet: Recombinant Human/Mouse/Rat BMP-2 Protein , RnD Systems , Cat#355-BM-010.

Techniques: Control, Recombinant, Blocking Assay, Irradiation, Enzyme-linked Immunosorbent Assay, Microarray, Software, Microscopy

Journal: Antiviral research

Article Title: Aberrant regulation of the Akt signaling network by human cytomegalovirus allows for targeting of infected monocytes.

doi: 10.1016/j.antiviral.2018.07.015

Figure Lengend Snippet: Primary monocytes were mock infected, HCMV infected, GM-CSF treated, or M-CSF treated for 24 hours. Monocytes were also pretreated with MK (an AKT inhibitor) for one hour prior to a 24 h infection. Cells were then lysed and subjected to protein microarray analysis from Fullmoon Biosystems. Data are the mean from 3 independent blood donors.

Article Snippet: Mock infection was performed by adding an equivalent volume of RPMI 1640 medium to monocytes, while GM-CSF or M-CSF treatment was performed by adding an equivalent volume of RPMI 1640 medium with recombinant human GM-CSF or M-CSF at 100 ng/ml (R&D Systems, Minneapolis, MI).

Techniques: Infection, Microarray

Journal: Antiviral research

Article Title: Aberrant regulation of the Akt signaling network by human cytomegalovirus allows for targeting of infected monocytes.

doi: 10.1016/j.antiviral.2018.07.015

Figure Lengend Snippet: (A-B) Peripheral blood monocytes were mock or HCMV infected, or treated with GM-CSF or M-CSF for (A) 24 h or (B) 30 m. (C-D) Monocytes were pretreated with LY294002 (a PI3K inhibitor), MK, or rapamycin (a mTOR inhibitor) for 1 h. Following treatment with inhibitors, cells were mock or HCMV infected for (C) 24 h or (D) 30 min. (A-D) Levels of p-S6K (T389), and S6K were detected by immunoblotting from whole cell lysates. Membranes were then reprobed for β-actin as a loading control. Data are representative of 3–6 independent blood donors.

Article Snippet: Mock infection was performed by adding an equivalent volume of RPMI 1640 medium to monocytes, while GM-CSF or M-CSF treatment was performed by adding an equivalent volume of RPMI 1640 medium with recombinant human GM-CSF or M-CSF at 100 ng/ml (R&D Systems, Minneapolis, MI).

Techniques: Infection, Western Blot

Journal: Antiviral research

Article Title: Aberrant regulation of the Akt signaling network by human cytomegalovirus allows for targeting of infected monocytes.

doi: 10.1016/j.antiviral.2018.07.015

Figure Lengend Snippet: (A) Monocytes were mock or HCMV infected, or treated with GM-CSF or M-CSF for 24 h. (B) Monocytes were infected with HCMV strain TB40E or Towne/E for 24 h. (C-D) Monocytes were pretreated with LY, MK, or rapamycin for 1 h. Cells were then mock or HCMV infected for 24 h. (A-D) Levels of p-XIAP and XIAP were determined by immunoblotting. Membranes were then reprobed for β-actin as a loading control. Data are representative of 3–6 independent blood donors.

Article Snippet: Mock infection was performed by adding an equivalent volume of RPMI 1640 medium to monocytes, while GM-CSF or M-CSF treatment was performed by adding an equivalent volume of RPMI 1640 medium with recombinant human GM-CSF or M-CSF at 100 ng/ml (R&D Systems, Minneapolis, MI).

Techniques: Infection, Western Blot

Journal: Antiviral research

Article Title: Aberrant regulation of the Akt signaling network by human cytomegalovirus allows for targeting of infected monocytes.

doi: 10.1016/j.antiviral.2018.07.015

Figure Lengend Snippet: (A-B) Peripheral blood monocytes were mock or HCMV infected, or treated with GM-CSF or M-CSF for (A) 30 m or (B) 24 h. (C) Monocytes were pretreated with 4EGI-1 (an eIF4E inhibitor) for 1 h, then mock or HCMV infected for 24 h. Levels of Mcl-1, HSP27, and XIAP expression were determined by immunoblotting. (D-E) Monocytes were pretreated with LY294002, MK, or rapamycin for 1 h. Following pretreatment, cells were mock or HCMV infected for (D) 30 m or (E) 24 h. 4E-BP1 and p-4E-BP1 expression were determined by immunoblotting. Electrophoresis of 4E-BP1 separates into three different forms (Constantinou and Clemens, 2005). The γ band corresponds to the most highly phosphorylated form of 4E-BP1, whereas the β and α bands represent the intermediate and least phosphorylated form, respectively. (A-E) Membranes were reprobed for β-actin as a loading control. Data are representative of 4–6 independent blood donors.

Article Snippet: Mock infection was performed by adding an equivalent volume of RPMI 1640 medium to monocytes, while GM-CSF or M-CSF treatment was performed by adding an equivalent volume of RPMI 1640 medium with recombinant human GM-CSF or M-CSF at 100 ng/ml (R&D Systems, Minneapolis, MI).

Techniques: Infection, Expressing, Western Blot, Electrophoresis

Journal: Antiviral research

Article Title: Aberrant regulation of the Akt signaling network by human cytomegalovirus allows for targeting of infected monocytes.

doi: 10.1016/j.antiviral.2018.07.015

Figure Lengend Snippet: (A-B) Monocytes were mock or HCMV infected, or treated with GM-CSF or M-CSF for (A) 30 m or (B) 24 h. (C-D) Monocytes were pretreated with LY294002, MK, or rapamycin for 1 h, after which cells were either mock or HCMV infected for (C) 30 m or (D) 24 h. (A-D) HSF1 and p-HSF1 expression were determined by immunoblotting. (E-F) Prior to 24 h mock or HCMV infection, monocytes were pretreated with KRIBB11 (a HSF1 inhibitor) for 1 h. (E) mTOR and p-mTOR or (F) HSF1, p-HSF1, Mcl-1, HSP-27, and XIAP expression were assessed by immunoblot. (A-F) Membranes were then reprobed for β-actin as a loading control. Data are representative of 3–5 independent blood donors.

Article Snippet: Mock infection was performed by adding an equivalent volume of RPMI 1640 medium to monocytes, while GM-CSF or M-CSF treatment was performed by adding an equivalent volume of RPMI 1640 medium with recombinant human GM-CSF or M-CSF at 100 ng/ml (R&D Systems, Minneapolis, MI).

Techniques: Infection, Expressing, Western Blot