Journal: Cells

Article Title: Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins

doi: 10.3390/cells10020274

Figure Lengend Snippet: Galectins induce CXCL10 in a cell type-specific manner. ( A ) SDS-polyacrylamide gel electrophoresis of recombinant human galectins (rhGal) stained with Coomassie Blue. Molecular weights of protein standards are indicated on the left. The hemagglutination assay was performed using serial two-fold dilutions of the recombinant protein. The bottom row contains protein incubated with β-lactose, a competitive carbohydrate inhibitor of galectin binding. ( B ) Cultures of human corneal fibroblasts, human corneal epithelial cells, THP-1 monocytes and human umbilical vein endothelial cells (HUVECs) were incubated with 50 µg/mL rhGal-1, -3 and -8 or 50 µg/mL IFN-γ. Quantitative real-time PCR was used to determine the levels of CXCL10 mRNA after 6 h of incubation ( n = 3 independent experiments), whereas ELISA was used to determine the levels of CXCL10 protein in cell culture supernatants after 24 h of incubation ( n = 6 independent experiments). The data represent the mean ± SEM.

Article Snippet: A mouse monoclonal antibody against CXCL10 (1.2 μg/mL; MAB266, R&D Systems) was added to the lower compartment of the chamber in neutralization studies.

Techniques: Polyacrylamide Gel Electrophoresis, Recombinant, Staining, Hemagglutination Assay, Incubation, Binding Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cells

Article Title: Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins

doi: 10.3390/cells10020274

Figure Lengend Snippet: Galectins differentially dictate fibroblast-dependent immune cell chemotaxis. ( A ) Recombinant human galectin (rhGal)-1, -3 and -8 were incubated with fibroblasts for 24 h. The conditioned media were pre-cleared with β-lactose Sepharose beads three times to remove galectins prior to chemotaxis assays. The presence of galectins in pre-cleared conditioned media was evaluated by immunoblotting. ( B ) Quantification by flow cytometry of the migration of CFSE-labeled THP-1 and Jurkat cells to conditioned media from fibroblasts treated for 24 h with increasing concentrations of recombinant galectins or BSA ( n = 3 independent experiments performed in duplicate). ( C ) Migration of CFSE-labeled THP-1 and Jurkat cells to conditioned media from fibroblasts treated for 24 h with 50 μg/mL of recombinant galectins or BSA. A CXCL10 neutralizing antibody was added to the conditioned media in neutralization studies ( n = 4–5 independent experiments in duplicate). The data in ( B ) represent the mean ± SEM. The box and whisker plots show the 25 and 75 percentiles (box), the median, and the minimum and maximum data values (whiskers). Significance was determined using Student’s t test. ** p < 0.01.

Article Snippet: A mouse monoclonal antibody against CXCL10 (1.2 μg/mL; MAB266, R&D Systems) was added to the lower compartment of the chamber in neutralization studies.

Techniques: Chemotaxis Assay, Recombinant, Incubation, Western Blot, Flow Cytometry, Migration, Labeling, Neutralization, Whisker Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Trophoblast-Induced Changes in C-X-C Motif Chemokine 10 Expression Contribute to Vascular Smooth Muscle Cell Dedifferentiation During Spiral Artery Remodeling

doi: 10.1161/atvbaha.112.300354

Figure Lengend Snippet: Figure 2. C-X-C motif chemokine 10 (CXCL10), stanniocalcin-1, and placental growth factor mRNA expression is significantly increased by trophoblast conditioned media (CM). Messenger RNA expression in vascular spheroids was measured by quantitative real-time PCR after stimulation with trophoblast CM for 24 hours (n=4). Data are expressed as mRNA expression relative to an external calibrator. A, CXCL10. B, Stanniocalcin-1. C, Placental growth factor. All data are presented as mean±SEM. *P<0.05.

Article Snippet: CXCL10 recombinant protein was purchased from R & D Systems (Abingdon, UK).

Techniques: Expressing, RNA Expression, Real-time Polymerase Chain Reaction

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Trophoblast-Induced Changes in C-X-C Motif Chemokine 10 Expression Contribute to Vascular Smooth Muscle Cell Dedifferentiation During Spiral Artery Remodeling

doi: 10.1161/atvbaha.112.300354

Figure Lengend Snippet: Figure 3. C-X-C motif chemokine 10 (CXCL10) protein is expressed by vascular spheroids stimulated with trophoblast con- ditioned media. A, Vascular spheroid lysates were examined by Western blot analysis for the presence of CXCL10. Tubulin was used as an internal loading control. The image shown is represen- tative of 3 independent experiments. Densitometric analysis of Western blots (n=3) with mean±SEM CXCL10/tubulin presented as a fold-change over control-treated spheroids. *P<0.05. Vascu- lar spheroids treated with control media (B) or trophoblast condi- tioned media (C) were cryosectioned and sections were examined by immunostaining for the presence of CXCL10. Sections were taken through approximately the center of the spheroid. Negative control incubated with rabbit IgG in place of primary antibody is inset. Scale bar,100 µm.

Article Snippet: CXCL10 recombinant protein was purchased from R & D Systems (Abingdon, UK).

Techniques: Western Blot, Control, Immunostaining, Negative Control, Incubation

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Trophoblast-Induced Changes in C-X-C Motif Chemokine 10 Expression Contribute to Vascular Smooth Muscle Cell Dedifferentiation During Spiral Artery Remodeling

doi: 10.1161/atvbaha.112.300354

Figure Lengend Snippet: Figure 4. C-X-C motif chemokine 10 (CXCL10) protein is expressed by first trimester decidua and dissected spiral arteries stimulated with trophoblast conditioned media. CXCL10 (A and C) and α-smooth muscle actin protein (D) expression was examined by immunohis- tochemistry in serially sectioned first trimester decidua (n=5; representative image shown). CXCL10 and α-smooth muscle actin protein colocalized to the same cells (indicated by arrowheads). Trophoblasts (labeled with CK7, B) were present at this stage of remodeling. Negative control (inset) was incubated with nonimmune IgG in place of primary antibody. Scale bar represents 100 µm or 50 µm in zoom. (E) Expression of CXCL10 (green) and (F) VWF, an endothelial cell marker (red), was examined in a dissected spiral artery treated with extravillous trophoblast (EVT) conditioned media (G, merge). EC indicates endothelial cells; and VSM, vascular smooth muscle. Negative control (inset) was incubated with nonimmune IgG in place of primary antibody. Scale bar, 50 µm.

Article Snippet: CXCL10 recombinant protein was purchased from R & D Systems (Abingdon, UK).

Techniques: Expressing, Labeling, Negative Control, Incubation, Marker

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Trophoblast-Induced Changes in C-X-C Motif Chemokine 10 Expression Contribute to Vascular Smooth Muscle Cell Dedifferentiation During Spiral Artery Remodeling

doi: 10.1161/atvbaha.112.300354

Figure Lengend Snippet: Figure 5. The role of IFN-γ in C-X-C motif chemokine 10 (CXCL10) expression. A, Recombinant IFN-γ induces CXCL10 expression in vascular spheroids. Control or rhIFN-γ was added to spheroids made of endothelial cell (EC) alone, vascular smooth muscle cell (VSMC) alone, or cocultured EC/VSMC. CXCL10 expression was measured by Western blot analysis (n=4). B, Neutralizing IFN-γ in extravillous trophoblast (EVT) conditioned media (CM) decreased vascular spheroid CXCL10 expression. Control media or EVT CM was incubated with IFN-γ–neutralizing antibody or corresponding IgG control and added to EC/VSMC spheroids. CXCL10 expression was determined by Western blot analysis. *P<0.05 (n=5).

Article Snippet: CXCL10 recombinant protein was purchased from R & D Systems (Abingdon, UK).

Techniques: Expressing, Recombinant, Control, Western Blot, Incubation

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Trophoblast-Induced Changes in C-X-C Motif Chemokine 10 Expression Contribute to Vascular Smooth Muscle Cell Dedifferentiation During Spiral Artery Remodeling

doi: 10.1161/atvbaha.112.300354

Figure Lengend Snippet: Figure 6. The effects of extravillous tro- phoblast (EVT) conditioned media (CM) and C-X-C motif chemokine 10 (CXCL10) on vascular smooth muscle cells (VSMCs). VSMCs were incubated for 72 hours in media containing 0.5% FCS, then a further 72 hours with indicated concentrations of EVT CM and recombi- nant human CXCL10 (n=at least 5 inde- pendent experiments). Expression of (A) α-smooth muscle actin and (B) calponin was examined by Western blot analysis. Time-lapse microscopy was used to analyze the effects of rhCXCL10 (C) and EVT CM (D) on VSMC motility during a 24-hour incubation. Data are displayed as the mean±SEM of a minimum of 3 pooled experiments. *P<0.05; **P<0.01.

Article Snippet: CXCL10 recombinant protein was purchased from R & D Systems (Abingdon, UK).

Techniques: Incubation, Expressing, Western Blot, Time-lapse Microscopy

Journal: Science Advances

Article Title: Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

doi: 10.1126/sciadv.aea7017

Figure Lengend Snippet: ( A and B ) Flow cytometry and statistical analysis of CD8 + CD45 + T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups ( n =3 per group). ( C and D ) Flow cytometry and statistical analysis of IFN-γ + CD8 + T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups ( n =3 per group). ( E and F ) Flow cytometry and statistical analysis of Granzyme B + CD8 + T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups ( n =3 per group). ( G ) The secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in the tail skin of mice in the vehicle, 6-OHDA, and 6-OHDA + NE groups were detected by ELISA ( n =4 per group). All vehicle mice were only treated with 0.1% ascorbic acid in 0.9% sterile NaCl. Error bars represent mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Sterility

Journal: Science Advances

Article Title: Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

doi: 10.1126/sciadv.aea7017

Figure Lengend Snippet: ( A ) Volcano plots illustrated the expression of adrenergic receptors on fibroblasts in human skin, including ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, and ADRB3. ( B ) Bubble map illustrated the up-regulation of ADRA2A expression on fibroblasts in the lesional skin of patients with vitiligo and the normal skin of healthy controls. ( C ) Representative immunofluorescence images of fibroblasts, ADRA2A, CXCL9, CXCL10, and CD8 + T cells from the normal skin of the healthy control and the lesional skin of the patient with vitiligo ( n =3 per group). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. ( D ) Immunofluorescence analysis of ADRA2A (red) expression in BJ cells after treatment with 5 μM NE for 48 hours. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( E ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in BJ cells were detected by ELISA with treatment with aposcopolamine (Apos) or NE ( n =3 per group). ( F ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in BJ cells with ADRA2A siRNA or control siRNA were detected by ELISA ( n =3 per group). Error bars represent mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

doi: 10.1126/sciadv.aea7017

Figure Lengend Snippet: ( A ) Volcano plots illustrated the expression of adrenergic receptors on keratinocytes in human skin, including ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, and ADRB3. ( B ) Bubble map illustrated the up-regulation of ADRB2 expression on keratinocytes in the lesional skin of patients with vitiligo and the normal skin of healthy controls. ( C ) Representative immunofluorescence images of keratinocytes, ADRB2, CXCL9, CXCL10, and CD8 + T cells from the normal skin of the healthy control and the lesional skin of the patient with vitiligo ( n =3 per group). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. ( D ) Immunofluorescence analysis of ADRB2 (red) expression in keratinocytes after treatment with 5 μM NE for 48 hours. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( E ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in keratinocytes were detected by ELISA with treatment with NE or ICI ( n =3 per group). ( F ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in keratinocytes with ADRB2 siRNA or control siRNA were detected by ELISA ( n =3 per group). Error bars represent mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Control, Staining, Enzyme-linked Immunosorbent Assay