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

Journal: International Journal of Biological Sciences

Article Title: CD44 3'-Untranslated Region Functions as a Competing Endogenous RNA to Enhance NK Sensitivity of Liver Cancer Stem Cell by Regulating ULBP2 Expression

doi: 10.7150/ijbs.35216

Figure Lengend Snippet: CSCs derived from liver cancer were susceptible to NK cell mediated cytotoxicity in correlation with CD44 expression. (A) CD44 high iCSC, CD44 int iCSC, HepG2, and Hep3B were used in the 4 hours NK cell cytotoxicity assay with NK-92 cells at different E:T ratios. Data are showed in means ± SD from three independent experiments which are performed in triplicate (ns: not significant, **: p < 0.01). (B) IFN-γ release in the supernatants of cytotoxic assay (Fig. A, E:T ratio = 15:1) was determined by ELISA. Data were presented as mean ± SD (n=3) (*: p<0.05, **: p<0.01). (C) CD44 transcript level of CD44 high iCSC/CD44 int iCSC stably expressing dCas9-KRAB and sgRNA against CD44 (CD44-knockdown, CD44-kd) was analyzed by qRT-PCR. Levels are represented relative to those found in control-infected cells as means ± SD (n=3) (**: p < 0.01). (D) Identical cells (Fig. C) were used in the 4 hours NK cell cytotoxicity assay with NK-92 cells at different E:T ratios. Data are showed in means ± SD from three independent experiments which are performed in triplicate (**: p < 0.01). (E) IFN-γ release in the supernatants of cytotoxic assay (Fig. D, E:T ratio = 15:1) was determined by ELISA. Data were presented as mean ± SD (n=3) (**: p<0.01).

Article Snippet: Concentrations of secreted IFN-γ were determined using Human Interferon gamma ELISA Kit (ab46048; Abcam, Cambridge, MA, USA).

Techniques: Derivative Assay, Expressing, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Knockdown, Quantitative RT-PCR, Control, Infection

Journal: International Journal of Biological Sciences

Article Title: CD44 3'-Untranslated Region Functions as a Competing Endogenous RNA to Enhance NK Sensitivity of Liver Cancer Stem Cell by Regulating ULBP2 Expression

doi: 10.7150/ijbs.35216

Figure Lengend Snippet: CD44 regulated ULBP2 expression, which then further influenced the susceptibility of CSCs to NK cell mediated cytotoxicity. (A ) MICA/B, ULBP1-3, CD48, CD112, CD155, and ICAM1 transcript levels of CD44 high iCSC/CD44 int iCSC stably expressing dCas9-KRAB and sgRNA against CD44 (CD44-knockdown, CD44-kd) were analyzed by qRT-PCR. Levels are represented relative to those found in control-infected cells as means ± SD (n=3) (**: p < 0.01). (B) CD44 and ULBP2 protein levels of identical cells (Fig. A) were analyzed by Western blotting. β-actin served as a loading control. (C) CD44 high iCSC and CD44 int iCSC were used in the 4 hours NK cell cytotoxicity assay with NK-92 cells at different E:T ratios. Assays were performed either in the presence of M311 mAb (anti-ULBP2) or control IgG. Data are showed in means ± SD from three independent experiments which are performed in triplicate (**: p < 0.01). (D) IFN-γ release in the supernatants of cytotoxic assay (Fig. C, E:T ratio = 15:1) was determined by ELISA. Data were presented as mean ± SD (n=3) (**: p<0.01).

Article Snippet: Concentrations of secreted IFN-γ were determined using Human Interferon gamma ELISA Kit (ab46048; Abcam, Cambridge, MA, USA).

Techniques: Expressing, Stable Transfection, Knockdown, Quantitative RT-PCR, Control, Infection, Western Blot, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cyclin G2 in macrophages triggers CTL-mediated antitumor immunity and antiangiogenesis via interferon-gamma

doi: 10.1186/s13046-022-02564-2

Figure Lengend Snippet: Cyclin G2 in macrophages regulates CTL chemotaxis and vascular endothelial cell tube formation via CXCL9. A CXCL9 levels in the supernatants of BMDMs from WT and Ccng2 −/− C57BL/6 mice treated with IFN-γ were determined by ELISA (representing 3 independent experiments). B , C CXCL9 levels in the supernatants of THP-1 stable cell lines (Nonsense, shcyclin G2#1, and shcyclin G2#2 and Vector and Flag-cyclin G2) treated with IFN-γ were determined by ELISA (representing 3 independent experiments). D , E CTL chemotaxis analyzed by treating conditioned medium from BMDMs isolated from WT and Ccng2 −/− C57BL/6 mice treated with or without recombinant CXCL9. Scale bar = 200 μm (representing 3 independent experiments). F Tube formation experiments showed the tube formation ability of SVEC4–10 cells treated with conditioned medium from BMDMs isolated from Ccng2 −/− C57BL/6 mice. The recombinant CXCL9 was added or not added to the conditioned medium. Scale bar = 500 μm (representing 3 independent experiments). G Tube formation experiments showed the tube formation ability of HUVECs treated with conditioned medium from a THP-1 stable cell line (shcyclin G2#1). The recombinant CXCL9 was added or not added to the conditioned medium. Scale bar = 200 μm (representing 3 independent experiments). (A–C, E–G) Data were analyzed with the unpaired Student’s t-test. Data are presented as the mean ± SD ** p < 0.01; *** p < 0.001; ns , not significant

Article Snippet: Cell culture supernatants were collected, and human and mouse CXCL9 levels in the supernatants were quantified using the human and mouse CXCL9 Enzyme-Linked Immunosorbent Assay (ELISA) Kits (E-EL-H6062 and E-EL-M0020c, Elabscience), respectively.

Techniques: Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Plasmid Preparation, Isolation, Recombinant

Journal: bioRxiv

Article Title: CD11c+ Tbet+ B cells constrain obesity- and vaccination-induced germinal center B cells and T helper cells

doi: 10.1101/2025.09.01.673552

Figure Lengend Snippet: Representative flow cytometry contour plots of PD-1 x CXCR5 expression within CD25-CD4+ T cells isolated from NCD (left) and HFD (right) mouse spleen (top) and liver (bottom) ( A ). Blue gates represent PD-1 HI CXCR5+ cells, yellow gates represent PD-1 HI CXCR5-cells, and black gates represent PD-1-cells ( A ). Frequency and absolute number of PD-1 HI CXCR5+ T FH cells ( B , blue), or PD-1 HI CXCR5-T PH cells ( C , yellow) within the CD25-CD4+ T cell compartments of spleen (top) and liver (bottom) of NCD (grey) and HFD (blue/yellow) mice. Quantification of Bcl-6 ( D ), ICOS ( E ), and T-bet ( F ) protein expression (gMFI) in splenic (left) or hepatic (right) CD25-CD4+ T cell subsets. Representative flow cytometry contour plots depicting relative expression of IL-21 and IFNg by PD-1 HI CXCR5+ T FH cells or PD-1 HI CXCR5-T PH cells from spleen (top) or liver (bottom) that are also CXCR3+ (blue/red) or CXCR3-(black) ( G ). Summary of frequency of IL-21 ( H,J; left) or IFNg ( I,K ; right) producing CD4+ PD-1 HI CXCR5+ T FH (blue) or PD-1 HI CXCR5-T PH (red) cells categorized by CXCR3+ expression as noted in spleen ( H, I ) or liver ( J,K ) from HFD-fed mice. Data pooled from 2 independent experiments with 4 female mice/group, Bar ± SEM; [ Student’s t-test (two-tailed) (B,C); one-way ANOVA (D-F; H-K)]; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: C57BL/6J WT female mice were injected IP with 0.25 mg of anti-mouse CXCR3 (CD183) and anti-mouse CXCL9 (MIG) or hamster IgG (BioXCell) every other day for 14 days after completing 12 weeks on HFD.

Techniques: Flow Cytometry, Expressing, Isolation, Two Tailed Test