Journal: Nature Communications

Article Title: CD163 interacts with TWEAK to regulate tissue regeneration after ischaemic injury

doi: 10.1038/ncomms8792

Figure Lengend Snippet: ( a ) TWEAK (100 ng ml −1 ) was added to VEGFR1, Fn14 or CD163-coated wells. Bound protein was measured by ELISA using an antibody to detect TWEAK. ( b ) Fn14 or CD163 were coated onto 96-well plates and incubated with increasing doses of untagged TWEAK. Bound protein was measured by ELISA. ( c ) Immunoblotting of MDEC stimulated with TWEAK at 200 ng ml −1 (which activates both canonical and non-canonical NF-κB signalling) and increasing doses of recombinant mouse sCD163 for the indicated proteins. ( d ) Immunoprecipitation of serum before and after femoral ligation using anti-TWEAK antibody with immunoblotting for CD163. (The figures show a representative blot with a total of four experiments conducted.) ( e ) Immunostaining for VE-cadherin and β-dystroglycan (red) in limbs of CD163 −/− mice treated for 14 days with saline (control), TWEAK administration (25 μg kg −1 day −1 administered via Alzet pump) alone or in combination with sCD163 administration (25 μg kg −1 day −1 administered by intraperitoneal injection) ( n =5 per group). The graphs show the number of VE-cadherin positive cells per myofibre and the average muscle fibre area per group. ( f ) Immunblotting of CD163 −/− limb after 14 days of TWEAK administration (25 μg kg −1 day −1 administered via Alzet pump) alone or in combination with sCD163 administration (25 μg kg −1 day −1 administered by intraperitoneal injection) ( n =5 per group). O.D., optical density. Bar graphs show quantitation of densitometry for phosphorylated to total p65, p52 and NICD. * P <0.05 versus other groups. For multiple group comparisons, we utilized a one-way ANOVA. If the variance ratio test ( F -test) was significant, a more detailed post hoc analysis of differences between groups was made using a Tukey–Kramer honest significance difference test. For binding experiments, average of at least four well per group shown with each experiment repeated at least two times with representative results shown.

Article Snippet: For experiments shown in , mouse recombinant TWEAK (untagged) was obtained from Kingfisher Biotech.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Recombinant, Immunoprecipitation, Ligation, Immunostaining, Saline, Control, Injection, Quantitation Assay, Binding Assay

Journal: The FASEB Journal

Article Title: CD163 deficiency increases foam cell formation and plaque progression in atherosclerotic mice

doi: 10.1096/fj.202000177r

Figure Lengend Snippet: FIGURE 6 CD163 prevents pro-inflammatory actions induced by TWEAK. A, Representative western blot analysis of p-p65 and p65 in VSMCs treated with 0% FBS (control), rTW (100 ng/mL) or rTW plus rCD (0.01-0.5 μg/mL). Right panel show the quantification of band densitometry values of p-p65 protein levels expressed in arbitrary units after correction for p65 (loading control). *P < .01 vs Control; †P < .01 vs rTW; Student's t test. B, Relative CCL2, CCL5, ICAM-1, MMP-2, and MMP-9 mRNA expression levels normalized to 18S rRNA of VSMCs treated with rTW (100 ng/mL) or rTW plus rCD (0.01-0.5 μg/mL) during 24 hours. The same cDNA was repeatedly tested for multiple genes. Data represent the mean ± SEM of three independent experiments. *P < .05 vs Control; †P < .05 vs rTW. Student's t test. C, Effect of CD163 on CCL2 and CCL5 secretion induced by TWEAK in VSMCs. VSMCs were incubated with rTW (100 ng/mL) or rTW plus rCD (0.01-0.5 μg/mL) during 24 hours and supernatants were tested by ELISA for CCL2 or CCL5. Data represent the mean ± SEM of six independent experiments; *P < .001 vs control; †P < .001 vs rTW; Student's t test. D, Peritoneal macrophages were seeded in the upper surface of chemotaxis chambers and stimulated with the supernatants of VSMCs treated with 0% FBS (control), rTWEAK (100 ng/mL) or rTW plus rCD (0.1-0.5 μg/mL). Quantification of migrated cells in 10 fields per condition is shown in the right panel. Data represent the mean ± SEM of four independent experiments; *P < .001 vs Control; †P < .001 vs rTW; Student's t test. Scale bars, 50 μm

Article Snippet: For experimental analysis, cells were made quiescent by 24-hour incubation in medium without FBS before stimulation with recombinant murine TWEAK (0.1 μg/mL; 1237-TW; R&D Systems) and preincubated or not with different concentrations of rCD163 (7435-CD; R&D Systems).

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

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Comparative transcriptome analysis of human and murine choroidal neovascularization identifies fibroblast growth factor inducible-14 as phylogenetically conserved mediator of neovascular age-related macular degeneration.

doi: 10.1016/j.bbadis.2022.166340

Figure Lengend Snippet: Fig. 4. FN14 decoy receptor modulates the cytokine profile in CNV and reduces neovascularization size. (A): Experimental setup. (B): Intravitreal injection of a FN14 decoy receptor compared to PBS (control) significantly reduced CNV size. For statistical analysis, Mann–Whitney U test was applied. Color fundus (CF) and fluo rescence angiography (FA) on d7 of representative eyes of the FN14 decoy receptor (upper panel) and the PBS group (lower panel) as well as confocal microscopy (CM) images of representative lesions (COL4 in red) the size of which approximately corresponds to the mean spot size within the respective group. (C): Protein concentrations of several cytokines on d5 in the RPE of unlasered controls, CNV injected with PBS (CNV) or with FN14 decoy receptor on d1 (CNV + decoy). Data are shown as mean with SEM. For statistical analysis, paired ANOVA adjusting for total protein concentration was applied. *: p < 0.05, ns: not significant.

Article Snippet: At d1 mice received an intravitreal injection of 4 μg FN14 decoy receptor (1610-TW, R&D Systems) solved in 1 μl PBS in one eye and the same volume of PBS in the other eye, as described above.

Techniques: Injection, Control, MANN-WHITNEY, Confocal Microscopy, Protein Concentration

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: TWEAK regulates the functions of hair follicle stem cells via the Fn14-Wnt/β-catenin-CXCR4 signalling axis.

doi: 10.1111/wrr.70032

Figure Lengend Snippet: FIGURE 1 TWEAK/Fn14 signalling upregulates the expressions of HFSC markers. (A) Immunofluorescence analysis of Fn14 and CXCR4 expression in mouse hair follicles. The white arrows indicate the Fn14- or CXCR4-positive cells; however, these positive cells were significantly diminished and even became indistinct in the Fn14-deficient specimen. Scale bar = 20 μm. (B) Histochemical analysis of Fn14, K19 and CD34 expression in mouse hair follicles. Scale bar = 20 μm. (C) Primary human HFSCs cultured in vitro. (D) Immunofluorescence analysis of β1 integrin and K15 expression in HFSCs stimulated with TWEAK (10 ng/mL). Scale bar = 10 μm. (e, f) Western blotting of K19, integrin β1 and K15 proteins. Data represent mean ± SEM from three independent experiments. *p < 0.05, compared with the 0 ng/mL TWEAK group. CXCR4, chemokine (C X C motif) receptor 4; Fn14, fibroblast growth factor-inducible 14; HFSC, hair follicle stem cells; K15/K19, keratins 15/19; TWEAK, tumour necrosis factor- like weak inducer of apoptosis.

Article Snippet: HFSCs were stimulated for 48 hours with recombinant human TWEAK (0–250 ng/mL; R&D Systems, Minneapolis, MN).

Techniques: Immunofluorescence, Expressing, Cell Culture, In Vitro, Western Blot

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: TWEAK regulates the functions of hair follicle stem cells via the Fn14-Wnt/β-catenin-CXCR4 signalling axis.

doi: 10.1111/wrr.70032

Figure Lengend Snippet: FIGURE 2 TWEAK induces proliferation, migration and cytokine production in HFSCs. Human HFSCs were cultured in vitro and stimulated with TWEAK (0–250 ng/mL). (A) Proliferation of HFSCs analysed via flow cytometry. n = 5 per group. (B) Cell migration assessed via scratch analysis. n = 5 per group. (C) mRNA expression levels of EGF, BFGF, TGF-β, NGF and VEGF measured via qRT-PCR. n = 3 per group. (D) Cytokine levels in the supernatants determined via ELISA. n = 3 per group. Data represent mean ± SEM from three to five independent experiments. *p < 0.05 compared with the 0 ng/mL group. #p < 0.05 compared with the 10 ng/mL group. HFSC, hair follicle stem cells; qRT-PCR, quantitative real-time PCR; SEM, standard error of the mean; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.

Article Snippet: HFSCs were stimulated for 48 hours with recombinant human TWEAK (0–250 ng/mL; R&D Systems, Minneapolis, MN).

Techniques: Migration, Cell Culture, In Vitro, Flow Cytometry, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: TWEAK regulates the functions of hair follicle stem cells via the Fn14-Wnt/β-catenin-CXCR4 signalling axis.

doi: 10.1111/wrr.70032

Figure Lengend Snippet: FIGURE 3 TWEAK upregulates the expressions of HFSC phenotype markers. Human HFSCs were cultured in vitro and stimulated with TWEAK (0–250 ng/mL). (A) Fn14 and TNFR2 expressions detected via immunofluorescence. (B) mRNA expression levels of FN14, TNFR2, IGFR and CXCR4 analysed via qRT-PCR. (C, D) Protein expressions of these markers determined via Western blotting, with quantification using ImageJ software. Data represent mean ± SEM from three independent experiments. *p < 0.05 compared with the 0 ng/mL group. #p < 0.05 compared with the 10 ng/mL group. Δp < 0.05 compared with the 50 ng/mL group. HFSC, hair follicle stem cells; qRT-PCR, quantitative real-time PCR; SEM, standard error of the mean; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.

Article Snippet: HFSCs were stimulated for 48 hours with recombinant human TWEAK (0–250 ng/mL; R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, In Vitro, Immunofluorescence, Expressing, Quantitative RT-PCR, Western Blot, Software, Real-time Polymerase Chain Reaction

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: TWEAK regulates the functions of hair follicle stem cells via the Fn14-Wnt/β-catenin-CXCR4 signalling axis.

doi: 10.1111/wrr.70032

Figure Lengend Snippet: FIGURE 4 TWEAK activates Wnt/β-catenin signalling in HFSCs. Human HFSCs were cultured in vitro and stimulated with TWEAK (10 ng/mL). (A) mRNA expression levels of WNT5A, CTNNB and GSK3B analysed via qRT-PCR. (B, C) Protein expressions of these markers detected via Western blotting, with quantification using ImageJ software. (D) mRNA expression levels of TNFR2, IGFR and CXCR4 analysed in cells pretreated with the β-catenin inhibitor XAV-939. (E, F) Protein expressions of these markers detected via Western blotting, with quantification using ImageJ software. Data represent mean ± SEM from three independent experiments. In (A, C), *p < 0.05 compared with the blank group. In (D, F), *p < 0.05 compared with the PBS alone group; #p < 0.05 compared with the XAV-939 group. Δp < 0.05 compared with the TWEAK alone group. HFSC, hair follicle stem cells; qRT-PCR, quantitative real-time PCR; TWEAK, tumour necrosis factor- like weak inducer of apoptosis.

Article Snippet: HFSCs were stimulated for 48 hours with recombinant human TWEAK (0–250 ng/mL; R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, In Vitro, Expressing, Quantitative RT-PCR, Western Blot, Software, Real-time Polymerase Chain Reaction

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: TWEAK regulates the functions of hair follicle stem cells via the Fn14-Wnt/β-catenin-CXCR4 signalling axis.

doi: 10.1111/wrr.70032

Figure Lengend Snippet: FIGURE 5 CXCR4 inhibitor suppresses the effect of TWEAK on HFSCs. Human HFSCs were cultured in vitro and treated with TWEAK (10 ng/mL) in the presence or absence of the CXCR4 inhibitor EPI-X4. (A) Proliferation of HFSCs assessed via flow cytometry. n = 5 per group. (B, C) Protein expressions of differentiation markers detected via Western blotting and quantitated using ImageJ software. n = 3 per group. (D). Cytokines synthesised by HFSCs and measured via ELISA in the culture supernatants. n = 3 per group. Data represent mean ± SEM from three to five independent experiments. *p < 0.05 compared with the blank group. #p < 0.05 compared with the TWEAK alone group. CXCR4, chemokine (C X C motif) receptor 4; HFSC, hair follicle stem cells; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.

Article Snippet: HFSCs were stimulated for 48 hours with recombinant human TWEAK (0–250 ng/mL; R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, In Vitro, Flow Cytometry, Western Blot, Software, Enzyme-linked Immunosorbent Assay