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R&D Systems human tweak

FIGURE 1 <t>TWEAK/Fn14</t> 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 <t>human</t> <t>HFSCs</t> 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.

Human Tweak, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/recombinant+human+tweak+tnfsf12/Recombinant+Human+TWEAK%2FTNFSF12+Protein%2C+CF/pm40325995-53-8-12
Average 93 stars, based on 3 article reviews

human tweak - by Bioz Stars, 2026-10

93/100 stars

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1) Product Images from "TWEAK regulates the functions of hair follicle stem cells via the Fn14-Wnt/β-catenin-CXCR4 signalling axis."

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

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

doi: 10.1111/wrr.70032

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

Recombinant:

Article Title: A Small Molecule PAI-1 Functional Inhibitor Attenuates Neointimal Hyperplasia and Vascular Smooth Muscle Cell Survival by Promoting PAI-1 Cleavage
Article Snippet: Endotoxin levels in full-length, stabilized recombinant 14-1b PAI-1 (FL-PAI-1), R76E-PAI-1 (LRP1-binding deficient mutant; Molecular Innovations, Novi, MI), human neutrophil elastase (Abcam, Cambridge, UK) and high-molecular weight two chain uPA (Seiksui Diagnostics, LLC, Lexington, MA) preparations were measured using the Limulus Amebocyte Lysate kit QCL-1000 (Lonza, Basel, Switzerland) and all found to be <0.14 EU/ml, the acceptable threshold [ 22 ]. .. Recombinant Human TWEAK/TNFSF12 was obtained from R&D Systems (Minneapolis, MN). .. siRNA designed against human PAI-1 mRNA (5′-AAGGATGAGATCAGCACCACA-3′) and a scrambled control (sc) siRNA (5′-AATTCTCCGAACGTGTCACGT-3′) [ 23 ] were obtained from QIAGEN Inc. (Valencia, CA).

Article Title: Effect of opioid receptor antagonist on mitigating tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-induced apoptolysis in pemphigus pathogenesis.
Article Snippet: HaCaT cells were conventionally maintained at 37 ◦C in a humidified atmosphere with 5 % (v/v) CO2 and cultured in RPMI 1640 culture medium (Sigma, Cat#MFCD00217820, Darmstadt, Germany) containing 10 % fetal bovine serum (Biological Industries, Cat#04-121-1A, Israel) (hereafter referred to as culture medium). .. For the TWEAK stimulation group, HEKs and HaCaT cells were treated with 100 ng/mL recombinant human TWEAK/TNFSF12 (100 ng/mL, R&D Systems, Cat#1090-TW, Minneapolis, USA) or the respective vehicle (0.1 % PBS buffer) at multiple time points. .. For caspase inhibition, the pan-caspase inhibitors Z-VAD-FMK-FMK (50 μM, Med Chem Express, Cat#HY-16658B, New Jersey, USA) and BOC-DFMK-FMK (50 μM, Cayman Chemical Company, Cat# 16118, Michigan, USA) were used.

Article Title: Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Promotes Hepatic Stellate Cells Migration via Canonical NF-κB/MMP9 Pathway
Article Snippet: LX-2 cells[ ] (#SCC064) were purchased from Merk Millipore, USA in December, 2015. .. Recombinant Human TWEAK/TNFSF12, 25 ug (1090-TW) was obtained from R&D system. .. BCA Protein Assay Kit was supplied by Keygen Biotech (Nanjing, China).

Image Search Results

Arg1 + MMP12 + macrophage-derived TWEAK licenses PDGFB production in fibroTECs via Fn14 signaling. (A) Ligand-receptor communication analysis between different cell types in IRI kidneys. (B) The major incoming signaling of different cell types. (C) Bubble plots showing the expression of specific ligand-receptor pairs in the interaction between myeloid cell subsets and TEC subsets. (D) Bubble plots showing the expression of specific ligand-receptor pairs in the interaction between TEC subsets and myeloid cell subsets. (E, F) WT/AMPKα1 −/− BMDMs were treated with IL-4 and IL-13, and the expression levels of Arg 1 , Mmp12 , and Tnfsf12 were analyzed by qPCR (n = 5). (G, H) TCMK-1 cells were treated with CM from IL-4 and IL-13-stimulated WT/AMPKα1 −/− BMDMs. The expression of Pdgfb , Cxcl1 , and Egfr in TCMK-1 cells was analyzed by qPCR (n = 5). (I, J) TCMK-1 cells were treated with CM from IL-4 and IL-13-stimulated WT/AMPKα1 −/− BMDMs or together with recombinant TWEAK (rTWEAK). The expression of Pdgfb in TCMK-1 cells was analyzed by qPCR (n = 5). (K, L) TCMK-1 cells were treated with rTWEAK or rTWEAK and an Fn14 inhibitor. The expression of Pdgfb in TCMK-1 cells was detected by qPCR (n = 5). (M) Immunoblots and quantification of IKK-β and nuclear NF-κB p65 in TCMK-1 cells treated with PBS or rTWEAK (n = 3). The results represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Journal: Redox Biology

Article Title: Macrophage AMPK activated by oxidative stress drives profibrotic crosstalk with tubular cells to accelerate renal fibrosis after ischemic and reperfusion injury

doi: 10.1016/j.redox.2025.104002

Figure Lengend Snippet: Arg1 + MMP12 + macrophage-derived TWEAK licenses PDGFB production in fibroTECs via Fn14 signaling. (A) Ligand-receptor communication analysis between different cell types in IRI kidneys. (B) The major incoming signaling of different cell types. (C) Bubble plots showing the expression of specific ligand-receptor pairs in the interaction between myeloid cell subsets and TEC subsets. (D) Bubble plots showing the expression of specific ligand-receptor pairs in the interaction between TEC subsets and myeloid cell subsets. (E, F) WT/AMPKα1 −/− BMDMs were treated with IL-4 and IL-13, and the expression levels of Arg 1 , Mmp12 , and Tnfsf12 were analyzed by qPCR (n = 5). (G, H) TCMK-1 cells were treated with CM from IL-4 and IL-13-stimulated WT/AMPKα1 −/− BMDMs. The expression of Pdgfb , Cxcl1 , and Egfr in TCMK-1 cells was analyzed by qPCR (n = 5). (I, J) TCMK-1 cells were treated with CM from IL-4 and IL-13-stimulated WT/AMPKα1 −/− BMDMs or together with recombinant TWEAK (rTWEAK). The expression of Pdgfb in TCMK-1 cells was analyzed by qPCR (n = 5). (K, L) TCMK-1 cells were treated with rTWEAK or rTWEAK and an Fn14 inhibitor. The expression of Pdgfb in TCMK-1 cells was detected by qPCR (n = 5). (M) Immunoblots and quantification of IKK-β and nuclear NF-κB p65 in TCMK-1 cells treated with PBS or rTWEAK (n = 3). The results represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: To examine the role of the TWEAK-Fn14 axis, cells were treated with recombinant TWEAK (MCE, HY-P7309, 100 ng/ml) and the Fn14 inhibitor, L524-0366 (Sigma, 509374, 10 μM).

Techniques: Derivative Assay, Expressing, Recombinant, Western Blot

Effects of TWEAK on the proliferation and apoptosis of PDLSCs. (A) Line chart depicting the effects of various concentrations of TWEAK (0, 1, 5, 20, 50 and 100 ng/ml) on PDLSC proliferation over 5 days, as assessed using the CCK-8 assay. (B) Statistical analysis of the CCK-8 data from day 5 of TWEAK stimulation shown in (A). (C) Statistical analysis of the average fluorescence intensity of TUNEL (green) following treatment with varying concentrations of TWEAK (0, 1, 5, 20, 50 and 100 ng/ml). (D) TUNEL assay detecting the effects of various concentrations (0, 1, 5, 20, 50 and 100 ng/ml) of TWEAK on PDLSC apoptosis. Scale bar, 200 μ m. Statistical analysis was performed using a one-way ANOVA. * P<0.05; **** P<0.0001. Data are presented as the mean ± SD (n=5 or 6). CCK-8, Cell Counting Kit-8; OD450, optical density at 450 nm; PDLSC, periodontal ligament stem cell; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Effects of TWEAK on the proliferation and apoptosis of PDLSCs. (A) Line chart depicting the effects of various concentrations of TWEAK (0, 1, 5, 20, 50 and 100 ng/ml) on PDLSC proliferation over 5 days, as assessed using the CCK-8 assay. (B) Statistical analysis of the CCK-8 data from day 5 of TWEAK stimulation shown in (A). (C) Statistical analysis of the average fluorescence intensity of TUNEL (green) following treatment with varying concentrations of TWEAK (0, 1, 5, 20, 50 and 100 ng/ml). (D) TUNEL assay detecting the effects of various concentrations (0, 1, 5, 20, 50 and 100 ng/ml) of TWEAK on PDLSC apoptosis. Scale bar, 200 μ m. Statistical analysis was performed using a one-way ANOVA. * P<0.05; **** P<0.0001. Data are presented as the mean ± SD (n=5 or 6). CCK-8, Cell Counting Kit-8; OD450, optical density at 450 nm; PDLSC, periodontal ligament stem cell; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Article Snippet: Recombinant human cynomolgus TWEAK/TNF superfamily member 12 (TNFSF12) protein (mFc tag) (cat. no. 90094-C04H; Sino Biological, Inc.) was dissolved in sterile water to obtain a stock solution concentration of 50 μ g/ml.

Techniques: CCK-8 Assay, Fluorescence, TUNEL Assay, Cell Counting

Effects of TWEAK on the migration and osteogenic differentiation of PDLSCs. (A) Effects of various concentrations of TWEAK (0, 1, 5, 20, 50 and 100 ng/ml) on the number of migrating PDLSCs, and (B) quantitative analysis of the number of migrating cells (n=6). Scale bar, 400 μ m. (C) Effects of various concentrations of TWEAK on PDLSC migration toward scratch wounds over 24 h, and (D) quantitative analysis of the percentage of wound area reduction (n=12). Scale bar, 1 mm. (E) Effects of various concentrations of TWEAK on ALP staining in PDLSCs, and (F) quantitative analysis of grayscale values from ALP staining (n=6). Scale bar, 200 μ m. (G) Alizarin Red staining revealed the effects of various concentrations of TWEAK on PDLSC mineralization, and (H) quantitative analysis of grayscale values from Alizarin Red staining (n=6). Scale bar, 200 μ m. (I) Reverse transcription-quantitative PCR was used to assess the mRNA expression levels of RUNX2 , SP7 , ALP and OPG in PDLSCs after TWEAK stimulation (n=4), with β-actin serving as the internal control. (J) Western blot analysis of RUNX2, SP7, ALP and OPG protein expression in PDLSCs after TWEAK induction, and (K) semi-quantitative analysis of the gel band intensity, using β-actin as the internal control. Statistical analysis was performed using a one-way ANOVA. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. Data are presented as the mean ± SD. ALP, alkaline phosphatase; IOD, integral optical density; ns, not significant; OPG, osteoprotegerin; PDLSC, periodontal ligament stem cell; RUNX2, runt-related transcription factor 2; SP7, Sp7 transcription factor; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Effects of TWEAK on the migration and osteogenic differentiation of PDLSCs. (A) Effects of various concentrations of TWEAK (0, 1, 5, 20, 50 and 100 ng/ml) on the number of migrating PDLSCs, and (B) quantitative analysis of the number of migrating cells (n=6). Scale bar, 400 μ m. (C) Effects of various concentrations of TWEAK on PDLSC migration toward scratch wounds over 24 h, and (D) quantitative analysis of the percentage of wound area reduction (n=12). Scale bar, 1 mm. (E) Effects of various concentrations of TWEAK on ALP staining in PDLSCs, and (F) quantitative analysis of grayscale values from ALP staining (n=6). Scale bar, 200 μ m. (G) Alizarin Red staining revealed the effects of various concentrations of TWEAK on PDLSC mineralization, and (H) quantitative analysis of grayscale values from Alizarin Red staining (n=6). Scale bar, 200 μ m. (I) Reverse transcription-quantitative PCR was used to assess the mRNA expression levels of RUNX2 , SP7 , ALP and OPG in PDLSCs after TWEAK stimulation (n=4), with β-actin serving as the internal control. (J) Western blot analysis of RUNX2, SP7, ALP and OPG protein expression in PDLSCs after TWEAK induction, and (K) semi-quantitative analysis of the gel band intensity, using β-actin as the internal control. Statistical analysis was performed using a one-way ANOVA. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. Data are presented as the mean ± SD. ALP, alkaline phosphatase; IOD, integral optical density; ns, not significant; OPG, osteoprotegerin; PDLSC, periodontal ligament stem cell; RUNX2, runt-related transcription factor 2; SP7, Sp7 transcription factor; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Techniques: Migration, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot

Transcriptome analysis of the effect of TWEAK treatment on PDLSCs. (A) Venn diagram illustrating differentially expressed genes in PDLSCs following TWEAK treatment. (B) Multipoint differential scatter plot showing differentially expressed genes in PDLSCs following TWEAK treatment. (C) GO enrichment analysis comparing PDLSCs and PDLSCs treated with 50 ng/ml TWEAK. (D) KEGG enrichment analysis comparing PDLSCs and PDLSCs treated with 50 ng/ml TWEAK. (E) GO enrichment analysis comparing PDLSCs and PDLSCs treated with 100 ng/ml TWEAK. (F) KEGG enrichment analysis comparing PDLSCs and PDLSCs treated with 100 ng/ml TWEAK. (G) Western blot analysis was conducted to detect the levels of Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs stimulated with 50 and 100 ng/ml TWEAK. (H) Statistical analysis of protein band intensities from (G) (n=3). (I) Western blot analysis of the levels of Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs after Fn14 was silenced using an shRNA. (J) Statistical analysis of protein band intensities from (I) (n=3). Statistical analysis was performed using (G and H) one-way ANOVA or (I and J) a two-tailed Student's t-test. * P<0.05; ** P<0.01; *** P<0.001. Data are presented as the mean ± SD. FC, fold change; Fn14, fibroblast growth factor-inducible 14; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NLRP3, NOD-like receptor thermal protein domain-associated protein 3; ns, not significant; P-, phosphorylated; PDLSC, periodontal ligament stem cell; shRNA/sh, short hairpin RNA; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Transcriptome analysis of the effect of TWEAK treatment on PDLSCs. (A) Venn diagram illustrating differentially expressed genes in PDLSCs following TWEAK treatment. (B) Multipoint differential scatter plot showing differentially expressed genes in PDLSCs following TWEAK treatment. (C) GO enrichment analysis comparing PDLSCs and PDLSCs treated with 50 ng/ml TWEAK. (D) KEGG enrichment analysis comparing PDLSCs and PDLSCs treated with 50 ng/ml TWEAK. (E) GO enrichment analysis comparing PDLSCs and PDLSCs treated with 100 ng/ml TWEAK. (F) KEGG enrichment analysis comparing PDLSCs and PDLSCs treated with 100 ng/ml TWEAK. (G) Western blot analysis was conducted to detect the levels of Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs stimulated with 50 and 100 ng/ml TWEAK. (H) Statistical analysis of protein band intensities from (G) (n=3). (I) Western blot analysis of the levels of Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs after Fn14 was silenced using an shRNA. (J) Statistical analysis of protein band intensities from (I) (n=3). Statistical analysis was performed using (G and H) one-way ANOVA or (I and J) a two-tailed Student's t-test. * P<0.05; ** P<0.01; *** P<0.001. Data are presented as the mean ± SD. FC, fold change; Fn14, fibroblast growth factor-inducible 14; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NLRP3, NOD-like receptor thermal protein domain-associated protein 3; ns, not significant; P-, phosphorylated; PDLSC, periodontal ligament stem cell; shRNA/sh, short hairpin RNA; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Techniques: Western Blot, shRNA, Two Tailed Test

Inhibition of Fn14 and NF-κB effectively blocks TWEAK-induced alterations in PDLSC characteristics. (A) Western blot analysis was conducted to assess the levels of Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs. (B) Statistical analysis of the intensities of the protein bands shown in (A) (n=3). (C) A CCK-8 assay was performed to generate the proliferation curve of PDLSCs. (D) Statistical analysis of the OD450 values of cells from each group on day 5 of the CCK-8 assay, as presented in (C) (n=6). (G) Results of ALP staining and (E) the corresponding statistical analysis of PDLSCs after osteogenic induction (n=6). Scale bar, 400 μ m. (H) Results of Alizarin Red staining and (F) the corresponding statistical analysis of PDLSCs following osteogenic induction (n=6). Scale bar, 400 μ m. (I) Reverse transcription-quantitative PCR was used to assess the mRNA expression levels of RUNX2 , SP7 , ALP and OPG in PDLSCs, with β-actin serving as an internal reference (n=4). (J) Western blot analysis was performed to detect the protein expression levels of RUNX2, SP7, ALP and OPG in PDLSCs, and (K) the grayscale values of the gel images were semi-quantitatively analyzed, with β-actin used as an internal reference (n=3). Statistical analysis was performed using a one-way ANOVA. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. Data are presented as the mean ± SD. ALP, alkaline phosphatase; CCK-8, Cell Counting Kit-8; Fn14, fibroblast growth factor-inducible 14; IOD, integral optical density; NLRP3, NOD-like receptor thermal protein domain-associated protein 3; ns, not significant; OD450, optical density at 450 nm; OPG, osteoprotegerin; P-, phosphorylated; PDLSC, periodontal ligament stem cell; RUNX2, runt-related transcription factor 2; sh, short hairpin RNA; SP7, Sp7 transcription factor; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Inhibition of Fn14 and NF-κB effectively blocks TWEAK-induced alterations in PDLSC characteristics. (A) Western blot analysis was conducted to assess the levels of Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs. (B) Statistical analysis of the intensities of the protein bands shown in (A) (n=3). (C) A CCK-8 assay was performed to generate the proliferation curve of PDLSCs. (D) Statistical analysis of the OD450 values of cells from each group on day 5 of the CCK-8 assay, as presented in (C) (n=6). (G) Results of ALP staining and (E) the corresponding statistical analysis of PDLSCs after osteogenic induction (n=6). Scale bar, 400 μ m. (H) Results of Alizarin Red staining and (F) the corresponding statistical analysis of PDLSCs following osteogenic induction (n=6). Scale bar, 400 μ m. (I) Reverse transcription-quantitative PCR was used to assess the mRNA expression levels of RUNX2 , SP7 , ALP and OPG in PDLSCs, with β-actin serving as an internal reference (n=4). (J) Western blot analysis was performed to detect the protein expression levels of RUNX2, SP7, ALP and OPG in PDLSCs, and (K) the grayscale values of the gel images were semi-quantitatively analyzed, with β-actin used as an internal reference (n=3). Statistical analysis was performed using a one-way ANOVA. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. Data are presented as the mean ± SD. ALP, alkaline phosphatase; CCK-8, Cell Counting Kit-8; Fn14, fibroblast growth factor-inducible 14; IOD, integral optical density; NLRP3, NOD-like receptor thermal protein domain-associated protein 3; ns, not significant; OD450, optical density at 450 nm; OPG, osteoprotegerin; P-, phosphorylated; PDLSC, periodontal ligament stem cell; RUNX2, runt-related transcription factor 2; sh, short hairpin RNA; SP7, Sp7 transcription factor; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Techniques: Inhibition, Western Blot, CCK-8 Assay, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Cell Counting, shRNA

Inhibition of Fn14 and NF-κB effectively blocks TWEAK-induced alterations in the microenvironmental regulatory potential of PDLSCs. (A) Expression levels of OPG (green) and RANKL (red) in PDLSCs were detected by immunofluorescence staining, followed by quantitative analysis of the MOD values for (B) RANKL and (C) OPG, and (D) the MOD ratio of RANKL/OPG (n=5). Scale bar, 200 μ m. (E) Expression levels of CD68 and CD163 in RAW264.7 macrophages were detected by immunofluorescence staining, followed by quantitative analysis of the MOD values for (F) CD68 and (G) CD163 (n=5). Scale bar, 200 μ m. Statistical analysis was performed using a one-way ANOVA. ** P<0.01; *** P<0.001; **** P<0.0001. Data are presented as the mean ± SD. Fn14, fibroblast growth factor-inducible 14; MOD, mean optical density; ns, not significant; OPGa, osteoprotegerin; PDLSC, periodontal ligament stem cell; RANKL, receptor activator of nuclear factor-κB ligand; sh, short hairpin RNA; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Inhibition of Fn14 and NF-κB effectively blocks TWEAK-induced alterations in the microenvironmental regulatory potential of PDLSCs. (A) Expression levels of OPG (green) and RANKL (red) in PDLSCs were detected by immunofluorescence staining, followed by quantitative analysis of the MOD values for (B) RANKL and (C) OPG, and (D) the MOD ratio of RANKL/OPG (n=5). Scale bar, 200 μ m. (E) Expression levels of CD68 and CD163 in RAW264.7 macrophages were detected by immunofluorescence staining, followed by quantitative analysis of the MOD values for (F) CD68 and (G) CD163 (n=5). Scale bar, 200 μ m. Statistical analysis was performed using a one-way ANOVA. ** P<0.01; *** P<0.001; **** P<0.0001. Data are presented as the mean ± SD. Fn14, fibroblast growth factor-inducible 14; MOD, mean optical density; ns, not significant; OPGa, osteoprotegerin; PDLSC, periodontal ligament stem cell; RANKL, receptor activator of nuclear factor-κB ligand; sh, short hairpin RNA; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Techniques: Inhibition, Expressing, Immunofluorescence, Staining, shRNA

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Inhibition of the TWEAK/Fn14/NF-κB/NLRP3 pathway enhances the functional properties of iPDLSCs. (A) Expression profile of surface markers in iPDLSCs quantified using flow cytometry. (B) Levels of TWEAK, Fn14, NF-κB, P-NF-κB and NLRP3 in PDLSCs, iPDLSCs, and iPDLSCs after the downregulation of Fn14, NF-κB and NLRP3, and (C) statistical analysis of the band density values (n=3). (D) Apoptosis levels in PDLSCs, iPDLSCs, and iPDLSCs after downregulation of Fn14, NF-κB and NLRP3 were detected using the TUNEL assay, and (E) statistical analysis of the average fluorescence intensity of TUNEL was performed (n=5). Scale bar, 200 μ m. (F) A Cell Counting Kit-8 assay was used to assess the proliferative potential of PDLSCs, iPDLSCs, and iPDLSCs after the downregulation of Fn14, NF-κB and NLRP3, and (G) statistical analysis of the OD450 values on day 5 of the experiment was performed (n=6). (H) Transwell migration assay evaluating the migratory potential of PDLSCs, iPDLSCs, and iPDLSCs after Fn14, NF-κB or NLRP3 downregulation, with (I) quantification of the number of migrated cells (n=6). Scale bar, 400 μ m. (J) Wound healing assay evaluating the migratory potential of PDLSCs, iPDLSCs, and iPDLSCs after Fn14, NF-κB or NLRP3 downregulation, with (K) quantification of the percentage of wound closure (%) (n=16). Scale bar, 1 mm. (L) ALP staining was used to evaluate the mineralization potential of PDLSCs, iPDLSCs, and iPDLSCs after downregulation of Fn14, NF-κB or NLRP3, with (M) quantification of the integral optical density of the ALP-stained images (n=6). Scale bar, 400 μ m. (N) Alizarin Red staining was used to evaluate the mineralization potential of PDLSCs, iPDLSCs, and iPDLSCs after downregulation of Fn14, NF-κB or NLRP3, with (O) quantification of the integral optical density of the Alizarin Red-stained images (n=6). Scale bar, 400 μ m. (P) Reverse transcription-quantitative PCR was used to evaluate the mRNA expression levels of RUNX2 , SP7 , ALP and OPG in PDLSCs, iPDLSCs, and iPDLSCs after the downregulation of Fn14, NF-κB and NLRP3 (n=4). (Q) Western blotting was used to detect the expression levels of RUNX2, SP7, ALP and OPG in PDLSCs, iPDLSCs, and iPDLSCs after the downregulation of Fn14, NF-κB and NLRP3, and (R) statistical analysis of the band density values was performed (n=3). Statistical analysis was performed using a one-way ANOVA. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. Data are presented as the mean ± SD. ALP, alkaline phosphatase; Fn14, fibroblast growth factor-inducible 14; IOD, integral optical density; iPDLSC, inflammatory PDLSC; NLRP3, NOD-like receptor thermal protein domain-associated protein 3; ns, not significant; OD450, optical density at 450 nm; OPG, osteoprotegerin; P-, phosphorylated; PDLSC, periodontal ligament stem cell; RUNX2, runt-related transcription factor 2; sh, short hairpin RNA; SP7, Sp7 transcription factor; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Techniques: Inhibition, Functional Assay, Expressing, Flow Cytometry, TUNEL Assay, Fluorescence, Cell Counting, Transwell Migration Assay, Wound Healing Assay, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, shRNA

Effects of TWEAK and TWEAK-Fn14-IN-1 on the progression of rat periodontitis. (A) Micro-CT images of the rat maxilla, with the distance between the two red short lines representing the CEJ-ABC distance on the buccal side, and with statistical analysis of the (B) distance of CEJ-ABC (n=6), and (C) BV/TV at the root bifurcation of the maxillary second molar (n=6). Scale bar, 1 mm. Images of (D) H&E and (E) Masson's trichrome staining of rat periodontal tissues. Scale bar, 1 mm (top) or 100 μ m (bottom). (F) Representative images of TRAP/alkaline phosphatase double staining in the periodontal tissue of the rat maxillary second molar, with (G) quantification and statistical analysis of osteoclast numbers (TRAP-positive, multinucleated cells located in the bone resorption lacunae) at the mesial root (n=6). Red triangles indicate osteoclasts. Scale bar, 50 μ m. (H) Immunofluorescence staining of CD163 (green) and CD68 (red) in periodontal tissues, with (J) quantification of the mean fluorescence intensity of CD163 and (K) quantification of the mean fluorescence intensity of CD68 (n=6). Scale bar, 100 μ m. (I) Immunofluorescence staining of RUNX2 (green) and Periostin (red) in periodontal tissues, with (L) quantification of the mean fluorescence intensity of RUNX2 and (M) quantification of the mean fluorescence intensity of Periostin (n=6). Scale bar, 100 μ m. Blank represents the unmodeled group, PBS refers to the control group where PBS was used instead of TWEAK or TWEAK-Fn14-IN-1 during modeling, and TWEAK and TWEAK-Fn14-IN-1 represent experimental groups where the recombinant TWEAK protein or TWEAK-Fn14-IN-1 inhibitor was applied, respectively. Statistical analysis was performed using a one-way ANOVA. * P<0.05; ** P<0.01; **** P<0.0001. Data are presented as the mean ± SD. ABC, alveolar bone crest; BV/TV, bone volume to total volume; CEJ, cementoenamel junction; Fn14, fibroblast growth factor-inducible 14; MOD, mean optical density; ns, not significant; RUNX2, runt-related transcription factor 2; TRAP, tartrate-resistant acid phosphatase; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Journal: International Journal of Molecular Medicine

Article Title: TWEAK modulates the characteristics of periodontal ligament stem cells via the Fn14/NF-κB pathway

doi: 10.3892/ijmm.2025.5679

Figure Lengend Snippet: Effects of TWEAK and TWEAK-Fn14-IN-1 on the progression of rat periodontitis. (A) Micro-CT images of the rat maxilla, with the distance between the two red short lines representing the CEJ-ABC distance on the buccal side, and with statistical analysis of the (B) distance of CEJ-ABC (n=6), and (C) BV/TV at the root bifurcation of the maxillary second molar (n=6). Scale bar, 1 mm. Images of (D) H&E and (E) Masson's trichrome staining of rat periodontal tissues. Scale bar, 1 mm (top) or 100 μ m (bottom). (F) Representative images of TRAP/alkaline phosphatase double staining in the periodontal tissue of the rat maxillary second molar, with (G) quantification and statistical analysis of osteoclast numbers (TRAP-positive, multinucleated cells located in the bone resorption lacunae) at the mesial root (n=6). Red triangles indicate osteoclasts. Scale bar, 50 μ m. (H) Immunofluorescence staining of CD163 (green) and CD68 (red) in periodontal tissues, with (J) quantification of the mean fluorescence intensity of CD163 and (K) quantification of the mean fluorescence intensity of CD68 (n=6). Scale bar, 100 μ m. (I) Immunofluorescence staining of RUNX2 (green) and Periostin (red) in periodontal tissues, with (L) quantification of the mean fluorescence intensity of RUNX2 and (M) quantification of the mean fluorescence intensity of Periostin (n=6). Scale bar, 100 μ m. Blank represents the unmodeled group, PBS refers to the control group where PBS was used instead of TWEAK or TWEAK-Fn14-IN-1 during modeling, and TWEAK and TWEAK-Fn14-IN-1 represent experimental groups where the recombinant TWEAK protein or TWEAK-Fn14-IN-1 inhibitor was applied, respectively. Statistical analysis was performed using a one-way ANOVA. * P<0.05; ** P<0.01; **** P<0.0001. Data are presented as the mean ± SD. ABC, alveolar bone crest; BV/TV, bone volume to total volume; CEJ, cementoenamel junction; Fn14, fibroblast growth factor-inducible 14; MOD, mean optical density; ns, not significant; RUNX2, runt-related transcription factor 2; TRAP, tartrate-resistant acid phosphatase; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Techniques: Micro-CT, Staining, Double Staining, Immunofluorescence, Fluorescence, Control, Recombinant

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

a. Violin plots display the expression of Tnfrsf12a and Tnfrsf1a in PT-S1 and DCT. The kinetic curves show Tnfrsf12a mRNA expression in total kidney samples in male (blue) and female (red) mice treated with FGF23 for 0, 1, 4, 12, and 24h. b. In vitro analysis highlights the expression of TNFRSF12A, EGR1 , and TNFRSF1A in HEK293 (black line) and HEK293-mKL (red line) cells treated with FGF23 for different times. c. Correlation analysis shows the positive interrelationship between EGR1 and TNFRSF12A. d. HEK293-mKL cells were treated with FGF23 (50 ng/ml) for four hours and RNAseq was performed. The heatmap graphic displays differential gene expression of TNFRs genes that change with FGF23 treatment. e. HEK293-mKL cells were treated with FGF23 (50 ng/ml) for 4 hours and ATACseq was performed. Representative ATACseq peaks of cells treated with vehicle (top track) compared to cells treated with FGF23 (lower track) highlights the increase of chromatin accessibility across the TNFRSF12A gene body. f. HEK293-mKL cells were pretreated with the MEK inhibitor U0126 (5, or 10 μM) for 1h prior to FGF23 administration (50 ng/ml) for 10 minutes; p-ERK immunoblot. g. HEK293-mKL cells were pretreated with U0126 (5, or 10 μM) for 1h prior to FGF23 (50 ng/ml) for 4 hours followed by RNA extraction and assessment of EGR1 and TNFRSF12A mRNAs by qPCR. h. HEK293-mKL cells were pretreated with BMS-34554 (10 μM) for 1h followed by FGF23 (50 ng/ml) for 4h then TNFRSF12A and CXCL8 mRNA expression analysis. i. HEK293-mKL cells were treated with 50 ng/ml of FGF23 for 0, 1, 2, 4, 8, and 24h. The mRNA expression of CCL5 , CXCL8, and IL6 obtained from in vitro studies were measured by qPCR. The red kinetic curve represents the HEK293-mKL cell studies whereas the black kinetic curve shows the data from HEK293 cell line experiments. j. HEK293-mKL cells were treated with the ligand of TNFRSF12A (TNFSF12, TWEAK 100 ng/ml) for 1h followed by FGF23 treatment for 4h and pERK immunoblot. k. HEK293-mKL cells were pretreated with TWEAK (100 ng/ml) for 1h followed by FGF23 (50 ng/ml) for 16h. EGR1 expression was then tested by qPCR to assess FGF23 bioactivity. l. In vivo studies were performed by treating C57BL/6 mice with recombinant mouse TWEAK for 4 days at the rate of one injection per day. On the fourth day mice received FGF23 treatment for 4h and Tnfrsf12a was assessed by qPCR. m-p. HEK293-mKL cells were pretreated with TNF (100 ng/ml) for 16 h followed with FGF23 (50 ng/ml) for 4 h. pNF-κB, total NF-κB, and β-actin were evaluated by immunoblot ( m ). The mRNA expression of NFKB ( n ), CXCL8 ( o ), and EGR1 ( p ) were assessed by qPCR.

Journal: bioRxiv

Article Title: Dynamic Single Cell Transcriptomics Defines Kidney FGF23/KL Bioactivity and Novel Segment-Specific Inflammatory Targets

doi: 10.1101/2024.05.24.595014

Figure Lengend Snippet: a. Violin plots display the expression of Tnfrsf12a and Tnfrsf1a in PT-S1 and DCT. The kinetic curves show Tnfrsf12a mRNA expression in total kidney samples in male (blue) and female (red) mice treated with FGF23 for 0, 1, 4, 12, and 24h. b. In vitro analysis highlights the expression of TNFRSF12A, EGR1 , and TNFRSF1A in HEK293 (black line) and HEK293-mKL (red line) cells treated with FGF23 for different times. c. Correlation analysis shows the positive interrelationship between EGR1 and TNFRSF12A. d. HEK293-mKL cells were treated with FGF23 (50 ng/ml) for four hours and RNAseq was performed. The heatmap graphic displays differential gene expression of TNFRs genes that change with FGF23 treatment. e. HEK293-mKL cells were treated with FGF23 (50 ng/ml) for 4 hours and ATACseq was performed. Representative ATACseq peaks of cells treated with vehicle (top track) compared to cells treated with FGF23 (lower track) highlights the increase of chromatin accessibility across the TNFRSF12A gene body. f. HEK293-mKL cells were pretreated with the MEK inhibitor U0126 (5, or 10 μM) for 1h prior to FGF23 administration (50 ng/ml) for 10 minutes; p-ERK immunoblot. g. HEK293-mKL cells were pretreated with U0126 (5, or 10 μM) for 1h prior to FGF23 (50 ng/ml) for 4 hours followed by RNA extraction and assessment of EGR1 and TNFRSF12A mRNAs by qPCR. h. HEK293-mKL cells were pretreated with BMS-34554 (10 μM) for 1h followed by FGF23 (50 ng/ml) for 4h then TNFRSF12A and CXCL8 mRNA expression analysis. i. HEK293-mKL cells were treated with 50 ng/ml of FGF23 for 0, 1, 2, 4, 8, and 24h. The mRNA expression of CCL5 , CXCL8, and IL6 obtained from in vitro studies were measured by qPCR. The red kinetic curve represents the HEK293-mKL cell studies whereas the black kinetic curve shows the data from HEK293 cell line experiments. j. HEK293-mKL cells were treated with the ligand of TNFRSF12A (TNFSF12, TWEAK 100 ng/ml) for 1h followed by FGF23 treatment for 4h and pERK immunoblot. k. HEK293-mKL cells were pretreated with TWEAK (100 ng/ml) for 1h followed by FGF23 (50 ng/ml) for 16h. EGR1 expression was then tested by qPCR to assess FGF23 bioactivity. l. In vivo studies were performed by treating C57BL/6 mice with recombinant mouse TWEAK for 4 days at the rate of one injection per day. On the fourth day mice received FGF23 treatment for 4h and Tnfrsf12a was assessed by qPCR. m-p. HEK293-mKL cells were pretreated with TNF (100 ng/ml) for 16 h followed with FGF23 (50 ng/ml) for 4 h. pNF-κB, total NF-κB, and β-actin were evaluated by immunoblot ( m ). The mRNA expression of NFKB ( n ), CXCL8 ( o ), and EGR1 ( p ) were assessed by qPCR.

Article Snippet: In some experiments, cells were pretreated with BMS-345541 (BMS; Selleck Chemicals, Houston, TX), U0126 (MAPK inhibitor), rTWEAK (1090-TW; R&D Systems), rTNFα (210-TA; R&D Systems) prior to FGF23 (2604-FG; R&D Systems) treatment.

Techniques: Expressing, In Vitro, Gene Expression, Western Blot, RNA Extraction, In Vivo, Recombinant, Injection

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