rabbit anti fn14  (Cell Signaling Technology Inc)

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.

Article Snippet: The following antibodies were used for western blotting: Rabbit anti-Fn14 antibody (cat. no. bs-2493R; BIOSS), rabbit anti-NF-κB p65 (D14E12) antibody (cat. no. 8242; Cell Signaling Technology, Inc.), rabbit anti-phospho-NFκB p65 (Ser536) antibody (cat. no. 3033; Cell Signaling Technology, Inc.), rabbit anti-NLRP3 antibody (cat. no. bs-41293R; BIOSS), mouse anti-RUNX2 antibody (cat. no. ab76956; Abcam), rabbit anti-Sp7/Osterix antibody [ EPR21034 ] (cat. no. ab209484; Abcam), rabbit anti-OPG antibody (cat. no. ab73400; Abcam), mouse anti-ALP antibody [2F4] (cat. no. ab126820; Abcam), rabbit anti-TWEAK antibody (cat. no. PK93318; Abmart Pharmaceutical Technology Co., Ltd.), mouse anti-β-actin antibody (cat. no. T200068-8F10; ZENBIO Biotechnology Co., Ltd.), goat anti-mouse IgG H&L (HRP) (cat. no. 511103; ZENBIO Biotechnology Co., Ltd.) and goat anti-rabbit IgG H&L (HRP) (cat. no. 511203; ZENBIO Biotechnology Co., Ltd.).

Techniques: Western Blot, shRNA, Two Tailed Test

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.

Article Snippet: The following antibodies were used for western blotting: Rabbit anti-Fn14 antibody (cat. no. bs-2493R; BIOSS), rabbit anti-NF-κB p65 (D14E12) antibody (cat. no. 8242; Cell Signaling Technology, Inc.), rabbit anti-phospho-NFκB p65 (Ser536) antibody (cat. no. 3033; Cell Signaling Technology, Inc.), rabbit anti-NLRP3 antibody (cat. no. bs-41293R; BIOSS), mouse anti-RUNX2 antibody (cat. no. ab76956; Abcam), rabbit anti-Sp7/Osterix antibody [ EPR21034 ] (cat. no. ab209484; Abcam), rabbit anti-OPG antibody (cat. no. ab73400; Abcam), mouse anti-ALP antibody [2F4] (cat. no. ab126820; Abcam), rabbit anti-TWEAK antibody (cat. no. PK93318; Abmart Pharmaceutical Technology Co., Ltd.), mouse anti-β-actin antibody (cat. no. T200068-8F10; ZENBIO Biotechnology Co., Ltd.), goat anti-mouse IgG H&L (HRP) (cat. no. 511103; ZENBIO Biotechnology Co., Ltd.) and goat anti-rabbit IgG H&L (HRP) (cat. no. 511203; ZENBIO Biotechnology Co., Ltd.).

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

Article Snippet: The following antibodies were used for western blotting: Rabbit anti-Fn14 antibody (cat. no. bs-2493R; BIOSS), rabbit anti-NF-κB p65 (D14E12) antibody (cat. no. 8242; Cell Signaling Technology, Inc.), rabbit anti-phospho-NFκB p65 (Ser536) antibody (cat. no. 3033; Cell Signaling Technology, Inc.), rabbit anti-NLRP3 antibody (cat. no. bs-41293R; BIOSS), mouse anti-RUNX2 antibody (cat. no. ab76956; Abcam), rabbit anti-Sp7/Osterix antibody [ EPR21034 ] (cat. no. ab209484; Abcam), rabbit anti-OPG antibody (cat. no. ab73400; Abcam), mouse anti-ALP antibody [2F4] (cat. no. ab126820; Abcam), rabbit anti-TWEAK antibody (cat. no. PK93318; Abmart Pharmaceutical Technology Co., Ltd.), mouse anti-β-actin antibody (cat. no. T200068-8F10; ZENBIO Biotechnology Co., Ltd.), goat anti-mouse IgG H&L (HRP) (cat. no. 511103; ZENBIO Biotechnology Co., Ltd.) and goat anti-rabbit IgG H&L (HRP) (cat. no. 511203; ZENBIO Biotechnology Co., Ltd.).

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.

Article Snippet: The following antibodies were used for western blotting: Rabbit anti-Fn14 antibody (cat. no. bs-2493R; BIOSS), rabbit anti-NF-κB p65 (D14E12) antibody (cat. no. 8242; Cell Signaling Technology, Inc.), rabbit anti-phospho-NFκB p65 (Ser536) antibody (cat. no. 3033; Cell Signaling Technology, Inc.), rabbit anti-NLRP3 antibody (cat. no. bs-41293R; BIOSS), mouse anti-RUNX2 antibody (cat. no. ab76956; Abcam), rabbit anti-Sp7/Osterix antibody [ EPR21034 ] (cat. no. ab209484; Abcam), rabbit anti-OPG antibody (cat. no. ab73400; Abcam), mouse anti-ALP antibody [2F4] (cat. no. ab126820; Abcam), rabbit anti-TWEAK antibody (cat. no. PK93318; Abmart Pharmaceutical Technology Co., Ltd.), mouse anti-β-actin antibody (cat. no. T200068-8F10; ZENBIO Biotechnology Co., Ltd.), goat anti-mouse IgG H&L (HRP) (cat. no. 511103; ZENBIO Biotechnology Co., Ltd.) and goat anti-rabbit IgG H&L (HRP) (cat. no. 511203; ZENBIO Biotechnology Co., Ltd.).

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

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: Effect of inhibition of the tumor necrosis factor-like weak inducer of apoptosis/Fn14/NF-κB/NLRP3 pathway on the microenvironmental regulatory ability of iPDLSCs. (A) Immunofluorescence staining was used to detect the expression levels of RANKL and OPG in PDLSCs, iPDLSCs, and iPDLSCs after the downregulation of Fn14, NF-κB and NLRP3, followed by statistical analysis of the average optical density values of (B) RANKL and (C) OPG, and (D) the RANKL/OPG ratio (n=5). Scale bar, 200 μ m. (E) Immunofluorescence staining was used to detect the expression levels of CD68 and CD163 in macrophages cocultured with PDLSCs, iPDLSCs, and iPDLSCs after the downregulation of Fn14, NF-κB and NLRP3, followed by statistical analysis of the average optical density values of (F) CD68 and (G) CD163 (n=5). Scale bar, 200 μ m. 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. Fn14, fibroblast growth factor-inducible 14; iPDLSC, inflammatory PDLSC; MOD, mean optical density; NLRP3, NOD-like receptor thermal protein domain-associated protein 3; ns, not significant; OPG, osteoprotegerin; PDLSC, periodontal ligament stem cell; RANKL, receptor activator of nuclear factor-κB ligand; sh, short hairpin RNA.

Article Snippet: The following antibodies were used for western blotting: Rabbit anti-Fn14 antibody (cat. no. bs-2493R; BIOSS), rabbit anti-NF-κB p65 (D14E12) antibody (cat. no. 8242; Cell Signaling Technology, Inc.), rabbit anti-phospho-NFκB p65 (Ser536) antibody (cat. no. 3033; Cell Signaling Technology, Inc.), rabbit anti-NLRP3 antibody (cat. no. bs-41293R; BIOSS), mouse anti-RUNX2 antibody (cat. no. ab76956; Abcam), rabbit anti-Sp7/Osterix antibody [ EPR21034 ] (cat. no. ab209484; Abcam), rabbit anti-OPG antibody (cat. no. ab73400; Abcam), mouse anti-ALP antibody [2F4] (cat. no. ab126820; Abcam), rabbit anti-TWEAK antibody (cat. no. PK93318; Abmart Pharmaceutical Technology Co., Ltd.), mouse anti-β-actin antibody (cat. no. T200068-8F10; ZENBIO Biotechnology Co., Ltd.), goat anti-mouse IgG H&L (HRP) (cat. no. 511103; ZENBIO Biotechnology Co., Ltd.) and goat anti-rabbit IgG H&L (HRP) (cat. no. 511203; ZENBIO Biotechnology Co., Ltd.).

Techniques: Inhibition, Immunofluorescence, Staining, Expressing, 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: 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.

Article Snippet: The following antibodies were used for western blotting: Rabbit anti-Fn14 antibody (cat. no. bs-2493R; BIOSS), rabbit anti-NF-κB p65 (D14E12) antibody (cat. no. 8242; Cell Signaling Technology, Inc.), rabbit anti-phospho-NFκB p65 (Ser536) antibody (cat. no. 3033; Cell Signaling Technology, Inc.), rabbit anti-NLRP3 antibody (cat. no. bs-41293R; BIOSS), mouse anti-RUNX2 antibody (cat. no. ab76956; Abcam), rabbit anti-Sp7/Osterix antibody [ EPR21034 ] (cat. no. ab209484; Abcam), rabbit anti-OPG antibody (cat. no. ab73400; Abcam), mouse anti-ALP antibody [2F4] (cat. no. ab126820; Abcam), rabbit anti-TWEAK antibody (cat. no. PK93318; Abmart Pharmaceutical Technology Co., Ltd.), mouse anti-β-actin antibody (cat. no. T200068-8F10; ZENBIO Biotechnology Co., Ltd.), goat anti-mouse IgG H&L (HRP) (cat. no. 511103; ZENBIO Biotechnology Co., Ltd.) and goat anti-rabbit IgG H&L (HRP) (cat. no. 511203; ZENBIO Biotechnology Co., Ltd.).

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

Journal: Cell reports

Article Title: Fn14 is an activity-dependent, Bmal1-regulated cytokine receptor that induces rod-like microglia and restricts neuronal activity in vivo

doi: 10.1016/j.celrep.2026.116926

Figure Lengend Snippet: (A and B) Confocal images of sagittal sections of the mouse brain at P28 (A) and P90 (B) subjected to single-molecule fluorescence in situ hybridization (smFISH) to label Fn14 mRNA (white). Scale bars, 1 mm. (C and D) High-resolution confocal images of the dLGN in coronal sections from a P28 (C) and a P90 (D) mouse brain probed for Fn14 (green) and the glutamatergic neuronal marker Vglut1 (magenta). DAPI is shown in blue. Scale bars, 20 μm. (E) Quantification of the percentage of Fn14 -expressing cells that also express Vglut1 in the dLGN at P28 and P90. Unpaired Student’s t test, p > 0.05; n = 3 mice/age. (F–I) Confocal images of CA1 (F), CA3 (G), and dentate gyrus (DG; H) subregions of the hippocampus in a coronal section from a P28 mouse brain probed for Fn14 (green) and Vglut1 (magenta). DAPI is shown in blue. Scale bars, 20 μm. Inset scale bar, 5 μm. (I–K) Confocal images of CA1 (I), CA3 (J), and DG (K) regions of the hippocampus in a coronal section from a P90 mouse brain probed for Fn14 (green) and Vglut1 (magenta). DAPI is shown in blue. Scale bar, 20 μm. Inset scale bar, 5 μm. (L) Quantification of Fn14 -expressing cells that are positive for Vglut1 in the hippocampus at both ages. Two-way ANOVA: region, p > 0.05; age, p > 0.05; and interaction, p > 0.05; n = 3 mice/age for each region. (M) Confocal image of Fn14 (green) and the inhibitory neuron marker Gad1 (magenta) in the DG at P90. Scale bar, 20 μm. Inset scale bar, 5 μm. (N) Scatterplot demonstrating the correlation between Fn14 expression ( x axis) and the expression of excitatory ( Vglut1 ) or inhibitory ( Gad1 ) neuron markers ( y axis) in the hippocampus. Linear regression with slope comparison, *** p < 0.001. (O and P) Confocal images of CA1 following smFISH for Fn14 (green), the pyramidal (PYR) neuron marker Camk2a (red), and the activity-dependent gene Fos (yellow) in mice exposed to vehicle (O) or kainate (P). Scale bars, 50 μm. Inset scale bars, 16 μm. (Q and R) Confocal images of CA1 following smFISH for Fn14 (green), the interneuron marker Gad2 (red), and the activity-dependent gene Fos (yellow) in mice exposed to vehicle (Q) or kainate (R). Scale bars, 50 μm. Inset scale bars, 16 μm. (S and T) Quantification of Fos (S) or Fn14 (T) expression in Camk2a + neurons in response to vehicle or kainate exposure; values are normalized to vehicle. (U and V) Quantification of Fos (U) or Fn14 (V) expression in Gad2 + interneurons in response to vehicle or kainate exposure; values are normalized to vehicle. Statistics for (S)–(V): unpaired Student’s t test, *** p < 0.001, ** p < 0.01, and * p < 0.05; n.s., p > 0.05; n = 4 mice/condition. (W) Quantification of Fn14 expression in Fos − and Fos + PYR neurons aggregated from both vehicle and kainate conditions. Unpaired Student’s t test, * p < 0.05. (X) Schematic of the environmental enrichment paradigm depicting 5 days of deprived housing followed by 24 h of enrichment. (Y and Z) Confocal images of CA1 following smFISH for Fn14 (green) and Fos (magenta) in deprived (Y) or enriched conditions (Z). Inset shown in the upper right. Scale bars, 50 μm. Inset scale bars, 10 μm. (AA) Quantification of Fn14 transcripts per cell in deprived and enriched conditions, normalized to deprived. Unpaired Student’s t test, * p < 0.05; n = 7 mice/condition. See also .

Article Snippet: Fn14 protein abundance in hippocampal lysates from mice exposed to kainate or water (vehicle) was measured by immunoprecipitating Fn14 using a rabbit anti-Fn14 antibody (Cell Signaling Technologies, 4403s) at 1:50.

Techniques: Fluorescence, In Situ Hybridization, Marker, Expressing, Comparison, Activity Assay

Journal: Cell reports

Article Title: Fn14 is an activity-dependent, Bmal1-regulated cytokine receptor that induces rod-like microglia and restricts neuronal activity in vivo

doi: 10.1016/j.celrep.2026.116926

Figure Lengend Snippet: (A) Diagram of the cued fear conditioning (CFC) paradigm. An auditory tone and a unique spatial context were initially paired with an aversive foot shock. The ability of mice to remember this association was later tested by exposing the mice to either the spatial context or the auditory tone in the absence of the shock. Freezing behavior, which mice exhibit when afraid, serves as a readout for how well the mice remember the association between conditioned and unconditioned stimuli. (B) Quantification of percentage of time spent freezing across all conditions. Repeated measures ANOVA: trial, p < 0.0001; genotype, p < 0.05; subject, trial × genotype, p < 0.0001. Bonferroni-corrected multiple comparisons, WT vs. KO: training, p > 0.05; context − tone, p = 0.089; novel context, p > 0.05; novel context + tone, p < 0.001. (C) Diagram of Morris water maze (MWM) training and probe trials. (D) Latency to goal platform swum during MWM trials. Repeated measures ANOVA with Šídák’s multiple comparisons test: platform is visible (V), genotype, p > 0.05, trial, p < 0.0001, trial × genotype, p > 0.05; platform is hidden (H), genotype, p > 0.05, trial, p < 0.0001, trial × genotype, p > 0.05; platform goal zone is reversed (R), genotype, p > 0.05, trial, p < 0.001, trial × genotype, p > 0.05. (E) Path length swum by Fn14 WT and KO mice during the MWM test. Repeated measures ANOVA with Šídák’s multiple comparisons test: V, genotype, p > 0.05, trial, p < 0.0001, trial × genotype, p > 0.05; H, genotype, p > 0.05, trial, p < 0.0001, trial × genotype, p > 0.05; R, genotype, p > 0.05, trial, p < 0.001, trial × genotype, p > 0.05. Same n as in (B). (F) Distance swum by mice in the target quadrant during the probe trial (cm). Unpaired Student’s t test, ** p < 0.01. (G) Time spent in target quadrant during probe trial (seconds). Unpaired Student’s t test, p = 0.12. ** p < 0.01 and *** p < 0.001. For all quantifications, n = 17 WT and 19 Fn14-KO mice. See also .

Article Snippet: Fn14 protein abundance in hippocampal lysates from mice exposed to kainate or water (vehicle) was measured by immunoprecipitating Fn14 using a rabbit anti-Fn14 antibody (Cell Signaling Technologies, 4403s) at 1:50.

Techniques:

Journal: Cell reports

Article Title: Fn14 is an activity-dependent, Bmal1-regulated cytokine receptor that induces rod-like microglia and restricts neuronal activity in vivo

doi: 10.1016/j.celrep.2026.116926

Figure Lengend Snippet: (A) Schematic of the experimental timeline with an example confocal image of GCaMP6f expression in CA1 and the optic fiber tract right above CA1. Scale bar, 100 μm. ZT, zeitgeber time (mouse’s subjective time of day). Blue bars, 10-min recording periods. M, months of age. H, hours. (B) Example 10-min binned calcium traces (ΔF/F) from a representative WT and Fn14-KO mouse, recorded every hour (ZT 0–23) over a single day. (C) Maximum ΔF/F signal over each time bin. Repeated measures two-way ANOVA: time, p > 0.05; genotype, p > 0.05; interaction, p > 0.05. (D) Analysis of maximum ΔF/F signal during the light and dark phases of the day plotted as Z scores. Repeated measures two-way ANOVA: time, p > 0.05; genotype, p > 0.05; interaction, p > 0.05. (E) Ca 2+ event frequency in WT and Fn14-KO mice over a 24-h recording period. Repeated measures two-way ANOVA: time, p > 0.05; genotype, p < 0.05; interaction, p > 0.05; with Tukey post hoc test: * p < 0.05 at ZT 11. (F) Quantification of the Ca 2+ event frequency during the light (ZT 0–11) and dark (ZT 12–23) phases of the day. Repeated measures ANOVA: time, p > 0.05; genotype, p < 0.01; interaction, p > 0.05; with Tukey post hoc test: ** p < 0.01 during the dark phase. For all fiber analyses, n = 36 traces from 3 mice per genotype. Line graphs and histograms show mean ± SEM, while histograms show both acquisitions (closed circles) and within-mouse averages (open circles). (G) Confocal images of smFISH in hippocampal CA1 probing against Fn14 (green) at ZT 6 (1:00 p.m., top) and ZT 18 (1:00 a.m., bottom). DAPI is in blue. Scale bar, 50 μm. Inset scale bar, 10 μm. (H) Quantification of Fn14 transcripts per cell in CA1 at ZT 6 and ZT 18 normalized to ZT 6. Unpaired Student’s t test, p = 0.1508; n = 5 mice/time point. Data points represent individual mice with mean ± SEM shown. (I) Schematic depicting predicted Bmal1 binding sites (magenta) within ±1,000 bp of the Fn14 transcription start site (TSS). Black arrow depicts the start of the Fn14 gene, gray rectangles represent Fn14 exons, black lines indicate 500-bp segments relative to the Fn14 TSS. (J) Confocal images of smFISH in hippocampal CA1 of Bmal1 conditional knockout (Bmal1 cKO ) mice, probing against Bmal1 (magenta) and Fn14 (green). Control hemisphere (top), CA1 neurons were transduced with eBFP2. Bmal1 cKO hemisphere (bottom), CA1 neurons were transduced with Cre-GFP to elicit ablation of Bmal1 (see related and for validations). DAPI is shown in blue. Scale bar, 50 μm. Inset scale bar, 10 μm. (K) Quantification of Fn14 transcripts per cell in control and Bmal1 cKO hemispheres. Paired t test, * p < 0.05. Data points represent individual mice, n = 3 mice, 3 hemispheres/condition. See also .

Article Snippet: Fn14 protein abundance in hippocampal lysates from mice exposed to kainate or water (vehicle) was measured by immunoprecipitating Fn14 using a rabbit anti-Fn14 antibody (Cell Signaling Technologies, 4403s) at 1:50.

Techniques: Expressing, Binding Assay, Knock-Out, Control, Transduction

Journal: Cell reports

Article Title: Fn14 is an activity-dependent, Bmal1-regulated cytokine receptor that induces rod-like microglia and restricts neuronal activity in vivo

doi: 10.1016/j.celrep.2026.116926

Figure Lengend Snippet: (A) Uniform manifold approximation and projection (UMAP) visualization of the hippocampal snRNA-seq dataset including cells from WT and Fn14-KO mice subjected to 24 h of environmental enrichment. The excitatory CA1 PYR neuron cluster (Exc-CA1) is circled. (B) Volcano plot showing differentially expressed genes (DEGs) in Exc-CA1 neurons between WT and Fn14-KO mice. Genes upregulated in the KO are shown in red, while downregulated genes are shown in blue. Genes of particular interest based upon their involvement in synapse assembly and organization are shown in orange. Significance thresholds were set at an average log 2 (1.2) fold change and false discovery rate (FDR) < 0.05. (C) Top 10 Gene Ontology (GO) terms enriched among DEGs downregulated in Exc-CA1 neurons from Fn14-KO mice compared to WT. (D) Confocal images of hippocampal CA1 from Fn14-KO and WT mice immunostained for the excitatory presynaptic marker vGluT1 (magenta) and the post-synaptic marker Homer1b/c (green). Rightmost column shows merge of vGluT1 and Homer1b/c with colocalizations colored in cyan. Scale bar, 5 μm. (E and F) Quantification of excitatory synapse (i.e., colocalized pre- and postsynaptic puncta) density (E) and volume (F). Data points represent individual mouse averages with mean ± SEM. Unpaired Student’s t test, n = 4 mice/genotype. (G) Representative confocal images of microglia (Iba1, green), excitatory synapses (vGluT1, magenta), and inhibitory synapses (vGaT, cyan) in WT and Fn14-KO mice. Scale bar, 10 μm. (H) Example reconstructions of microglia (Iba1, green) in contact with excitatory synapses (vGluT1, magenta) and inhibitory synapses (vGaT, cyan) in hippocampal CA1. Signals were reconstructed from fluorescence images shown in (G). (I–L) Quantification of the number and volume of excitatory synapses contacted by microglia (I and J), with quantification of the number and volume of inhibitory synapses contacted by microglia shown in (K) and (L). Data are represented on a log scale to best fit the distribution of the data. For (I)–(L), individual datapoints indicate cells, while open circles indicate mouse averages; n = 45/50 microglia from 3 WT/4 Fn14-KO mice. Mann-Whitney test, * p < 0.05, ** p < 0.01, and *** p < 0.001. (M) Example reconstructions of microglia (Iba1) in WT and Fn14-KO mice depicting concentric rings for Sholl analysis. Scale bar, 5 μm. (N) Quantification of microglial complexity based upon Sholl analysis. Line graph depicts microglial intersections per Sholl radius with mean (dark line) ± SEM (shaded region). Repeated measures two-way ANOVA: radii, p < 0.0001; genotype, p < 0.05; interaction, p < 0.01. Inset plot shows area under the curve (AUC) averaged across all cells ± SEM. Unpaired Student’s t test, ** p < 0.01; n = 16 cells/genotype, 3 mice/genotype. See also and .

Article Snippet: Fn14 protein abundance in hippocampal lysates from mice exposed to kainate or water (vehicle) was measured by immunoprecipitating Fn14 using a rabbit anti-Fn14 antibody (Cell Signaling Technologies, 4403s) at 1:50.

Techniques: Marker, Fluorescence, MANN-WHITNEY

Journal: Cell reports

Article Title: Fn14 is an activity-dependent, Bmal1-regulated cytokine receptor that induces rod-like microglia and restricts neuronal activity in vivo

doi: 10.1016/j.celrep.2026.116926

Figure Lengend Snippet: (A) Schematic representation of the AAV construct for driving Fn14 overexpression (OE) in CA1 neurons. The construct consists of the human Synaptophysin promoter (hSyn, gray arrow), N-terminal HA-tagged Fn14 (HA-Fn14, magenta arrow), internal ribosome entry site (IRES, gray rectangle), and cytosolic eGFP as a marker of transduction (green arrow). (B) Illustration of experimental paradigm showing bilateral injections into CA1 of Fn14-KO mice with control AAV9-hSyn-eBFP2 into the left hemisphere and AAV9-hSyn-HA-Fn14-IRES-eGFP (i.e., Fn14 OE) into the right hemisphere. (C) Example confocal images of CA1 showing Fn14 (magenta, immunostained), eGFP (green), and eBFP2 (cyan) in control (i.e., Fn14 KO, top) and Fn14-OE (bottom) mice. Images were taken from the same animal. Scale bar, 50 μm. (D) Quantification of Fn14 fluorescence intensity in Fn14-KO and -OE hemispheres from the same mice. Paired t test, * p < 0.05; n = 3 mice, 1 hemisphere/condition/mouse. (E) Plot of eGFP by Fn14 fluorescence intensity for each cell analyzed, depicting a positive correlation. Pearson correlation coefficients, r 2 = 0.07397, **** p < 0.0001; n = 122 cells. Data points show individual cells with line of best fit and 95% confidence bands (teal region). (F) Confocal images of microglia (Iba1, magenta) from Fn14-KO and Fn14-OE hemispheres within the same animal. Scale bar, 10 μm. (G) Quantification of total Iba1 volume within CA1. Data points represent individual mice. (H) Quantification of dominant angle of Iba1 signal relative to dendrites. Data points represent individual mice. (I) Colocalization channel of microglia (Iba1) and virally transduced dendrites from Fn14-KO and -OE conditions (top row). Bottom row, left: Iba1 (magenta) and eGFP (green). Bottom row, right: colocalization channel showing where Iba1 and eGFP overlap. Scale bar (top), 50 μm. Inset scale bar (bottom), 10 μm. (J and K) Quantification of total number (J) and volume (K) of Iba1-dendrite colocalizations. Data points represent individual mice. (L) Left: example confocal images of microglia (Iba1, magenta) and excitatory synapses (vGluT1, yellow) from Fn14-KO (top) and Fn14-OE (bottom) hemispheres from the same mouse. Right: 3D reconstructions of Iba1 (magenta) and vGluT1-Iba1 contacts (yellow) based upon the images on the left. (M and N) Quantification of the total number of vGluT1 puncta per image volume (M) and the number of vGluT1-Iba1 contacts per image volume (N) with data points representing individual mice. Statistical analysis for (G)–(N): paired t test; * p < 0.05 and ** p < 0.01; n = 4 mice, 1 hemisphere/condition/mouse. See also .

Article Snippet: Fn14 protein abundance in hippocampal lysates from mice exposed to kainate or water (vehicle) was measured by immunoprecipitating Fn14 using a rabbit anti-Fn14 antibody (Cell Signaling Technologies, 4403s) at 1:50.

Techniques: Construct, Over Expression, Marker, Transduction, Control, Fluorescence

Journal: Cell reports

Article Title: Fn14 is an activity-dependent, Bmal1-regulated cytokine receptor that induces rod-like microglia and restricts neuronal activity in vivo

doi: 10.1016/j.celrep.2026.116926

Figure Lengend Snippet: (A) Actograms of representative WT and Fn14-KO mice under normal 12:12 light/dark conditions (top) and in constant darkness (bottom). Normalized wheel-running activity is represented in dark blue. (B) Periodicity of wheel-running activity under normal 12:12 light conditions. Unpaired Student’s t test, p > 0.05; n = 11 WT and 4 Fn14-KO mice. (C) Innate free-running period of Fn14-KO and WT mice during constant darkness, representative of the mouse’s internal circadian cycle. Unpaired Student’s t test, * p < 0.05; n = 11 WT and 4 Fn14-KO mice. (D–F) Quantification of electroencephalogram/electromyography (EEG/EMG) analysis of sleep and activity states during a 24-h recording period. Line, mean; shaded area, SEM. Repeated measures two-way ANOVA with Šídák’s multiple comparisons; for (D) and (E), time, genotype, and interaction: p > 0.05; for (F), time, p > 0.05, genotype, * p < 0.05, and interaction, p > 0.05. (G) REM bout duration (seconds) during the day (sleep phase), during the night (wake phase), and over the full 24-h period (total). (H) Quantification of bout number during the day, during the night, and over the full 24-h period. (I) Mean wake bout duration (seconds) during the day, during the night, and over the full 24-h period. (J) Mean number of wake bouts during the day, during the night, and over the full 24-h period. (K) Low (light gray) and high (dark gray) theta frequency bands following sleep deprivation in WT mice. (L) Low (light teal) and high (dark teal) theta frequency bands following sleep deprivation in Fn14-KO mice. (M) Quantification of the ratio of low and high theta frequency in WT and Fn14-KO mice. Statistics for (G)–(J) and (M): unpaired Student’s t test, * p < 0.05. (D)–(M) n = 6 mice/genotype. See also .

Article Snippet: Fn14 protein abundance in hippocampal lysates from mice exposed to kainate or water (vehicle) was measured by immunoprecipitating Fn14 using a rabbit anti-Fn14 antibody (Cell Signaling Technologies, 4403s) at 1:50.

Techniques: Activity Assay

Journal: Cell reports

Article Title: Fn14 is an activity-dependent, Bmal1-regulated cytokine receptor that induces rod-like microglia and restricts neuronal activity in vivo

doi: 10.1016/j.celrep.2026.116926

Figure Lengend Snippet: (A) Schematic of EEG electrode placement and the experimental timeline. (B) Example EEG traces from WT (gray) and Fn14-KO (teal) mice after pentylenetetrazol (PTZ) injection (black arrow). Red triangles indicate the onset of general tonic-clonic (GTC) seizures (WT, latency = 311 s, duration = 19.8 s; Fn14 KO, latency = 159 s, duration = 35 s). The Fn14-KO mouse died shortly after GTC seizures, demonstrated by the elimination of signal following the seizure. (C) Percentage of mice that had GTC seizures relative to the time course of the experiment. WT, median = 311 s; Fn14 KO, median = 159 s. Log-rank test, * p < 0.05. (D) Latency between PTZ injection and GTC onset. Mann-Whitney test, ** p < 0.01. (E) Duration of GTCs. Unpaired Student’s t test, * p < 0.05. (F) Mortality rate of Fn14-KO and WT mice following PTZ administration. Log-rank test, * p < 0.05. (G) Number of PTZ-induced myoclonic seizures. Mann-Whitney test, p > 0.05. (H) The fraction of mice presenting with electrophysiological spikes (white), myoclonic seizures (gray), GTCs (teal), or death as their worst PTZ-induced outcome. Data are presented as the mean ± SEM, with data points representing individual mice or as the percentage of subjects where applicable. (B)–(H) n = 13 mice/genotype.

Article Snippet: Fn14 protein abundance in hippocampal lysates from mice exposed to kainate or water (vehicle) was measured by immunoprecipitating Fn14 using a rabbit anti-Fn14 antibody (Cell Signaling Technologies, 4403s) at 1:50.

Techniques: Injection, MANN-WHITNEY

Journal: Cellular & molecular immunology

Article Title: Synergistic effects of LCN2 and TWEAK on the progression of psoriasis.

doi: 10.1038/s41423-025-01292-9

Figure Lengend Snippet: Fig. 1 TWEAK, Fn14, and LCN2 are overexpressed in skin lesions of psoriasis patients and IMQ induced psoriatic mice model. A Single cell data from psoriasis was used for correlation analysis between TWEAK, Fn14 and LCN2. B Immunostaining of LCN2, TWEAK and Fn14 in the skin of healthy control (HC) and patient with psoriasis (Brown). Normal control skin samples = 4, patient skin samples = 4. Nuclei were stained with hematoxylin (Bar = 200 μm). C Immunostaining of LCN2, TWEAK and Fn14 were performed on wild-type mouse skin paraffin sections. Stained epidermal region areas were quantitated by ImageJ software. The relationship between the IOD of TWEAK (or Fn14) and LCN2 staining was assessed by Spearman’s rank correlation. Number of control mice = 3, Number of IMQ induced WT mice = 4. Representative images are shown (Bar = 200 μm). D Volcano Plot showed the RNA-seq analysis of wild-type control mice and wild-type imiquimod model mice. E RNA-seq analysis of wild-type mice and Lcn2–/– mice treated with imiquimod. F Immunostaining of TWEAK, Fn14 and Ly6G were performed on Lcn2 knockout mice skin paraffin sections. The relationship between the IOD of TWEAK (or Fn14) and number of Ly6G positive cell staining was assessed by Spearman’s rank correlation. Number of control mice = 3, Number of IMQ induced WT mice = 4, Number of IMQ induced Lcn2–/–

Article Snippet: For Western blotting, a rabbit anti-human Fn14 antibody (1:1000, CST, MA, USA) was used as the primary antibody.

Techniques: Immunostaining, Control, Staining, Software, RNA Sequencing, Knock-Out

Journal: Cellular & molecular immunology

Article Title: Synergistic effects of LCN2 and TWEAK on the progression of psoriasis.

doi: 10.1038/s41423-025-01292-9

Figure Lengend Snippet: Fig. 2 Expression localization of TWEAK and LCN2 and the interaction between LCN2 and Fn14. A Tissue mIHC results showing the cellular localization of TWEAK expression and its relationship with CD68-labeled macrophages; B tissue mIHC results showing the cellular localization of LCN2 expression and its relationship with CD66b-labeled neutrophils (Bar = 200 μm); C cellular immunofluorescence results showing the colocalization of LCN2 and Fn14; D the binding affinity of LCN2 conjugate to the Fn14 molecule was determined by SPR analysis. E Co-IP experiment detecting the interaction between LCN2 and Fn14 after stimulating Hacat cells cultured in vitro with LCN2

Article Snippet: For Western blotting, a rabbit anti-human Fn14 antibody (1:1000, CST, MA, USA) was used as the primary antibody.

Techniques: Expressing, Labeling, Binding Assay, Co-Immunoprecipitation Assay, Cell Culture, In Vitro

Journal: Cellular & molecular immunology

Article Title: Synergistic effects of LCN2 and TWEAK on the progression of psoriasis.

doi: 10.1038/s41423-025-01292-9

Figure Lengend Snippet: Fig. 3 LCN2 induces the differentiation and secretion of TWEAK in macrophages. A, B Tnfsf12, Tnfrsf12a, and Il6 were detected by RT-qPCR after treating with 0, 10, 50 ng/mL LCN2. 24p3r, Mc4r, inos, and Il10 mRNA in J774A.1 cell were detected by RT-qPCR after treated with 50 ng/mL LCN2. C The intracellular levels of Fn14, TWEAK, p-NFκB P65, p-TRAF2, and TRAF2 proteins were detected by Western blotting after stimulation of J774A.1 cell with LCN2 at concentrations of 0, 10, 50 ng/mL, respectively. D, E TWEAK and CD163 was detected by immunofluorescence in J774A.1 cell after LCN2 treatment. Bar = 100 μm. F Elisa was used to detect the concentration of TWEAK in cell supernatants 24 h after LCN2 stimulation of J774A.1. Data from immunofluorescence assays and Elisa assays are representative of three or more independent experiments are presented as the means ± SEM of triplicate wells. qPCR data are presented as the means ± SEM for triplicate wells from three or more independent experiments. ANOVA was used for comparison between groups: *P < 0.05, **P < 0.01, and ***P < 0.001. ns not significant

Article Snippet: For Western blotting, a rabbit anti-human Fn14 antibody (1:1000, CST, MA, USA) was used as the primary antibody.

Techniques: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison

Journal: Cellular & molecular immunology

Article Title: Synergistic effects of LCN2 and TWEAK on the progression of psoriasis.

doi: 10.1038/s41423-025-01292-9

Figure Lengend Snippet: Fig. 4 LCN2 regulates the expression of TWEAK and Fn14 and MAPK pathway in keratinocytes. A Immunofluorescence was used to determine TWEAK level after treatment with LCN2 in HFKs. Bar = 50 μm. B Western blotting was used to detect the expression of p-ERK1/2, p-JNK, p-p38, p-TRAF2 and Fn14 in primary keratinocytes after treating the cells with 0, 100, 250 and 500 ng/mL LCN2, respectively. C After the addition of 500 ng/mL LCN2 stimulation, keratinocytes were collected at 0, 0.5, 1, 2, 6, 12, 24 h for WB assay to detect p-ERK1/2, p-JNK, p-p38, p-TRAF2 and Fn14 expression over time. Data from immunofluorescence assays and immunoblot assays are representative of three or more independent experiments are presented as the means ± SEM of triplicate wells

Article Snippet: For Western blotting, a rabbit anti-human Fn14 antibody (1:1000, CST, MA, USA) was used as the primary antibody.

Techniques: Expressing, Western Blot

Journal: Cellular & molecular immunology

Article Title: Synergistic effects of LCN2 and TWEAK on the progression of psoriasis.

doi: 10.1038/s41423-025-01292-9

Figure Lengend Snippet: Fig. 5 TWEAK promotes neutrophil expression of LCN2 but enhances inflammation in keratinocytes by inducing MMP9 expression. A Relative mRNA expression level of Lcn2, Tnfrsf12a, Tgfb1, Il6, Tnfa, Il1b, Krt5, Krt10, Krt17, 24p3r, Mc4r in mouse skin treated with TWEAK was detected by RT-qPCR. B Immunostaining of LCN2 and Ly6G were performed on paraffin sections of IMQ mice model with or without dorsally topical application of rmTWEAK (20 μg/mL, prepared in PBS). Unpaired two-tailed t test. C Immunofluorescence assays were used to detect LCN2 expression in neutrophils after TWEAK stimulation. Bar = 20 μm. D RT-qPCR was conducted to detect Lcn2 mRNA level in neutrophils treated with TWEAK. E LCN2 levels in neutrophil supernatants after TWEAK stimulation for 24 h were measured by ELISA. F Proteomic analysis was used to further investigate the protein changes and possible pathways following TWEAK action on keratinocytes. G After treating with TWEAK on primary keratinocyte with or without M5 cytokines for 48 h, LCN2 and 24P3R protein levels in keratinocytes were detected by Western blotting. H After the TWEAK stimulus, proteins such as MMP9 and TRAF2 were detected by Western blotting. Data from immunofluorescence assays and ELISA assay are representative of three or more independent experiments are presented as the means ± SEM of triplicate wells. ANOVA was used for comparison between groups: *P < 0.05, **P < 0.01, and ***P < 0.001. ns not significant

Article Snippet: For Western blotting, a rabbit anti-human Fn14 antibody (1:1000, CST, MA, USA) was used as the primary antibody.

Techniques: Expressing, Quantitative RT-PCR, Immunostaining, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Western Blot, Comparison

Journal: Cellular & molecular immunology

Article Title: Synergistic effects of LCN2 and TWEAK on the progression of psoriasis.

doi: 10.1038/s41423-025-01292-9

Figure Lengend Snippet: Fig. 6 The synergistic interaction between LCN2 and TWEAK in keratinocytes. A Heatmap showing differential expressed genes after co- stimulating with TWEAK and LCN2 in keratinocytes. B Relative expression levels of IL6, TNFa, IL17A, CCL5, CXCL5, CXCL10 and 24P3R were detected from HFKs using RT-qPCR. C, D Western blot was used to detect p-ERK1/2, p-JNK, p-p38, p-TRAF2, 24P3R and Fn14 protein level in keratinocytes after stimulation with LCN2, TWEAK, or both. One-way ANOVA was used for comparison between groups: ns (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: For Western blotting, a rabbit anti-human Fn14 antibody (1:1000, CST, MA, USA) was used as the primary antibody.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Comparison

Journal: Cellular & molecular immunology

Article Title: Synergistic effects of LCN2 and TWEAK on the progression of psoriasis.

doi: 10.1038/s41423-025-01292-9

Figure Lengend Snippet: Fig. 7 Lcn2–/– mice attenuate the pro-proliferative and inflammatory effects of IMQ on the epidermis. A After being treated with imiquimod and LCN2, the expression levels of Fn14, p-ERK1/2 and in the skin of wild-type mice were analyzed by Western blotting (n = 3 per group). B Relative expression levels of Tnfsf12(Tweak), Tnfrsf12a(Fn14), Il6, Tnfa, Il1b, Tgfb1, Krt5, Krt10, and Krt17 from mice skin were detected using RT- qPCR. C, D Photographs of skin lesions in mice and the mouse psoriasis severity score difference between wild-type imiquimod mice and Lcn2–/– imiquimod model mice. Body weight changes in mice modeled with imiquimod for six consecutive days. E Mouse serum levels of TWEAK were assayed by ELISA. F Western blotting was used to detect KRT1, KRT5, KRT6, KRT10, KRT14, KRT16, KRT17, Loricrin, Fn14, CXCL10, LCN2, p-ERK1/2, p-JNK, p-p38 in wild-type and Lcn2–/– mice after treated with imiquimod (n = 3 per group). G Relative mRNA expression levels of Lcn2, Tnfsf12, Tnfrsf12a, Il6, Tnfa, Tgfb1, Krt5, Krt10, Krt17, 24p3r, Mc4r, Il1b were detected by RT-qPCR (n = 3–5 per group). H Immunostaining of KRT5 and KRT14 were performed on paraffin sections. Data are shown as mean ± SEM (n = 3–5 per group). ANOVA was used for comparison between groups: ns (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: For Western blotting, a rabbit anti-human Fn14 antibody (1:1000, CST, MA, USA) was used as the primary antibody.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunostaining, Comparison

Journal: Cellular & molecular immunology

Article Title: Synergistic effects of LCN2 and TWEAK on the progression of psoriasis.

doi: 10.1038/s41423-025-01292-9

Figure Lengend Snippet: Fig. 8 LCN2 acts on the downstream MAPK signaling pathway by binding to Fn14, and the deletion of Fn14 can counteract the effect of TWEAK or LCN2. A KRT6, KRT16, LCN2 were detected by WB in the skin of wild-type mice as well as in the Fn14–/– mouse imiquimod model. (n = 3 per group). B Immunohistochemistry was used to detect the expression of KRT5 and KRT14 in the skin of wild-type mice and Fn14–/– mice (Brown). Nuclei were stained with hematoxylin (Bar = 200 μm). C WB detected p-ERK1/2, p-JNK and p-p38 expression in wild-type mice and Fn14–/– mouse. (n = 3 per group). D Tnfsf12, Tnfrsf12a, Lcn2, Il1b, Il6, Krt5, Krt16, and Krt17 mRNA expression levels were detected by using RT-qPCR methods. E Immunohistochemistry was used to detect the levels of LCN2 and Ly6G in the epidermis of Fn14–/– mouse imiquimod model after administration of TWEAK. F After treated by LCN2 or TWEAK, Western blot was used to detect the expression of KRT5, KRT6, KRT14, and KRT16 in the Fn14–/– mice skin tissue. (n = 3 per group) Data were pooled from three independent experiments. Data are shown as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. SPR surface plasmon resonance

Article Snippet: For Western blotting, a rabbit anti-human Fn14 antibody (1:1000, CST, MA, USA) was used as the primary antibody.

Techniques: Binding Assay, Immunohistochemistry, Expressing, Staining, Quantitative RT-PCR, Western Blot, SPR Assay