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 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: Neural Regeneration Research

Article Title: Tranylcypromine upregulates Sestrin 2 expression to ameliorate NLRP3-related noise-induced hearing loss

doi: 10.4103/NRR.NRR-D-24-00130

Figure Lengend Snippet: TCP increases SESN2 levels in NE mice cochleae. (A) Representative images of the medial cochlear turn in the control, NE+saline, and NE+TCP groups ( n = 3 cochleae from three mice per group). Hair cells were visualized by immunofluorescent staining for phalloidin (red), and the tissues were also stained for SESN2 (green), and nuclei (DAPI; blue). (B) Hair cells were visualized by immunofluorescent staining for phalloidin (red), and the tissues were also stained for NLRP3 (green), and nuclei (DAPI; blue) (n=3 cochleae from three mice per group). Scale bars: 20 μm in A and B. (C) Quantification of SESN2- and NLRP3-positive cells among the three groups. (D) SESN2 expression in mice cochleae ( n = 3 cochleae from three mice per group). All experiments were replicated thrice. Data are presented as mean ± standard of the mean. * P < 0.05, ** P < 0.01, **** P < 0.0001 (one-way analysis of variance with Bonferroni’s multiple comparison test). Ctrl: Control; DAPI: 4,6-diamidino-2-phenylindole; NE: noise exposure; ns: not significant; TCP: tranylcypromine.

Article Snippet: First, the membranes were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal anti-SESN2 (1:200, sc-393195, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-NLRP3 (1:400, NBP2-12446, Novus Biologicals, Centennial, CO, USA), rabbit polyclonal anti-LC3B (1:250, ab192890, Abcam, Cambridge, UK), rat monoclonal anti-LAMP1 (1:200, 14-1071-82, Invitrogen, Waltham, MA, USA), and mouse monoclonal 4-hydroxynonenal (4-HNE) (1:250, MA5-27570, Invitrogen).

Techniques: Control, Saline, Staining, Expressing, Comparison

Journal: Cell Death Discovery

Article Title: pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

doi: 10.1038/s41420-024-02285-9

Figure Lengend Snippet: A qPCR showed increased TLR4 expression in the 216911KO cell line. ** p < 0.01 by student T tests. B The diagram presented two pir-hsa-216911 binding sites in 3ʹUTR of TLR4. C The dual luciferase assay revealed that pir-has-216911 could bind both TLR4 binding sites TLR4S1 and TLR4S2. Plasmids carrying TLR4S1 and TLR4S2 generate a higher Firefly/Renilla Luciferase ratio in the 216911KO cell line, which pir-hsa-216911 was absent. **p < 0.01; ***p < 0.001 by student T tests. D TCGA-LIHC survival analysis found low expression of TLR4 to be unfavorable to the survival of HCC patients. E Western blot analysis revealed pir-hsa-216911 modulating the TLR4/NFκB/NLRP3 signaling pathway. TLR4, p-p65, and NLRP3 levels were elevated in the 216911KO cell line, which pir-hsa-216911 was absent. F Western blot analysis revealed pir-hsa-216911 modulating the TLR4/NFκB/NLRP3 signaling pathway through silencing TLR4. After silencing TLR4 with siRNA, pir-hsa-216911 absent could not elevate p-p65, and NLRP3 levels were elevated in the 216911KO cell line.

Article Snippet: The antibodies used in this study were Cleaved Caspase-1 Rabbit mAb (4199, Cell Signaling Technology, USA), Cleaved Gasdermin D Rabbit mAb (1:2000 dilute; 36425 Cell Signaling Technology, USA), anti-TLR4 rabbit polyclonal antibody (1:500 dilute; D121751, Sangon Biotech, China), anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (1:500 dilute; D155006, Sangon Biotech, China), anti-NLRP3 rabbit polyclonal antibody (1:250 dilute; D120143, Sangon Biotech, China), anti-ACTB rabbit polyclonal antibody (1:6000 dilute; D110001, Sangon Biotech, China), anti-GAPDH rabbit polyclonal antibody (1:4000 dilute; D110016, Sangon Biotech, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, China).

Techniques: Expressing, Binding Assay, Luciferase, Western Blot

Journal: Cell Death Discovery

Article Title: pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

doi: 10.1038/s41420-024-02285-9

Figure Lengend Snippet: A The diagram presented the xenograft modeling process. B Euthanized mouse to display the subcutaneous xenografted tumors. C 216911KO group resulted in much smaller tumors in size compared to the control group. D 216911KO group resulted in much smaller tumors in weight compared to the control group. The histogram represents the mean tumor weight of each group. ***p < 0.001 by student T tests. E Western blot analysis revealed pir-hsa-216911 modulating the TLR4/NFκB/NLRP3 signaling pathway in vivo. Both TLR4 and p-p65 levels were elevated in tumors formed in the 216911KO group compared to the control group.

Article Snippet: The antibodies used in this study were Cleaved Caspase-1 Rabbit mAb (4199, Cell Signaling Technology, USA), Cleaved Gasdermin D Rabbit mAb (1:2000 dilute; 36425 Cell Signaling Technology, USA), anti-TLR4 rabbit polyclonal antibody (1:500 dilute; D121751, Sangon Biotech, China), anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (1:500 dilute; D155006, Sangon Biotech, China), anti-NLRP3 rabbit polyclonal antibody (1:250 dilute; D120143, Sangon Biotech, China), anti-ACTB rabbit polyclonal antibody (1:6000 dilute; D110001, Sangon Biotech, China), anti-GAPDH rabbit polyclonal antibody (1:4000 dilute; D110016, Sangon Biotech, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, China).

Techniques: Control, Western Blot, In Vivo