Journal: Bone research

Article Title: ZBP1 (DAI/DLM-1) promotes osteogenic differentiation while inhibiting adipogenic differentiation in mesenchymal stem cells through a positive feedback loop of Wnt/β-catenin signaling.

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: Fig. 1 ZBP1 is upregulated during the osteogenic differentiation but downregulated during the adipogenic differentiation of mBMSCs. a RT-qPCR analysis and western blot analysis of ZBP1 expression during the osteogenic differentiation of mBMSCs. b RT-qPCR analysis and western blot analysis of ZBP1 expression during the adipogenic differentiation of mBMSCs. c IHC analysis of ZBP1 expression in the mouse femur. Macrophages (stars); osteoblasts on the trabecular bone surface (black arrows). n = 6. Bars indicate 30 μm. BM, bone marrow; TB, trabecular bone; MA, marrow adipocytes. d Semiquantitative quantification of ZBP1 immunostaining intensities in osteoblasts, osteocytes, marrow adipocytes, macrophages, and other marrow cells. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: For the ectopic overexpression of ZBP1, lentivirus particles expressing mouse ZBP1 and human ZBP1 were obtained from OriGene Technologies. mBMSCs and hMSCs were infected and then selected with 1 μg·mL−1 puromycin for 72 h. Luciferase assay mBMSCs were seeded into 12-well plates and infected with lentiviruses carrying TOPflash luciferase reporters.

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

Journal: Bone research

Article Title: ZBP1 (DAI/DLM-1) promotes osteogenic differentiation while inhibiting adipogenic differentiation in mesenchymal stem cells through a positive feedback loop of Wnt/β-catenin signaling.

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: Fig. 2 Depletion of ZBP1 suppresses the osteogenic differentiation but promotes the adipogenic differentiation of mBMSCs. a The siRNA- mediated depletion of ZBP1 was assessed by RT-qPCR and western blot analysis. b ALP activity assays of ZBP1-depleted mBMSCs and control cells after 7 days of osteogenic induction. c ARS staining of ZBP1-depleted mBMSCs and control cells after 2 weeks of osteogenic induction. d RT-qPCR analysis of osteogenesis-related genes (Runx2, sp7, Ibsp, and Bglap) in ZBP1-depleted mBMSCs and control cells. e H&E staining of transplant sections generated from ZBP1-depleted mBMSCs and control cells in HA/TCP scaffolds. Bar, 60 μm. n = 6. f, g Oil Red O staining of ZBP1-depleted mBMSCs and control cells after 3 weeks of adipogenic induction. Bar, 100 μm. h RT-qPCR analysis of adipogenic markers (CD36, Cebpa, Lpl, and Pparg) in ZBP1-depleted mBMSCs and control cells after 7 days of adipogenic induction. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: For the ectopic overexpression of ZBP1, lentivirus particles expressing mouse ZBP1 and human ZBP1 were obtained from OriGene Technologies. mBMSCs and hMSCs were infected and then selected with 1 μg·mL−1 puromycin for 72 h. Luciferase assay mBMSCs were seeded into 12-well plates and infected with lentiviruses carrying TOPflash luciferase reporters.

Techniques: Quantitative RT-PCR, Western Blot, Activity Assay, Control, Staining, Generated

Journal: Bone research

Article Title: ZBP1 (DAI/DLM-1) promotes osteogenic differentiation while inhibiting adipogenic differentiation in mesenchymal stem cells through a positive feedback loop of Wnt/β-catenin signaling.

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: Fig. 3 Overexpression of ZBP1 enhances the osteogenic differentiation but inhibits the adipogenic differentiation of mBMSCs. a The overexpression of ZBP1 was determined by RT-qPCR and western blot analysis. b ALP activity assays of ZBP1-overexpressing mBMSCs and control cells after 7 days of osteogenic induction. c ARS staining of ZBP1-overexpressing mBMSCs and control cells after 2 weeks of osteogenic stimulation. d RT-qPCR analysis of osteogenesis-related genes (Runx2, sp7, Ibsp, and Bglap) in ZBP1-overexpressing mBMSCs and control cells. e H&E staining of transplant sections generated from ZBP1-overexpressing mBMSCs and control cells in HA/TCP scaffolds. Bar, 60 μm. n = 6. f Oil Red O staining of ZBP1-overexpressing mBMSCs and control cells after 3 weeks of adipogenic stimulation. Bar, 100 μm. g RT-qPCR analysis of adipogenic markers (CD36, Cebpa, Lpl, and Pparg) in ZBP1-overexpressing mBMSCs and control cells after 7 days of adipogenic induction. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: For the ectopic overexpression of ZBP1, lentivirus particles expressing mouse ZBP1 and human ZBP1 were obtained from OriGene Technologies. mBMSCs and hMSCs were infected and then selected with 1 μg·mL−1 puromycin for 72 h. Luciferase assay mBMSCs were seeded into 12-well plates and infected with lentiviruses carrying TOPflash luciferase reporters.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Activity Assay, Control, Staining, Generated

Journal: Bone research

Article Title: ZBP1 (DAI/DLM-1) promotes osteogenic differentiation while inhibiting adipogenic differentiation in mesenchymal stem cells through a positive feedback loop of Wnt/β-catenin signaling.

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: Fig. 4 Overexpression of ZBP1 enhances the osteogenic differentiation but inhibits the adipogenic differentiation of hMSCs. a The overexpression of ZBP1 was determined by RT-qPCR and western blot analysis in hMSCs. b ALP activity assays of ZBP1-overexpressing hMSCs and control cells after 7 days of osteogenic induction. c ARS staining of ZBP1-overexpressing hMSCs and control cells after 3 weeks of osteogenic induction. d RT-qPCR analysis of osteogenic markers (RUNX2, SP7, COL1A1, and SPP1) in ZBP1-overexpressing hMSCs and control cells. e Oil Red O staining of ZBP1-overexpressing hMSCs and control cells after 4 weeks of adipogenic induction. Bar, 100 μm. f RT-qPCR analysis of adipogenic markers (CD36, CEBPA, LPL, and PPARG) in ZBP1-overexpressing hMSCs and control cells after 7 days of adipogenic induction. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: For the ectopic overexpression of ZBP1, lentivirus particles expressing mouse ZBP1 and human ZBP1 were obtained from OriGene Technologies. mBMSCs and hMSCs were infected and then selected with 1 μg·mL−1 puromycin for 72 h. Luciferase assay mBMSCs were seeded into 12-well plates and infected with lentiviruses carrying TOPflash luciferase reporters.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Activity Assay, Control, Staining

Journal: Bone research

Article Title: ZBP1 (DAI/DLM-1) promotes osteogenic differentiation while inhibiting adipogenic differentiation in mesenchymal stem cells through a positive feedback loop of Wnt/β-catenin signaling.

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: Fig. 5 Restoring ZBP1 expression rescues the osteogenic potential of ZBP1-depleted mBMSCs. a RT-qPCR and western blot analysis of the restoration of ZBP1 in ZBP-depleted mBMSCs. ALP activity (b) and ECM mineralization (c) were rescued by restoring ZBP1 expression in ZBP- depleted mBMSCs. d The expression of osteogenic markers (sp7, Ibsp, and Bglap) was rescued by restoring ZBP1 expression in ZBP1-depleted mBMSCs. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: For the ectopic overexpression of ZBP1, lentivirus particles expressing mouse ZBP1 and human ZBP1 were obtained from OriGene Technologies. mBMSCs and hMSCs were infected and then selected with 1 μg·mL−1 puromycin for 72 h. Luciferase assay mBMSCs were seeded into 12-well plates and infected with lentiviruses carrying TOPflash luciferase reporters.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Activity Assay

Journal: Bone research

Article Title: ZBP1 (DAI/DLM-1) promotes osteogenic differentiation while inhibiting adipogenic differentiation in mesenchymal stem cells through a positive feedback loop of Wnt/β-catenin signaling.

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: Fig. 6 ZBP1 is required for Wnt/β-catenin signaling. a GO analysis of genes downregulated (over 1.5-fold) in ZBP1-depleted mBMSCs. The top 8 ontology terms are shown. b GSEA of Wnt signaling-related genes in ZBP1-depleted mBMSCs compared with controls. The normalized enrichment score (NES) = −1.41, P < 0.05, and false discovery rate (FDR) < 0.05. c Topflash luciferase reporter assays of Wnt signaling activity in ZBP1-depleted mBMSCs and control cells after treatment with 100 ng·mL−1 Wnt3a for 24 h. RT-qPCR analysis of the expression of Axin2 (d) and Ccnd1 (e) in ZBP1-depleted mBMSCs and control cells after treatment with 100 ng·mL−1 Wnt3a for 4 h. f, g Endogenous IP reveals the interaction between ZBP1 and β-catenin in mBMSCs treated with Wnt3a (100 ng·mL−1) for 2 h. h Western blot analysis of the nuclear extract (NE) and cytoplasmic extract (CE) from ZBP1-depleted mBMSCs and control cells treated with 100 ng·mL−1 Wnt3a for 2 h. ChIP assay of the occupancy of β-catenin at the promoters of Runx2 (i) and Sp7 (j) in ZBP1-knockdown mBMSCs and control cells after treatment with 100 ng·mL−1 Wnt3a for 4 h. Arrows indicate primer annealing sites. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: For the ectopic overexpression of ZBP1, lentivirus particles expressing mouse ZBP1 and human ZBP1 were obtained from OriGene Technologies. mBMSCs and hMSCs were infected and then selected with 1 μg·mL−1 puromycin for 72 h. Luciferase assay mBMSCs were seeded into 12-well plates and infected with lentiviruses carrying TOPflash luciferase reporters.

Techniques: Luciferase, Activity Assay, Control, Quantitative RT-PCR, Expressing, Western Blot, Knockdown

Journal: Bone research

Article Title: ZBP1 (DAI/DLM-1) promotes osteogenic differentiation while inhibiting adipogenic differentiation in mesenchymal stem cells through a positive feedback loop of Wnt/β-catenin signaling.

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: Fig. 7 Zbp1 is a target of Wnt/β-catenin signaling. a RT-qPCR analysis of Zbp1 expression in mBMSCs treated with 100 ng·mL−1 Wnt3a for 4 h. b Western blot analysis of ZBP1 expression in mBMSCs treated with 100 ng·mL−1 Wnt3a for 24 h. c RT-qPCR analysis of Zbp1 expression in mBMSCs that were pretreated with 100 ng·mL−1 DKK1 for 2 h followed by 100 ng·mL−1 Wnt3a treatment for 2 h. d ChIP assay analysis of the occupancy of β-catenin at the promoter of Zbp1 after treatment with 100 ng·mL−1 Wnt3a for 4 h in mBMSCs. Arrows indicate primer annealing sites. WRE, Wnt/β-catenin response element. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: For the ectopic overexpression of ZBP1, lentivirus particles expressing mouse ZBP1 and human ZBP1 were obtained from OriGene Technologies. mBMSCs and hMSCs were infected and then selected with 1 μg·mL−1 puromycin for 72 h. Luciferase assay mBMSCs were seeded into 12-well plates and infected with lentiviruses carrying TOPflash luciferase reporters.

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Experimental & molecular medicine

Article Title: Porphyromonas gingivalis induces Zbp1-mediated macrophages PANoptosis in periodonitis pathophysiology.

doi: 10.1038/s12276-025-01443-y

Figure Lengend Snippet: Fig. 3 P. gingivalis infection activated Zbp1 in BMDMs. a Volcano plot of 1,068 DEGs (500 upregulated, 568 downregulated) in BMDMs treated with PBS or P. gingivalis (n = 4, log2FC ≥1.5 and P < 0.05). b Eighteen upregulated DEGs associated with cell death. c qRT–PCR showing significant upregulation of Zbp1 expression in P. gingivalis-infected BMDMs. n = 3. GO (d) and KEGG (e) enrichment analyses revealing cell death-related pathways abnormalities. f FISH and IF staining showing increased Zbp1 expression in P. gingivalis-infected BMDMs. n = 3. Scale bar, 10 μm. g Higher Zbp1 signals in P. gingivalis-induced BMDMs, displaying both dose- and time-dependent regulation. n = 3. h–j qRT–PCR analysis revealing elevated IL-1β, TNF-α and ZBP1 expression in periodontitis tissues. n = 5. *P < 0.05. k Western blotting analyses of Zbp1 expression in gingival tissues from healthy and periodontitis individuals. n = 5. l, m IHC and IF assays confirming upregulated Zbp1 expression in CD68+ and F4/80+ macrophages. n = 5. Scale bar, 50 μm. All data were derived from independent experiments. *P < 0.05.

Article Snippet: Zbp1−/− mice were established by Cyagen Biosciences using the CRISPR–Cas9 technology.

Techniques: Infection, Quantitative RT-PCR, Expressing, Staining, Western Blot, Derivative Assay

Journal: Experimental & molecular medicine

Article Title: Porphyromonas gingivalis induces Zbp1-mediated macrophages PANoptosis in periodonitis pathophysiology.

doi: 10.1038/s12276-025-01443-y

Figure Lengend Snippet: Fig. 5 Deletion of Zbp1 protected against PANoptosis activation and periodontal tissue destruction in P. gingivalis-induced mouse periodontitis. a IHC staining confirming Zbp1 deficiency in periodontal tissues of Zbp1−/−mice. Scale bar, 50 μm. Decreased apoptosis, necrosis (b) and LDH release (c) in BMDMs from Zbp1−/−mice. n = 3. Scale bar, 50 μm. d Establishment of a mouse periodontitis model in WT and Zbp1−/−mice. n = 5. e Representative micro-CT images revealing diminished periodontal tissue destruction in Zbp1−/−mice. n = 5. Diminished periodontal tissue destruction (f) and reduced osteoclastogenesis (g) in Zbp1−/−mice after P. gingivalis challenge. n = 5. Scale bar, 50 μm. h Reduced cleaved Gsdmd, cleaved Caspase3 and p-Mlkl expression in periodontitis lesions of Zbp1−/−mice. Scale bar, 50 μm. i, j Protein analysis from periodontal tissues showing Zbp1 deficiency alleviating upregulation of p-Ripk3, cleaved Caspase8, cleaved Caspase3, Nlrp3, cleaved Gsdmd and p-Mlkl induced by P. gingivalis infection. n = 3. All data were derived from independent experiments. *P < 0.05.

Article Snippet: Zbp1−/− mice were established by Cyagen Biosciences using the CRISPR–Cas9 technology.

Techniques: Activation Assay, Immunohistochemistry, Micro-CT, Expressing, Infection, Derivative Assay

Journal: Experimental & molecular medicine

Article Title: Porphyromonas gingivalis induces Zbp1-mediated macrophages PANoptosis in periodonitis pathophysiology.

doi: 10.1038/s12276-025-01443-y

Figure Lengend Snippet: Fig. 6 P. gingivalis activated Zbp1 in BMDMs via the Tlr2/4–JNK pathways. a A heat map showing Tlr gene expression profiles in BMDMs stimulated with P. gingivalis. b IHC assays revealing increased levels of Tlr2 and Tlr4 in P. gingivalis-induced periodontal tissues. n = 5. Scale bar, 50 μm. Tlr2 and Tlr4 inhibitors effectively counteracted Zbp1 gene (c) and protein expression (d) in the infected BMDMs. n = 3. e P. gingivalis- infected mouse periodontitis model with daily intraperitoneal administration of C29 (5 mg/kg) and TAK-242 (3 mg/kg). n = 5. Micro-CT (f), IHC (g) and IF staining (h) showing reduced alveolar bone resorption and Zbp1 activation with C29 and TAK-242 treatment. n = 5. Scale bar, 50 μm. i Western blotting showing increased phosphorylation levels of p65, ERK, JNK and p38 in BMDMs after P. gingivalis stimulation. n = 3. j The JNK inhibitor dose-dependently inhibited Zbp1 expression in the infected BMDMs. n = 3. All data were derived from independent experiments. ns, no significance, *P < 0.05.

Article Snippet: Zbp1−/− mice were established by Cyagen Biosciences using the CRISPR–Cas9 technology.

Techniques: Gene Expression, Expressing, Infection, Micro-CT, Staining, Activation Assay, Western Blot, Phospho-proteomics, Derivative Assay

Journal: Experimental & molecular medicine

Article Title: Porphyromonas gingivalis induces Zbp1-mediated macrophages PANoptosis in periodonitis pathophysiology.

doi: 10.1038/s12276-025-01443-y

Figure Lengend Snippet: Fig. 8 MNs delivery of Sal B alleviated P. gingivalis-induced periodontal resorption and promoted mucosal wound healing. a The chemical structure of Sal B. b Molecular docking simulations revealing Sal B binds favorably to Zbp1. c YO-PRO-1/PI staining showing inhibited apoptosis and necrosis with Sal B intervention. n = 3. Scale bar, 50 μm. d Sal B treatment significantly reduced Zbp1 and p-Ripk3 levels. n = 3. e Encapsulation of Sal B (0.5 mg or 1 mg) into PVA MN patches. f Sal B@MNs demonstrating mechanical strength exceeding 1 N per needle. g Dissolution of the modified MN tips in PBS. h Delivery efficiency of Sal B-1@MN and Sal B-2@MN. n = 3. i P. gingivalis-infected mouse periodontitis and hard palate wound healing models with daily administration of Sal B@MNs. n = 5. Representative images and HE staining illustrating reduced periodontal tissue resorption (j, k) and and near-complete wound healing (l, m) in mice treated with Sal B@MNs. n = 5. Scale bar, 50 μm. IHC images showing lower Zbp1 expression in the periodontitis (n) and hard palate wound healing (o) tissues of Sal B@MNs- treated mice. n = 5. Scale bar, 50 μm. All data were derived from independent experiments. *P < 0.05.

Article Snippet: Zbp1−/− mice were established by Cyagen Biosciences using the CRISPR–Cas9 technology.

Techniques: Staining, Encapsulation, Dissolution, Infection, Expressing, Derivative Assay

Journal: Journal of Ovarian Research

Article Title: Fisetin-induced cell death in human ovarian cancer cell lines via zbp1-mediated necroptosis

doi: 10.1186/s13048-022-00984-4

Figure Lengend Snippet: Cell necroptotic pathways regulate fisetin-induced cell death. (A) In A2780 cells, the expression of HMGB1, ZBP1, RIP3 and MLKL in the control and fisetin + z-VAD treatment groups was measured by Western blot. (B) Quantification and comparison of the levels of HMGB1, ZBP1, RIP3 and MLKL expression. (C, D) Western blot results and quantification of HMGB1, ZBP1, RIP3 and MLKL in OVCAR-3 cells under the respective interventions. *, p < 0.05

Article Snippet: ZBP1 siRNAs (Santa Cruz Biotechnology, Santa Cruz, CA) were introduced to block endogenous ZBP1 gene expression.

Techniques: Expressing, Control, Western Blot, Comparison

Journal: Journal of Ovarian Research

Article Title: Fisetin-induced cell death in human ovarian cancer cell lines via zbp1-mediated necroptosis

doi: 10.1186/s13048-022-00984-4

Figure Lengend Snippet: ZBP1 knockdown increased the migration of A2780 cells, blocking fisetin-induced apoptosis. (A) Western blotting of both the ZBP1 knockdown (ZBP1 −/− ) group and the control group, in which A2780 cells were treated with fisetin + z-VAD. (B) Quantification of ZBP1 expression with or without ZBP siRNA administration. (C) In vitro invasion assay between the respective groups. (D) MTT assay indicated differences in cell migration between the indicated groups. (E) Western blotting and (F) quantitative expression levels targeting RIP3 and MLKL in both ZBP1 −/− and normal A2780 cells, both of which were treated with fisetin + z-VAD. *, p < 0.05

Article Snippet: ZBP1 siRNAs (Santa Cruz Biotechnology, Santa Cruz, CA) were introduced to block endogenous ZBP1 gene expression.

Techniques: Knockdown, Migration, Blocking Assay, Western Blot, Control, Expressing, In Vitro, Invasion Assay, MTT Assay

Journal: Journal of Ovarian Research

Article Title: Fisetin-induced cell death in human ovarian cancer cell lines via zbp1-mediated necroptosis

doi: 10.1186/s13048-022-00984-4

Figure Lengend Snippet: ZBP1 knockdown increased the migration of OVCAR-3 cells, blocking fisetin-induced apoptosis. A Western blotting of both the ZBP1 knockdown (ZBP1 −/− ) group and the control group, in which OVCAR-3 cells were treated with fisetin + z-VAD. ( B ) Quantification of ZBP1 expression with or without ZBP siRNA administration. ( C ) In vitro invasion assay between the respective groups. ( D ) MTT assay indicated differences in cell migration between the indicated groups. ( E ) Western blotting and ( F ) quantitative expression levels targeting RIP3 and MLKL in both ZBP1 −/− and normal OVCAR-3 cells, both of which were treated with fisetin + z-VAD. *, p < 0.05

Article Snippet: ZBP1 siRNAs (Santa Cruz Biotechnology, Santa Cruz, CA) were introduced to block endogenous ZBP1 gene expression.

Techniques: Knockdown, Migration, Blocking Assay, Western Blot, Control, Expressing, In Vitro, Invasion Assay, MTT Assay