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: Molecules

Article Title: The RHIM of the Immune Adaptor Protein TRIF Forms Hybrid Amyloids with Other Necroptosis-Associated Proteins

doi: 10.3390/molecules27113382

Figure Lengend Snippet: ( A ) Domain architecture of human RHIM-containing proteins, which contain multiple globular domains that serve diverse functional roles. RHIMs are present within disordered protein regions. Z: Zα domain; DD: death domain; NTD: N-terminal domain; TIR: Toll/interleukin-1 (IL-1) receptor domain. ( B ) Alignment of RHIM sequences from human and virus proteins. ZBP1 contains at least two RHIMs, presented as RHIM-A and RHIM-B. The conserved core tetrad is indicated in the boxed region. ( C ) Design of recombinant RHIM-containing fusion proteins with RHIM-containing regions required for necroptosis (light grey) and ubiquitin or fluorescent partner domain.

Article Snippet: Synthetic gene sequences encoding RHIM-containing regions of human RIPK1, RIPK3, TRIF and ZBP1 were purchased from Genscript and cloned into modified pET15b vectors to generate genes for His-tagged fusion proteins with YPet, mCherry or ubiquitin as N-terminal fusion partners for the RHIM domains (as illustrated in ).

Techniques: Functional Assay, Virus, Recombinant, Ubiquitin Proteomics