Journal: bioRxiv

Article Title: Epigenetic Regulation of Inflammation by Dopamine in Primary Human Macrophages

doi: 10.64898/2026.01.21.700899

Figure Lengend Snippet: Primary human monocyte-derived macrophages (hMDM) from sixteen donors were treated with dopamine (10 -6 M) for 3 hours with or without pre-treatment using hypomethylating agents 5-aza-2’-deoxycytidine (dAZA; 10 -6 M), 5-azacytidine (AZA; 10 -6 M), or vehicle control. RNA and DNA were isolated for analysis of IL-1β gene expression by qPCR and IL-1β DNA methylation, respectively. (A) Dopamine increased IL-1β mRNA expression, an effect inhibited by dAZA (n= 7-8 donors, *p<0.05). (B) AZA pretreatment produced a similar inhibitory effect on dopamine-induced IL-1β expression, trending toward statistical significance (n=5-6, p=0.0513). (C) Dopamine treatment significantly increased methylated IL-1β levels compared to vehicle control (n=16, p<0.05). (D) Lipopolysaccharide (LPS) increased IL-1β DNA methylation; however, this effect did not reach statistical significance in this donor cohort (n=16). (E) LPS-induced changes in IL-1β DNA methylation were significantly correlated with dopamine-induced changes within the same donors, indicating consistent donor-specific responsiveness across stimuli (n=16, **p<0.01).

Article Snippet: Both high-methylated and low-methylated human genomic DNA (80-8061-HGHM5 and 80-8062-HGUM5 from EpigenDx) were bisulfite treated and used as controls for the primer sets.

Techniques: Derivative Assay, Control, Isolation, Gene Expression, DNA Methylation Assay, Expressing, Produced, Methylation

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: Chemical research in toxicology

Article Title: Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing.

doi: 10.1021/acs.chemrestox.2c00172

Figure Lengend Snippet: Figure 7. 3′-terminus produced by hyTDG-lyase cannot be extended by DNA Pol β but is resolved by APE1. In lane 1, A 5′-FAM-labeled 79 base oligonucleotide containing a U in a U:G mispair (2.5 pmol, 0.2 μM). In lane 2, the oligonucleotide was incubated with UDG (2.5 U, 0.84 pmol, 0.07 μM) for 1 h at 37 °C in CutSmart buffer, and subsequently, the abasic site was cleaved by incubating with APE1 (5 U, 0.18 pmol, 0.01 μM) for an additional 30 min. In lane 3, Pol β (6.2 pmol, 0.5 μM) and E. coli ligase (5 U, 4 pmol, 0.32 μM), dCTP (250 pmol, 20 μM), and NAD+ (325 pmol, 26 μM) were added for an additional 1 h to simulate short-patch BER. Lane 4 was otherwise identical to lane 2, but hyTDG-lyase was used instead of APE1 at 37 °C for 30 min. Lane 5 was otherwise identical to lane 3, except hyTDG-lyase (26.9 pmol, 2.15 μM) was used instead of APE1 at 37 °C. In lane 6, we similarly generated an abasic site that was then cleaved by hyTDG-lyase for 30 min at 37 °C. Then APE1, Pol β, dCTP, and E. coli ligase were added and incubated for an additional 1 h. Lane 6 demonstrates that the AP endonuclease activity of APE1 can clean up the PUA-βME 3′-end produced by the hyTDG-lyase. The complementary strand is labeled with a 5′-Cy5 fluorophore (red). The overlap between the full-length 5′-FAM-labeled oligo and its 5′-Cy5 complement is depicted as yellow.

Article Snippet: Human 8-oxoguanine DNA glycosylase (hOGG1, #NBP1-453180.1mg) and DNA polymerase β (Pol β, #NBP1-72434-0.5mg) were purchased from Novus Biologicals.

Techniques: Produced, Labeling, Incubation, Generated, Activity Assay

Journal: Oncotarget

Article Title: Eradication of LIG4-deficient glioblastoma cells by the combination of PARP inhibitor and alkylating agent

doi: 10.18632/oncotarget.26409

Figure Lengend Snippet: ( A ) mRNA expression level of 15 indicated genes in primary human glioblastoma cell lines (H3, H6 and H7) was normalized to the expression of reference gene – 18S rRNA. Data are presented as a fold change in reference to normal human astrocytes (NHA). Results represent mean value ± SD from 3 independent experiments each performed in triplicates. ( B ) Protein expression level presented as fold change in comparison to NHA where expression was set as 1. The expression level was normalized to the reference protein, GAPDH. Mean ± SD was calculated form 3 independent experiments. Representative Western protein expression analysis of LIG4 and GAPDH (loading control) is shown.

Article Snippet: Glioblastoma H6 cells were transfected with pCMV6-AC-GFP plasmid containing human LIG4 cDNA (OriGene Technologies).

Techniques: Expressing, Comparison, Western Blot, Control

Journal: Oncotarget

Article Title: Eradication of LIG4-deficient glioblastoma cells by the combination of PARP inhibitor and alkylating agent

doi: 10.18632/oncotarget.26409

Figure Lengend Snippet: ( A ) The effect of BMN673 in combination with TMZ on H7 cells transfected with GFP (Control) or GFP-LIG4 expression plasmid. Results represent mean % of viable cells ± SD from 3 independent experiments, * p < 0.05 comparing with Control. ( B ) Representative Western blot of LIG4 expression in H7 cells transfected with control GFP plasmid and with vector carrying GFP-LIG4. GAPDH is included as a loading control.

Article Snippet: Glioblastoma H6 cells were transfected with pCMV6-AC-GFP plasmid containing human LIG4 cDNA (OriGene Technologies).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Western Blot