Journal: Experimental and therapeutic medicine

Article Title: Luteolin suppresses TNF-α-induced inflammatory injury and senescence of nucleus pulposus cells via the Sirt6/NF-κB pathway.

doi: 10.3892/etm.2022.11396

Figure Lengend Snippet: Figure 1. Luteolin enhances the viability of TNF‑α‑induced HNPCs. (A) Chemical structure of luteolin. (B) Effect of different concentrations (1, 2 and 4 µM) of luteolin on HNPC viability. (C) Effect of luteolin on TNF‑α‑suppressed HNPC viability. ***P<0.001 vs. control. ##P<0.01 vs. TNF‑α. TNF‑α, tumor necrosis factor‑α; HNPCs, human nucleus pulposus cells.

Article Snippet: Immortalized human nucleus pulposus cells (HNPCs) were obtained from AcceGen Biotechnology (cat. no. ABI‐TC102D).

Techniques: Control

Journal: Experimental and therapeutic medicine

Article Title: Luteolin suppresses TNF-α-induced inflammatory injury and senescence of nucleus pulposus cells via the Sirt6/NF-κB pathway.

doi: 10.3892/etm.2022.11396

Figure Lengend Snippet: Figure 2. Luteolin inhibits TNF‑α‑induced HNPC inflammatory injury. (A and B) Expression levels of inflammatory cytokines IL‑1β (A) and IL‑6 (B) were detected by ELISA. (C) TUNEL staining was used to detect the effect of luteolin on TNF‑α‑induced apoptosis. (D) Expression levels of Bcl‑2, Bax and cleaved caspase‑3 protein were detected by western blot analysis. ***P<0.001 vs. control. #P<0.05, ##P<0.01 and ###P<0.001 vs. TNF‑α. TNF‑α, tumor necrosis factor‑α; HNPCs, human nucleus pulposus cells.

Article Snippet: Immortalized human nucleus pulposus cells (HNPCs) were obtained from AcceGen Biotechnology (cat. no. ABI‐TC102D).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Western Blot, Control

Journal: Experimental and therapeutic medicine

Article Title: Luteolin suppresses TNF-α-induced inflammatory injury and senescence of nucleus pulposus cells via the Sirt6/NF-κB pathway.

doi: 10.3892/etm.2022.11396

Figure Lengend Snippet: Figure 3. Luteolin suppresses TNF‑α‑induced senescence of HNPCs. (A) Senescence β‑galactosidase staining kit was used to detect the activity level of β‑galactosidase in HNPCs. (B) ELISA kit was performed to detect the activity of telomerase. (C) The expression levels of senescence related proteins (p16 and p21) were examined via western blot analysis. (D) Expression of p53 was detected by western blot analysis. ***P<0.001 vs. control. #P<0.05, ##P<0.01 and ###P<0.001 vs. TNF‑α. TNF‑α, tumor necrosis factor‑α; HNPCs, human nucleus pulposus cells.

Article Snippet: Immortalized human nucleus pulposus cells (HNPCs) were obtained from AcceGen Biotechnology (cat. no. ABI‐TC102D).

Techniques: Staining, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control

Journal: Experimental and therapeutic medicine

Article Title: Luteolin suppresses TNF-α-induced inflammatory injury and senescence of nucleus pulposus cells via the Sirt6/NF-κB pathway.

doi: 10.3892/etm.2022.11396

Figure Lengend Snippet: Figure 4. Luteolin regulates the Sirt6/NF‑κB pathway. (A) The expression levels of Sirt6/NF‑κB pathway‑related proteins (Sirt6, p‑NF‑κB and p‑NF‑κB p65) were examined by western blot analysis. (B) Western blot analysis was used to examine histone acetylation‑related H3K9ac expression level. (C) Effect of luteolin on TNF‑α‑induced Sirt6 activity was detected by SIRT6 activity assay kit. (D) Interaction between Sirt6 and luteolin was predicted by molecular docking. ***P<0.001 vs. control. #P<0.05, ##P<0.01 and ###P<0.001 vs. TNF‑α. TNF‑α, tumor necrosis factor‑α; HNPCs, human nucleus pulposus cells.

Article Snippet: Immortalized human nucleus pulposus cells (HNPCs) were obtained from AcceGen Biotechnology (cat. no. ABI‐TC102D).

Techniques: Expressing, Western Blot, Activity Assay, Control

Journal: Experimental and therapeutic medicine

Article Title: Luteolin suppresses TNF-α-induced inflammatory injury and senescence of nucleus pulposus cells via the Sirt6/NF-κB pathway.

doi: 10.3892/etm.2022.11396

Figure Lengend Snippet: Figure 5. Sirt6 knockdown partially reverses the inhibitory effect of luteolin on TNF‑α‑induced inflammatory damage of HNPCs. (A and B) The Sirt6 protein (A) and mRNA (B) expression levels were detected by western blot analysis and RT‑qPCR, respectively. (C‑E) Effects of Sirt6 knockdown on cell viability (C) and intracellular IL‑1β (D) and IL‑6 (E) expression levels. (F) TUNEL staining was used to detect the effect of Sirt6 knockdown on HNPC apoptosis. (G) Expression levels of Bcl‑2, Bax and cleaved caspase 3 protein were detected by western blot analysis. ***P<0.001 vs. control or si‑NC. ##P<0.01 and ###P<0.001 vs. TNF‑α. +P<0.05 ++P<0.01 and +++P<0.001 vs. TNF‑α + luteolin. @@P<0.01 and @@@P<0.001 vs. TNF‑α + luteolin + si‑NC. TNF‑α, tumor necrosis factor‑α; HNPCs, human nucleus pulposus cells; Sirt6, sirtuin 6.

Article Snippet: Immortalized human nucleus pulposus cells (HNPCs) were obtained from AcceGen Biotechnology (cat. no. ABI‐TC102D).

Techniques: Knockdown, Expressing, Western Blot, TUNEL Assay, Staining, Control

Journal: Experimental and therapeutic medicine

Article Title: Luteolin suppresses TNF-α-induced inflammatory injury and senescence of nucleus pulposus cells via the Sirt6/NF-κB pathway.

doi: 10.3892/etm.2022.11396

Figure Lengend Snippet: Figure 6. Knockdown of Sirt6 partially reverses the inhibitory effect of luteolin on TNF‑α‑induced senescence of HNPCs. (A) The activity level of β‑galactosidase in HNPCs was assayed by senescence β‑galactosidase staining. (B) ELISA kit was performed to detect the activity of telomerase. (C) Expression levels of senescence‑related proteins (p16 and p21) were examined via western blot analysis. (D) Expression of p53 was detected by western blot analysis. ***P<0.001 vs. control. ###P<0.001 vs. TNF‑α. +++P<0.001 vs. TNF‑α + luteolin. @@P<0.01, @@@P<0.001 vs. TNF‑α + luteolin + si‑NC. TNF‑α, tumor necrosis factor‑α; HNPCs, human nucleus pulposus cells; Sirt6, sirtuin 6.

Article Snippet: Immortalized human nucleus pulposus cells (HNPCs) were obtained from AcceGen Biotechnology (cat. no. ABI‐TC102D).

Techniques: Knockdown, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control

Journal: Redox Biology

Article Title: Differential endothelial signaling responses elicited by chemogenetic H 2 O 2 synthesis

doi: 10.1016/j.redox.2020.101605

Figure Lengend Snippet: Representative images of localized HyPer-DAAO expression. The upper panels show representative widefield ratiometric HyPer images of EA.hy926 endothelial cells transduced with adenoviral AV5 vectors encoding HyPer-DAAO constructs targeted to ( A ) the cell cytosol (HyPerDAAO-NES); ( B ) the cell nucleus (HyPerDAAO-NLS) and ( C ) plasmalemmal caveolae (HyPerDAAO-Cav). The lower panels ( D – F ) show representative HyPer responses quantitated in real time after treatment with H 2 O 2 (25 μM, black lines) or d -alanine (10 mM, red curves) of EA.926hy cells transduced with recombinant AV5 constructs expressing HyPer1-DAAO in different subcellular regions, as indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Plasmid transfection: EA.hy926 cells at ~70% confluence were transfected with 1 μg plasmid DNA encoding HyPer7.2-DAAO targeted to the cell cytosol, nucleus or caveolae [ ] in serum-free culture medium, using the transfection reagent PolyJet according to the manufacturer's instructions (SignaGen Laboratories).

Techniques: Expressing, Transduction, Construct, Recombinant

Journal: Redox Biology

Article Title: Differential endothelial signaling responses elicited by chemogenetic H 2 O 2 synthesis

doi: 10.1016/j.redox.2020.101605

Figure Lengend Snippet: Simultaneous generation and detection of intracellular H 2 O 2 in different subcellular locales using differentially-targeted HyPer7.2-DAAO . ( A-C ) Average curves HyPer7.2 ratio measured in response to 25 μM H 2 O 2 (black curves) or 10 mM d -alanine (red curves) of EA.hy926 expressing HyPer7.2-DAAO in ( A ) cell cytosol (Cyto), ( B ) nucleus (Nuc) or ( C ) plasmalemmal caveolae (Cav). ( D ) Zoom in overlaid average curves presented in A-C after calculation of maximum responses of all individual cells treated with either H 2 O 2 or d -Alanine defined as 100%. Time point 0 indicates time of application. ( E ) Statistical evaluation of average slopes expressed in %/s within the 10–70% interval of maximum cell responses to extracellular H 2 O 2 or endogenously generated H 2 O 2 by DAAO after adding d -alanine, in the cytosol (white bar, n = 8/18 and gray bar, n = 8/12); nucleus (white bar, n = 8/14 and gray bar, n = 9/21); or caveolae (white bar, n = 9/23 and gray bar, n = 8/18). ( F ) Columns represent maximal responses after application of exogenous H 2 O 2 (white bars) vs. d -alanine (gray bars) of same cells and subcellular locales analyzed in E. All values are presented as mean ± S.D; **P < 0.01 , ***P < 0.001. Responses to d -Alanine vs. extracellular H 2 O 2 application was analyzed by one-way ANOVA and Tukey's multiple comparison test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Plasmid transfection: EA.hy926 cells at ~70% confluence were transfected with 1 μg plasmid DNA encoding HyPer7.2-DAAO targeted to the cell cytosol, nucleus or caveolae [ ] in serum-free culture medium, using the transfection reagent PolyJet according to the manufacturer's instructions (SignaGen Laboratories).

Techniques: Expressing, Generated, Comparison

Journal: Redox Biology

Article Title: Differential endothelial signaling responses elicited by chemogenetic H 2 O 2 synthesis

doi: 10.1016/j.redox.2020.101605

Figure Lengend Snippet: Schematic model for eNOS phosphorylation pathways via differentially-targeted H 2 O 2 . This figure presents a model showing pathways for modulation of eNOS phosphorylation pathways by H 2 O 2 generated in different subcellular compartments (cytosol vs. nucleus vs. caveolae) in endothelial cells. We propose that H 2 O 2 generated in the endothelial cell nucleus, but not in the cytosol or caveolae, leads to eNOS phosphorylation via AMPK. Extracellular H 2 O 2 has only a nominal effect on these phosphorylation pathways. See text for details.

Article Snippet: Plasmid transfection: EA.hy926 cells at ~70% confluence were transfected with 1 μg plasmid DNA encoding HyPer7.2-DAAO targeted to the cell cytosol, nucleus or caveolae [ ] in serum-free culture medium, using the transfection reagent PolyJet according to the manufacturer's instructions (SignaGen Laboratories).

Techniques: Phospho-proteomics, Generated

Journal: Cell Communication and Signaling : CCS

Article Title: Therapeutic control and resistance of the EGFR-driven signaling network in glioblastoma

doi: 10.1186/s12964-015-0098-6

Figure Lengend Snippet: Schematics of EGFR signaling via PI3K and AKT. Graphics depicting cell membrane, nucleus and transcription taken from motifolio.com. This is an oversimplified view of the network. In reality, for example, crosstalks between different canonical pathways, such as between RAS and PI3K, and multiple feedback loops are also observed as discussed in the next sections.

Article Snippet: Graphics depicting cell membrane, nucleus and transcription taken from motifolio.com.

Techniques: Membrane

Journal: JOR Spine

Article Title: An Extracellular Matrix Hydrogel Restores Disc Volume and Alleviates Axial Hypersensitivity in a Rat Model of Disc‐Associated Pain

doi: 10.1002/jsp2.70073

Figure Lengend Snippet: Study overview. (A) The central proposal of this work was that a treatment for disc‐associated pain could be fabricated from a mixture of decellularized healthy porcine NP tissue, collagen, and genipin. This therapeutic would theoretically be tissue integrating, spontaneously fibrillogenic, cytocompatible, and biomechanically restorative, leading to pain‐like behavior remission. (B) The first arm of this work entailed testing decellularized nucleus pulposus gels supplemented with 6.0 mg/mL collagen and genipin from 0 to 20 mM. Testing of these gels (dNPs) included gelation kinetics, rheology, cytotoxicity, and ex vivo capacity to restore injured disc mechanics. (C) The outcomes of the first arm determined that the optimal formulation to test in vivo was 6.0 mg/mL dNP + 6.0 mg/mL collagen +2.5 mM genipin, referred to in this manuscript as dNP+. To test this therapeutic, disc degeneration was induced in female Sprague Dawley rats and allowed to progress for 8 weeks. At 9 weeks post‐injury, half of the injured animals were treated with dNP+ and the other half with 1X PBS. Throughout the in vivo arm, disc volume and pain‐like behavioral metrics were collected to monitor the effects of disc injury and treatment.

Article Snippet: NP cells were plated on top of the treatments at a density of 7500 cells/cm 2 and cultured for 48 h in phenol‐free Complete Nucleus Pulposus Cell Media (4801‐prf, ScienCell).

Techniques: Ex Vivo, Formulation, In Vivo