Journal: Biofactors (Oxford, England)

Article Title: Fabp5 Is the Key Regulator Mediating γ‐ CEHC Differentiation in Osteoblasts and Osteoclasts

doi: 10.1002/biof.70079

Figure Lengend Snippet: γ‐CEHC alleviates bone loss in OVX mice. (A) Administration of γ‐CEHC treatment in OVX mice. (B) Trabecular bone morphology of the femur in mice. (C) Analysis of trabecular morphological parameters, including BV/TV, Tb.N, Tb.Th, and Tb.Sp ( n = 6). (D) Images of TRAP staining (Magnification, 100×) of rat femoral tissue, with black arrows indicating osteoclasts. (E) Histomorphometric analysis of osteoclasts, including N.Oc/B.Pm and Oc.S/BS ( n = 6). (F) Plasma levels of the bone resorption marker CTX‐1 in mice ( n = 6). (G) Plasma levels of the bone formation marker P1NP in mice ( n = 6). Data were presented as means ± SD ( # p < 0.05, ## p < 0.01, ### p < 0.001 indicate statistically significant differences between the OVX group compared to the Sham group; * p < 0.05, ** p < 0.01, *** p < 0.001 indicate statistically significant differences between the different concentrations of γ‐CEHC treatment groups versus the OVX group).

Article Snippet: Horseradish peroxidase‐conjugated secondary antibodies were obtained from Agilent Technologies Inc., including γ‐CEHC and α‐CEHC (MedChemExpress, Monmouth Junction, NJ, USA) and dissolved in DMSO (Wako Pure Chemical Industries Ltd., Osaka, Japan).

Techniques: Staining, Clinical Proteomics, Marker

Journal: Biofactors (Oxford, England)

Article Title: Fabp5 Is the Key Regulator Mediating γ‐ CEHC Differentiation in Osteoblasts and Osteoclasts

doi: 10.1002/biof.70079

Figure Lengend Snippet: Effects of γ‐CEHC on osteoblast and osteoclast differentiation. (A) Cell viability of MC3T3‐E1 cells following 24‐h co‐culture with γ‐CEHC, determined by CCK‐8 assay. (B) Cell viability of RAW264.7 cells after a 24‐h co‐culture with γ‐CEHC, determined via CCK‐8 assay. (C) Expression of osteoclast marker genes ( Ctsk , C‐fos , Nfatc1 , Scr , and Acp5 ) detected by qRT‐PCR. (D) Expression of osteoblast marker genes ( Ocn , Runx2 , and Col1 ) detected by qRT‐PCR. (E) Protein expression levels of osteoclast markers (Ctsk, C‐fos, and Nfatc1) analyzed via Western blot. (F) Protein expression levels of osteoblast markers (Ocn, Runx2, and Col1) analyzed by Western blot. All measurements are presented as means ± SD for three biological replicates ( # p < 0.05, ## p < 0.0 1 , ### p < 0.001 indicate statistically significant differences between the LPS‐alone group versus the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 indicate statistically significant differences between the γ‐CEHC treatment group versus the LPS‐alone group).

Article Snippet: Horseradish peroxidase‐conjugated secondary antibodies were obtained from Agilent Technologies Inc., including γ‐CEHC and α‐CEHC (MedChemExpress, Monmouth Junction, NJ, USA) and dissolved in DMSO (Wako Pure Chemical Industries Ltd., Osaka, Japan).

Techniques: Co-Culture Assay, CCK-8 Assay, Expressing, Marker, Quantitative RT-PCR, Western Blot, Control

Journal: Biofactors (Oxford, England)

Article Title: Fabp5 Is the Key Regulator Mediating γ‐ CEHC Differentiation in Osteoblasts and Osteoclasts

doi: 10.1002/biof.70079

Figure Lengend Snippet: Isothermal TPP and label‐free quantitative proteomics reveal target profiles of γ‐CEHC intervention throughout osteoblast and osteoclast differentiation. (A, B) Volcano plot (A) and heatmap (B) of binding targets identified by isothermal TPP in MC3T3‐E1 cells. (C, D) Volcano plot (C) and heatmap (D) of binding targets identified by isothermal TPP in RAW264.7 cells. (E) KEGG pathway enrichment analysis of binding targets identified via isothermal TPP in RAW264.7 cells.

Article Snippet: Horseradish peroxidase‐conjugated secondary antibodies were obtained from Agilent Technologies Inc., including γ‐CEHC and α‐CEHC (MedChemExpress, Monmouth Junction, NJ, USA) and dissolved in DMSO (Wako Pure Chemical Industries Ltd., Osaka, Japan).

Techniques: Quantitative Proteomics, Binding Assay

Journal: Biofactors (Oxford, England)

Article Title: Fabp5 Is the Key Regulator Mediating γ‐ CEHC Differentiation in Osteoblasts and Osteoclasts

doi: 10.1002/biof.70079

Figure Lengend Snippet: Validation of γ‐CEHC targets. (A) Thermal shift curves of γ‐CEHC targets (including Smarcal1 and Fabp5) in MC3T3‐E1 cells. (B) Thermal shift curves of γ‐CEHC targets (including Rhob and Fabp5) in RAW264.7 cells. (C) Thermal shift curves of γ‐CEHC targets (including Smarcal1 and Fabp5) in hFOB1.19 cells. (D) Thermal shift curves of γ‐CEHC targets (including Rhob and Fabp5) in THP‐1 cells. Blue curves are the γ‐CEHC‐treated group, while black curves are the DMSO‐treated group (control). Data are presented as means ± SD ( n = 3).

Article Snippet: Horseradish peroxidase‐conjugated secondary antibodies were obtained from Agilent Technologies Inc., including γ‐CEHC and α‐CEHC (MedChemExpress, Monmouth Junction, NJ, USA) and dissolved in DMSO (Wako Pure Chemical Industries Ltd., Osaka, Japan).

Techniques: Biomarker Discovery, Control

Journal: Biofactors (Oxford, England)

Article Title: Fabp5 Is the Key Regulator Mediating γ‐ CEHC Differentiation in Osteoblasts and Osteoclasts

doi: 10.1002/biof.70079

Figure Lengend Snippet: Molecular docking and SPR confirm the specificity of γ‐CEHC binding to Fabp5. (A) Molecular docking indicating the binding of γ‐CEHC to the β‐barrel domain of Fabp5. (B) Three‐dimensional simulation and binding energy of γ‐CEHC and Fabp5 interaction. (C) SPR sensorgrams indicate interactions between γ‐CEHC concentration gradients and FABP ligand 6 protein (left panel) versus Fabp5 protein (right panel). (D) SPR sensorgrams demonstrate the interaction between α‐CEHC concentration gradients and Fabp5 protein. (E) SPR sensorgrams demonstrate the interaction between γ‐CEHC concentration gradients and Fabp5 protein following treatment with oleic acid. (F) CETSA competition assays display thermal shift curves of Fabp5. Blue curves indicate the γ‐CEHC‐treated group, green curves indicate the γ‐CEHC + anti‐Fabp5 antibody group, while black curves represent the DMSO‐treated group (control). The green ΔTm values represent the ΔTm of the γ‐CEHC + anti‐Fabp5 antibody group relative to the DMSO‐treated group. In contrast, the blue ΔTm values represent the ΔTm of the γ‐CEHC group relative to the DMSO‐treated group.

Article Snippet: Horseradish peroxidase‐conjugated secondary antibodies were obtained from Agilent Technologies Inc., including γ‐CEHC and α‐CEHC (MedChemExpress, Monmouth Junction, NJ, USA) and dissolved in DMSO (Wako Pure Chemical Industries Ltd., Osaka, Japan).

Techniques: Binding Assay, Concentration Assay, Control

Journal: Biofactors (Oxford, England)

Article Title: Fabp5 Is the Key Regulator Mediating γ‐ CEHC Differentiation in Osteoblasts and Osteoclasts

doi: 10.1002/biof.70079

Figure Lengend Snippet: γ‐CEHC restores M1/M2 polarization balance by downregulating Fabp5. (A) Expression of M1 markers ( iNOS , TNF‐α , and IL‐6 ) analyzed by qRT‐PCR. (B) Expression of M2 markers ( CD206 , IL‐10 , and Arg1 ) analyzed by qRT‐PCR. (C) ROS levels labeled with fluorescent probes in each group as detected via flow cytometry. (D) Statistical results of ROS levels. All measurements are presented as means ± SD for three biological replicates (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 indicates statistically significant differences of the γ‐CEHC treatment group relative to the LPS‐alone group; # p < 0.05, ## p < 0.01, ### p < 0.001 indicate statistically significant differences of the γ‐CEHC + LPS + empty vector group compared to the γ‐CEHC + LPS + si‐Fabp5 group).

Article Snippet: Horseradish peroxidase‐conjugated secondary antibodies were obtained from Agilent Technologies Inc., including γ‐CEHC and α‐CEHC (MedChemExpress, Monmouth Junction, NJ, USA) and dissolved in DMSO (Wako Pure Chemical Industries Ltd., Osaka, Japan).

Techniques: Expressing, Quantitative RT-PCR, Labeling, Flow Cytometry, Plasmid Preparation

Journal:

Article Title: ?-Tocopherol and its major metabolite, in contrast to ?-tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells

doi:

Figure Lengend Snippet: Differential effects of αT, γT, and γ-CEHC on PGE2 synthesis in macrophage and epithelial cells. (A) RAW264.7 cells were preincubated with αT, γT, or vehicle, ethanol (0.1–0.2%) for 8–14 h in 0.5% FBS-DMEM and then treated with LPS (0.1 μg/ml) for 14 h. γ-CEHC was added 1 h before LPS treatment. (B) A549 epithelial cells were preincubated with αT, γT, or ethanol (0.1–0.2%) for 8–14 h and then treated with IL-1β (10 ng/ml) for 24 h. γ-CEHC was added 1 h before IL-1β treatment. (C) A549 cells were treated with γT and IL-1β as described in B. After IL-1β treatment, the supernatant was removed and cells were resuspended with fresh medium containing 10 μM AA for 10 min at 37°C. The supernatant was collected and PGE2 was measured as described in Materials and Methods. Basal was the amount of PGE2 produced from cells not treated with LPS or IL-1β. *, P < 0.05 and **, P < 0.01, control vs. tocopherol supplementation.

Article Snippet: αT (99%) and γT (95–97%) were purchased from Acros Organics (Summerville, NJ) or Fluka. γ-CEHC (≥98%) and lipid hydroperoxide-free AA were from Cayman Chemicals (Ann Arbor, MI).

Techniques: Produced

Journal:

Article Title: ?-Tocopherol and its major metabolite, in contrast to ?-tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells

doi:

Figure Lengend Snippet: Inhibition of COX activity by postincubation with γT and γ-CEHC in COX-2-preinduced A549 cells. A549 cells were pretreated with IL-1β (10 ng/ml) for 24 h. (A) Cells were washed and incubated with fresh medium containing vehicle or γ-CEHC for 1 h. AA at final concentrations of 5 or 15 μM was added and incubated at 37°C for 10 min. (B) For γT, the postincubation period was extended to 24 h. Medium was then removed and replaced with a fresh one containing 10 or 30 μM AA at 37°C for 10 min. (C) Cells were postincubated with γT (10 μM) for 1, 8, or 24 h and COX-2 activity was measured as described in B. All reactions were stopped by the addition of 0.5 mM aspirin and PGE2 in the medium was measured. COX activity (%) is expressed as the ratio of PGE2 produced in the presence of γT or γ-CEHC to that with vehicle alone. Numbers within brackets indicate the final concentrations of AA. *, P < 0.05 and **, P < 0.01, control vs. tocopherol supplementation.

Article Snippet: αT (99%) and γT (95–97%) were purchased from Acros Organics (Summerville, NJ) or Fluka. γ-CEHC (≥98%) and lipid hydroperoxide-free AA were from Cayman Chemicals (Ann Arbor, MI).

Techniques: Inhibition, Activity Assay, Incubation, Produced

Journal:

Article Title: ?-Tocopherol and its major metabolite, in contrast to ?-tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells

doi:

Figure Lengend Snippet: Effects on COX-2 expression. (A) Western blot of RAW264.7 macrophages. Cells were treated with vehicle (lane 1); LPS (0.1 μg/ml, lane 2); or LPS (0.1 μg/ml) and γT (10 μM, lane3; and 40 μM, lane 4); αT (10 μM, lane 5; and 40 μM, lane 6) or γ-CEHC (10 μM, lane7; and 40 μM, lane 8) for 14 h. (B) Western blot of A549 cells; cells were treated with vehicle (lane 1); IL-1β (10 ng/ml, lane 2); or IL-1β and γT (10 μM, lane 3; and 40 μM, lane 4), and γ-CEHC (10 μM, lane 5; and 40 μM, lane 6) for 24 h. (C) Northern blot of RAW264.7 macrophages. The sample sequence was the same as specified in A.

Article Snippet: αT (99%) and γT (95–97%) were purchased from Acros Organics (Summerville, NJ) or Fluka. γ-CEHC (≥98%) and lipid hydroperoxide-free AA were from Cayman Chemicals (Ann Arbor, MI).

Techniques: Expressing, Western Blot, Northern Blot, Sequencing

Journal:

Article Title: ?-Tocopherol and its major metabolite, in contrast to ?-tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells

doi:

Figure Lengend Snippet: Effect of tocopherols on nitrite accumulation and iNOS expression

Article Snippet: αT (99%) and γT (95–97%) were purchased from Acros Organics (Summerville, NJ) or Fluka. γ-CEHC (≥98%) and lipid hydroperoxide-free AA were from Cayman Chemicals (Ann Arbor, MI).

Techniques: Expressing