Journal: Journal of Cellular Physiology

Article Title: Curcumin attenuates proangiogenic and proinflammatory factors in human eutopic endometrial stromal cells through the NF‐κB signaling pathway

doi: 10.1002/jcp.27360

Figure Lengend Snippet: List of antibodies used for western blot analysis

Article Snippet: Inhibitor of nuclear factor κ‐B kinase subunit β (IKKβ) , Anti‐IKKβ (IKKβ) , Cell Signaling, Beverly, MA , Rabbit , AB_11024092 , 1:1,000.

Techniques: Western Blot

Journal: Journal of Cellular Physiology

Article Title: Curcumin attenuates proangiogenic and proinflammatory factors in human eutopic endometrial stromal cells through the NF‐κB signaling pathway

doi: 10.1002/jcp.27360

Figure Lengend Snippet: Effects of CUR on phosphorylation and total expression of IKKα, IKKβ, and NF‐κB proteins in human NESCs and cells derived from EESCs subjects. Cells were treated with or without curcumin (CUR, 5 µg/ml or 10 µg/ml) for 24 and 48 hr in DMEM/Ham’s F‐12 media with 5% exosome‐depleted fetal bovine serum. Total protein was isolated, followed by equal amounts of protein (25 μg) from each sample separated by one‐dimensional gel electrophoresis and analyzed for phospho‐IKKα, phospho‐IKKβ, and phospho‐NF‐κB; and total IKKα, IKKβ, and NF‐κB protein. (a) Representative western blot analysis of protein for phospho‐ and total IKKα, IKKβ, and NF‐κB levels in NESCs and EESCs treated with or without CUR. α Tubulin was used as an internal constitutive control. (b) Bar diagrams represent the densitometric analyses of protein in WBs of three independent experiments ( n = 3) as mean ± SEM that were performed for each individual group. The bar graphs represent the ratios of phospho‐IKKα, phospho‐IKKβ, and phospho‐NF‐κB protein levels normalized to total IKKα, IKKβ, and NF‐κB, respectively, and the ratios of total IKKα, IKKβ, and NF‐κB protein levels, normalized to α tubulin. The superscript “a” represents significant differences ( p ≤ 0.05) in EESCs groups compared with respective NESCs groups at 24 and 48 hr. Star (*) represents significant differences ( p ≤ 0.05) in EESCs groups treated with CUR compared with respective NESCs groups treated with CUR at 24 and 48 hr. CUR: curcumin; DMEM: Dulbecco’s modified Eagle’s medium; EESCs: eutopic endometriotic stromal cells; IKKα/β: inhibitor of nuclear factor kappa‐B kinase subunit α/β; JNK: c‐Jun N‐terminal kinases; NESCs: normal endometrial stromal cells; NF‐κB: nuclear factor κ‐light‐chain‐enhancer of activated B cells; STAT3: signal transducer and activator of transcription 3; WBs: western blots

Article Snippet: Inhibitor of nuclear factor κ‐B kinase subunit β (IKKβ) , Anti‐IKKβ (IKKβ) , Cell Signaling, Beverly, MA , Rabbit , AB_11024092 , 1:1,000.

Techniques: Phospho-proteomics, Expressing, Derivative Assay, Isolation, Nucleic Acid Electrophoresis, Western Blot, Control, Modification

Journal: Journal of Cellular Physiology

Article Title: Curcumin attenuates proangiogenic and proinflammatory factors in human eutopic endometrial stromal cells through the NF‐κB signaling pathway

doi: 10.1002/jcp.27360

Figure Lengend Snippet: Two‐way ANOVA analysis of CUR dose and duration effects and dose–duration interaction on proinflammatory signaling molecules in human normal endometrial stromal cells (NESCs) and cells derived from eutopic endometrium of endometriosis subjects (EESCs) in vitro

Article Snippet: Inhibitor of nuclear factor κ‐B kinase subunit β (IKKβ) , Anti‐IKKβ (IKKβ) , Cell Signaling, Beverly, MA , Rabbit , AB_11024092 , 1:1,000.

Techniques: Derivative Assay

Journal: Journal of Cellular Physiology

Article Title: Curcumin attenuates proangiogenic and proinflammatory factors in human eutopic endometrial stromal cells through the NF‐κB signaling pathway

doi: 10.1002/jcp.27360

Figure Lengend Snippet: A schematic model showing the antiangiogenic and anti‐inflammatory functions of CUR in human ESCs. Schematic representation of CUR‐dependent inhibition of proangiogenic and proinflammatory chemokine and cytokines may be through inhibited or eliminated phosphorylated forms of JNK and STAT3, along with IKKα, IKKβ, and NF‐κB in human ESCs. CUR: curcumin; ESCs: endometrial stromal cells; IKKα/β: inhibitor of nuclear factor κ‐B kinase subunit α/β; JNK: c‐Jun N‐terminal kinases; NF‐κB: nuclear factor κ‐light‐chain‐enhancer of activated B; STAT3: signal transducer and activator of transcription 3; p—phosphorylated form; arrow represents promotion and blunt arrow represents inhibition [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Inhibitor of nuclear factor κ‐B kinase subunit β (IKKβ) , Anti‐IKKβ (IKKβ) , Cell Signaling, Beverly, MA , Rabbit , AB_11024092 , 1:1,000.

Techniques: Inhibition

Journal: Molecular medicine reports

Article Title: Protectin D1 reduces concanavalin A-induced liver injury by inhibiting NF-κB-mediated CX3CL1/CX3CR1 axis and NLR family, pyrin domain containing 3 inflammasome activation.

doi: 10.3892/mmr.2016.4980

Figure Lengend Snippet: Figure 5. PD1 inhibits NF‑κB‑stimulated CX3CL1/CX3CR1‑dependent inflammatory pathway activation and generation of the inflammasome. Western blot analysis for NF‑κB and CX3CL1/CX3CR1 activity was used to demonstrate the effect of PD1 on Con A‑induced liver injury and the underlying mechanisms. (A and B) Bands and quantification of TLR4, p‑NF‑κB, IκBα, IKKβ and MyD88 protein. (C and D) Bands and quantification of CX3CL1 and CX3CR1 protein. (E) Bands and quantification of NLRP3 and caspase‑1 protein. The bars indicate means ± standard error of the mean (n=10). ##P<0.01 vs. control group; *P<0.05 vs. Con A (30 mg/kg). PD1, protectin D1; Con A, concanavalin A; HPD1, 20 µg/kg PD1 pretreatment group; LPD1, 10 µg/kg PD1 pretreatment group; TLR4, Toll‑like receptor 4; NF‑κB, nuclear factor κ‑light‑chain‑enhancer of activated B cells; p‑NF‑κB, phosphorylated NF‑κB; IκBα, nuclear factor of κ light polypeptide gene enhancer in B‑cells inhibitor, α; IKKβ, inhibitor of nuclear factor κ‑B kinase subunit β; MyD88, myeloid differentiation primary response gene 88; CX3CL1, chemokine (C‑X3‑C motif) ligand 1; CX3CR1, chemokine (C‑X3‑C motif) receptor 1; NLRP3, NLR family, pyrin domain containing 3; IL‑1β, interleukin‑1β.

Article Snippet: Immunoblotting was conducted using the following primary polyclonal rabbitantibodies: Rabbit anti-GAPDH (dilution, 1:1,000; cat. no. 5174), NF-κB (dilution, 1:1,000; cat. no. 8242), phosphorylated-NF-κB (dilution, 1:1,000; cat. no. 4887), TLR4 (dilution, 1:1,000; cat. no. 14358), nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα) (dilution, 1:1,000; cat. no. 4814), inhibitor of nuclear factor κ-B kinase subunit beta (IKKβ) (dilution, 1:1,000; cat. no. 8943), myeloid differentiation primary response gene 88 (MyD88) (dilution, 1:2,000; cat. no. 4283), NLRP3 (dilution, 1:1,000; cat. no. 13158), IL-1β (dilution, 1:1,000; cat. no. 12426), caspase-1 (dilution, 1:1,000; cat. no. 3866) (Cell Signaling Technology, Inc., Danvers, MA, USA), CX3CR1 (cat. no. ab8021) and CX3CL1 (dilution, 1:1,000; cat. no. ab25088) (Abcam, Cambridge, MA, USA).

Techniques: Activation Assay, Western Blot, Activity Assay, Control

Journal: Molecular medicine reports

Article Title: Protectin D1 reduces concanavalin A-induced liver injury by inhibiting NF-κB-mediated CX3CL1/CX3CR1 axis and NLR family, pyrin domain containing 3 inflammasome activation.

doi: 10.3892/mmr.2016.4980

Figure Lengend Snippet: Figure 9. Inhibitory effect of Con A (20 µg/ml) PD1 (20 nM) on the NLRP3 signaling pathway. Total RNA extraction was conducted using TRIzol reagent (48 h). Quantification of mRNA expression levels of (A) NLRP3, (B) IL‑1β and (C) pro‑caspase‑1 in Con A‑induced lymphocytes. (D) Western blot and (E‑H) quanti fication of NLRP3, pro‑/mature caspase‑1 and mature IL‑1β protein. The bars indicate the mean ± standard error of the mean (n=10). #P<0.05 vs. control group. *P<0.05 vs. Con A. PD1, protectin D1; NLRP3, NLR family, pyrin domain containing 3; Con A, concanavalin A; IL‑1β, interleukin‑1β; mRNA, messenger RNA.

Article Snippet: Immunoblotting was conducted using the following primary polyclonal rabbitantibodies: Rabbit anti-GAPDH (dilution, 1:1,000; cat. no. 5174), NF-κB (dilution, 1:1,000; cat. no. 8242), phosphorylated-NF-κB (dilution, 1:1,000; cat. no. 4887), TLR4 (dilution, 1:1,000; cat. no. 14358), nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα) (dilution, 1:1,000; cat. no. 4814), inhibitor of nuclear factor κ-B kinase subunit beta (IKKβ) (dilution, 1:1,000; cat. no. 8943), myeloid differentiation primary response gene 88 (MyD88) (dilution, 1:2,000; cat. no. 4283), NLRP3 (dilution, 1:1,000; cat. no. 13158), IL-1β (dilution, 1:1,000; cat. no. 12426), caspase-1 (dilution, 1:1,000; cat. no. 3866) (Cell Signaling Technology, Inc., Danvers, MA, USA), CX3CR1 (cat. no. ab8021) and CX3CL1 (dilution, 1:1,000; cat. no. ab25088) (Abcam, Cambridge, MA, USA).

Techniques: RNA Extraction, Expressing, Western Blot, Control

Journal: Molecular medicine reports

Article Title: Protectin D1 reduces concanavalin A-induced liver injury by inhibiting NF-κB-mediated CX3CL1/CX3CR1 axis and NLR family, pyrin domain containing 3 inflammasome activation.

doi: 10.3892/mmr.2016.4980

Figure Lengend Snippet: Figure 5. PD1 inhibits NF‑κB‑stimulated CX3CL1/CX3CR1‑dependent inflammatory pathway activation and generation of the inflammasome. Western blot analysis for NF‑κB and CX3CL1/CX3CR1 activity was used to demonstrate the effect of PD1 on Con A‑induced liver injury and the underlying mechanisms. (A and B) Bands and quantification of TLR4, p‑NF‑κB, IκBα, IKKβ and MyD88 protein. (C and D) Bands and quantification of CX3CL1 and CX3CR1 protein. (E) Bands and quantification of NLRP3 and caspase‑1 protein. The bars indicate means ± standard error of the mean (n=10). ##P<0.01 vs. control group; *P<0.05 vs. Con A (30 mg/kg). PD1, protectin D1; Con A, concanavalin A; HPD1, 20 µg/kg PD1 pretreatment group; LPD1, 10 µg/kg PD1 pretreatment group; TLR4, Toll‑like receptor 4; NF‑κB, nuclear factor κ‑light‑chain‑enhancer of activated B cells; p‑NF‑κB, phosphorylated NF‑κB; IκBα, nuclear factor of κ light polypeptide gene enhancer in B‑cells inhibitor, α; IKKβ, inhibitor of nuclear factor κ‑B kinase subunit β; MyD88, myeloid differentiation primary response gene 88; CX3CL1, chemokine (C‑X3‑C motif) ligand 1; CX3CR1, chemokine (C‑X3‑C motif) receptor 1; NLRP3, NLR family, pyrin domain containing 3; IL‑1β, interleukin‑1β.

Article Snippet: Immunoblotting was conducted using the following primary polyclonal rabbitantibodies: Rabbit anti-GAPDH (dilution, 1:1,000; cat. no. 5174), NF-κB (dilution, 1:1,000; cat. no. 8242), phosphorylated-NF-κB (dilution, 1:1,000; cat. no. 4887), TLR4 (dilution, 1:1,000; cat. no. 14358), nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα) (dilution, 1:1,000; cat. no. 4814), inhibitor of nuclear factor κ-B kinase subunit beta (IKKβ) (dilution, 1:1,000; cat. no. 8943), myeloid differentiation primary response gene 88 (MyD88) (dilution, 1:2,000; cat. no. 4283), NLRP3 (dilution, 1:1,000; cat. no. 13158), IL-1β (dilution, 1:1,000; cat. no. 12426), caspase-1 (dilution, 1:1,000; cat. no. 3866) (Cell Signaling Technology, Inc., Danvers, MA, USA), CX3CR1 (cat. no. ab8021) and CX3CL1 (dilution, 1:1,000; cat. no. ab25088) (Abcam, Cambridge, MA, USA).

Techniques: Activation Assay, Western Blot, Activity Assay, Control

Journal: Molecular medicine reports

Article Title: Protectin D1 reduces concanavalin A-induced liver injury by inhibiting NF-κB-mediated CX3CL1/CX3CR1 axis and NLR family, pyrin domain containing 3 inflammasome activation.

doi: 10.3892/mmr.2016.4980

Figure Lengend Snippet: Figure 8. Effects of Con A (20 µg/ml) and PD1 (20 nM) on NF‑κB activated CX3CL1/CX3CR1 signaling pathway. IP was used to investigate (A) TLR4/MyD88, (B) NF‑κB/IκBα and (C) CX3CL1/CX3CR1 complex formation. The bars indicate the mean ± standard error of the mean. (n=10). IP, immunoprecipita tion; Ab, antibody; PD1, protectin D1; Con A, concanavalin A; TLR4, Toll‑like receptor 4; MyD88, myeloid differentiation primary response gene 88; NF‑κB, nuclear factor κ‑light‑chain‑enhancer of activated B cells; IκBα, nuclear factor of κ light polypeptide gene enhancer in B‑cells inhibitor; CX3CL1, che mokine (C‑X3‑C motif) ligand 1; CX3CR1, chemokine (C‑X3‑C motif) receptor 1.

Article Snippet: Immunoblotting was conducted using the following primary polyclonal rabbitantibodies: Rabbit anti-GAPDH (dilution, 1:1,000; cat. no. 5174), NF-κB (dilution, 1:1,000; cat. no. 8242), phosphorylated-NF-κB (dilution, 1:1,000; cat. no. 4887), TLR4 (dilution, 1:1,000; cat. no. 14358), nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα) (dilution, 1:1,000; cat. no. 4814), inhibitor of nuclear factor κ-B kinase subunit beta (IKKβ) (dilution, 1:1,000; cat. no. 8943), myeloid differentiation primary response gene 88 (MyD88) (dilution, 1:2,000; cat. no. 4283), NLRP3 (dilution, 1:1,000; cat. no. 13158), IL-1β (dilution, 1:1,000; cat. no. 12426), caspase-1 (dilution, 1:1,000; cat. no. 3866) (Cell Signaling Technology, Inc., Danvers, MA, USA), CX3CR1 (cat. no. ab8021) and CX3CL1 (dilution, 1:1,000; cat. no. ab25088) (Abcam, Cambridge, MA, USA).

Techniques: