Journal: Molecules

Article Title: Application of Cinnamomum burmannii Essential Oil in Promoting Wound Healing

doi: 10.3390/molecules29092080

Figure Lengend Snippet: BEO attenuated the inflammatory response in RAW 264.7 cells stimulated by LPS. ( A ) qPCR of IL-1β, IL-6 and TNF-α levels in RAW 264.7 cells that were stimulated by LPS at 24 h from different groups. ( B ) The level of IL-6 and TNF-α secreted by RAW 264.7 cells treated with BEO was determined by ELISA. ( C ) Effects of 150 μg/mL, 200 μg/mL, 250 μg/mL and 300 μg/mL BEO on the expression of NF-κB/p-IκBα protein. ( D ) The cell typing of RAW 264.7 was detected by flow cytometry. Polarization rate ( D ) and sectional images ( E , F ) of each group are shown. n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: Monoclonal antibodies against β-actin, and NF-κB p65 were purchased from MedChemExpress Inc. (MCE, Monmouth Junction, NJ, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Brazilin blocks catabolic processes in human osteoarthritic chondrocytes via inhibition of NFKB1/p50.

doi: 10.1002/jor.24013

Figure Lengend Snippet: Figure 4. Brazilin inhibits NFKB1/p50. (a) Primary human chondrocytes (n ¼ 4 donors) were left un- treated (C), treated with 10 ng/ml IL-1b for 6 h, or pre-incubated with 10 mg/ml brazilin for 1 h prior to stimulation with 10 ng/ml IL-1b for 6 h. NFKB1 mRNA levels were analyzed by RT-qPCR and expressed as relative quantities (mean SD) com- pared to controls. Significant differences compared to untreated cells (p < 0.05; n ¼ 4; Quade test). #Sig- nificant differences compared to cells treated only with IL-1b (p < 0.05; n ¼ 4; Quade test). (b) Primary human chondrocytes (n ¼ 4 donors) were left un- treated (C) or were treated with 10 ng/ml IL-1b for 15 min, 1 h, 4 h, 16 h, and 32 h. Protein extracts were subjected to Western blot analysis for the detection of p50, p65, and phosphorylated p65. b-Actin and a- tubulin were used as loading controls. The blot of one representative experiment is shown. (c) Primary chondrocytes (n ¼ 4) were left untreated (C), treated with 10 ng/ml IL-1b for 24 h, or pre-treated with 10 mg/ml brazilin for 1 h prior to stimulation with 10 ng/ml IL-1b for 24 h. Protein extracts were sub- jected to Western blot analysis for the detection of p50. b-Actin was used as loading control. The blot of one representative experiment is shown. (d) Primary chondrocytes (n ¼ 4) were left untreated (C), treated with 10 ng/ml IL-1b for 1 h, or pre-treated with 10 mg/ml brazilin for 1 h prior to stimulation with 10 ng/ml IL-1b for 1 h. Protein extracts were subjected to Western blot analysis for the detection of p65 and phosphorylated p65. a-Tubulin was used as loading control. The blot of one representative experiment is shown. (e–h) Primary chondrocytes (n ¼ 3 donors) were left untreated as controls, treated with 10 ng/ml IL-1b for 6 h, or pre-treated with 2.5, 5, and 10 mg/ml brazilin or 5, 10, and 20 mg/ml CAPE for 1 h prior to stimulation with 10 ng/ml IL-1b for 6 h. The mRNA levels of (e) IL1B, (f) TNF, (g) PTGS2, and (h) MMP13 were analyzed by RT-qPCR and expressed as percent relative to cells treated with IL-1b (100%). Significant differences compared to cells treated only with IL-1b (p < 0.05; n ¼ 3; Friedman test with many- to-one post-hoc test).

Article Snippet: After blocking, the membranes were probed for 2h with primary antibodies: Anti-NF-kB p105/p50 (1:400, ab7971, rabbit polyclonal; Abcam, Cambridge, MA), anti-NF-kB p65 (1:1,000, rabbit monoclonal; Cell Signaling Technology, Danvers, MA), anti-phospho-NF-kB p65 (Ser536, 1:1,000, rabbit monoclonal; Cell Signaling Technology), anti-a-tubulin (1:1,000, mouse monoclonal; Cell Signaling Technology) and anti-b-actin (1:5,000, mouse monoclonal; Sigma-Aldrich).

Techniques: Incubation, Quantitative RT-PCR, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: In Vitro and In Vivo Validation of Endothelium-Derived Potential Therapeutics for Myocardial Ischemia/Reperfusion Injury Identified by an AI-Enhanced Single-Cell and Virtual-Cell Paradigm

doi: 10.3390/ijms27062743

Figure Lengend Snippet: Andrographolide inhibits the S100A8 downstream IL-17 signaling pathway and reduces the inflammatory response of H/R-induced CMECs. ( A , B ) The expressions of IL-17A, IL-17RA, Act1, and NF-κB proteins were determined by Western blot ( n = 3). ( C – E ) The levels of IL-1β, IL-6, and TNF-α were determined through ELISA. ( F – H ) The expressions of IL-17A, IL-17RA and Act1 proteins were determined by Western blot in co-transfection with the overexpression S100A8 plasmid ( n = 3). Data are presented as mean ± SD ( n = 6). “Con or C” stands for the control group, “H/R” or “H” indicates the H/R group, and “H + AG or H + A” means the H/R group treated with AG. “H + A + S” means the H/R group treated with AG and overexpression of S100A8. ** p < 0.01 compared to the Con group; # p < 0.05, ## p < 0.01 compared to the H/R or H group, @ p < 0.05, @@ p < 0.01 compared to the H + A + S group.

Article Snippet: The membranes were blocked for 1 h at room temperature with 5% skimmed milk before being incubated overnight at 4 °C with the following primary antibodies: S100A8 (A15315, Abclonal, Wuhan, China), IL-17A (A12454, Abclonal, Wuhan, China), IL-17RA (A10052, Abclonal, Wuhan, China), Act1 (A6776, Abclonal, Wuhan, China), p-NF-κB p65 (1:500, AP0124, Abclonal, Wuhan, China), NF-κB p65 (1:500, A00284-1, Boster, Wuhan, China), β-actin (1:2000, TA-09, Zhongshan Golden Bridge, Beijing, China), and GAPDH (1:2000, TA-08, Zhongshan Golden Bridge, Beijing, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Cotransfection, Over Expression, Plasmid Preparation, Control

Journal: Acta biochimica et biophysica Sinica

Article Title: Therapeutic effect of artemisinin on lupus nephritis mice and its mechanisms.

doi: 10.1093/abbs/gmq101

Figure Lengend Snippet: Figure 2 Expression of NF-kBp65 protein, NF-kB, and TGF-b1 mRNA in renal tissue (A) Activity of NF-kB. (B) NF-kBp65 protein expression. (C) TGF-b1 mRNA expression. In the three figures, (1) stands for the mice in group (i), (2) for the mice in group (ii), (3) for the mice in group (iii), and (4) for the mice in group (iv).

Article Snippet: The membrane was incubated with mouse anti-NF-kBp65 monoclonal antibody (Wuhan Boster Biological Technology, Ltd., Wuhan, China) (1: 200) and then with HRP-conjugated anti-mouse IgG secondary antibody (Wuhan Boster Biological Technology, Ltd.) (1: 1000).

Techniques: Expressing, Activity Assay

Journal: Scientific Reports

Article Title: Promotion of the inflammatory response in mid colon of complement component 3 knockout mice

doi: 10.1038/s41598-022-05708-8

Figure Lengend Snippet: Expression levels of members in the NF-κB signaling pathway. ( A ) Expression levels of NF-κB-p65 and IκB-α proteins were determined by Western blot analysis using specific primary antibody and HRP-labeled anti-rabbit IgG antibody. ( B ) Band intensities were determined using an imaging densitometer, and protein expressions were calculated relative to the intensity of β-actin. Tissue samples were collected from 3 to 5 mice per group, and each lysate was analyzed in duplicate for Western blot (final n = 6–10). Data are reported as the mean ± SD. * indicates p < 0.05 compared to the WT mice.

Article Snippet: Proteins (30 μg) were then separated by 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h, and subsequently transferred to nitrocellulose membranes for 3 h at 40 V. Each membrane was then incubated separately, overnight at 4 °C, with the following primary antibodies: anti-C3 (ab200999, Abcam Com.), anti-C3aR (bs-2955R, Bioss Inc.), anti-CR1 (LS-C777464, LSBio Inc.), anti-COX-2 (12282, Cell Signaling Technology Inc., Danvers, MA, USA), anti-NLRP3 (15101, Cell Signaling Technology Inc.), anti-Cas 1 (24232, Cell Signaling Technology Inc.), anti-ASC (67824, Cell Signaling Technology Inc.), anti-iNOS (PA3-0304, Thermo Fisher Scientific Inc.), anti-ERK1/2 (9102, Cell Signaling Technology Inc.), anti-p-ERK (E-4) (9101, Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-JNK (9252, Cell Signaling Technology Inc.), anti-p-JNK (9251, Cell Signaling Technology Inc.), anti-p38 (9212, Cell Signaling Technology Inc.), anti-p-p38 (9211, Cell Signaling Technology Inc.), anti-p-NF-κB-p65 (A00284T254, Boster Bio Inc., Pleasanton, CA, USA), anti-IκB (9242, Cell Signaling Technology Inc.), anti-p-IκB (9246S, Cell Signaling Technology Inc.), anti-IL-6 (SC-1265, Santa Cruz Biotechnology Inc.), anti-C5 (ab11898, Abcam Com.), anti-thrombin (ab92621, Abcam Com.), anti-E-cadherin (24E10, Cell Signaling Technology Inc.) or anti-β-actin (4967, Sigma-Aldrich Co.).

Techniques: Expressing, Western Blot, Labeling, Imaging

Journal: Drug Design, Development and Therapy

Article Title: Cyclooxygenase inhibitors potentiate receptor tyrosine kinase therapies in bladder cancer cells in vitro

doi: 10.2147/DDDT.S158518

Figure Lengend Snippet: Expression profile of RTKs and COXs signaling pathway proteins in human and primary canine TCC cells by WB analysis. Cells were grown in the absence (−) or presence (+) of FBS for 24 hours. The expression of c-Kit, PDGFRα, p-ERK1/2, ERK1/2, p-Akt (Ser473), Akt1/2/3, COX-1, COX-2, and NF-κB (specific band labeled with arrow) proteins was evaluated by WB analysis. Actin was used as a loading control. Tested K9TCC cells had higher expression of RTK and COX proteins as compared to h-T24, 5637, and UM-UC-3 cells. Abbreviations: COX, cyclooxygenase; RTK, receptor tyrosine kinase; WB, Western blot; TCC, transitional cell carcinoma; PDGFR, platelet-derived growth factor receptor; NF-κB, nuclear factor kappa-light-chain-enhance of activated B cells; ERK1/2, extracellular signal regulated kinases 1/2; Akt, V-akt murine thymoma oncogene homolog 1; p-ERK1/2, phosphorylated ERK1/2; p-Akt, phosphorylated Akt; FBS, fetal bovine serum; MW, molecular weight; ns, non-specific.

Article Snippet: The antibodies for COX-2 (C-20), COX-1 (H-62), PDGFRα (C-20), phosphorylated PDGFRα (p-PDGFRα) (Tyr720), phosphorylated extracellular signal regulated kinases 1/2 (p-ERK1/2) (E-4), phosphorylated V-akt murine thymoma oncogene homolog 1 (p-Akt) (Ser473), c-Kit (961-976), p-c-Kit (Tyr721), Akt1/2/3 (H136), ERK1/2 (K-23), caspase-3 (H-277), actin, and donkey anti-goat secondary antibodies were purchased from Santa Cruz Biotechnology Inc (Dallas, TX, USA); antibody for p65 (nuclear factor kappa-light-chain-enhance of activated B cells [NF-κB]) was purchased from BD Biosciences (San Jose, CA, USA); poly (ADP-ribose) polymerase (PARP) (recognize total and cleaved PARP [cPARP]), cleaved caspase-3 (cCaspase-3) (D175), secondary anti-rabbit and anti-mouse antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Labeling, Western Blot, Derivative Assay, Molecular Weight

Journal: Drug Design, Development and Therapy

Article Title: Cyclooxygenase inhibitors potentiate receptor tyrosine kinase therapies in bladder cancer cells in vitro

doi: 10.2147/DDDT.S158518

Figure Lengend Snippet: Axitinib and AB1010 increased COX-2 expression in a dose-dependent manner in tested TCC cells by WB analysis. ( A ) h-5637 and K9TCC#1Lillie cells were treated with axitinib and AB1010 at doses of 0, 1, 5, and 10 μM for 24 hours. Actin was used as a loading control. Axitinib and AB1010 increased COX-2, but not NF-κB (specific band labeled with arrow) expression in h-5637 cells. AB1010 increased COX-2 and NF-κB protein expression in a dose-dependent manner in K9TCC#1Lillie cells. ( B ) Densitometry analysis of COX-2 and p-Akt protein bands from WB analysis was performed using VisionWorks analysis software. Values represent the mean ± SE of the measured densitometry of each protein band from three independent experiments. Paired Student’s t -test was used to compare axitinib and AB1010 treatments to controls (* p <0.05, ** p <0.01, *** p <0.001). Abbreviations: COX, cyclooxygenase; WB, Western blot; TCC, transitional cell carcinoma; NF-κB, nuclear factor kappa-light-chain-enhance of activated B cells; Akt, V-akt murine thymoma oncogene homolog 1; p-Akt, phosphorylated Akt; MW, molecular weight; ns, non-specific.

Article Snippet: The antibodies for COX-2 (C-20), COX-1 (H-62), PDGFRα (C-20), phosphorylated PDGFRα (p-PDGFRα) (Tyr720), phosphorylated extracellular signal regulated kinases 1/2 (p-ERK1/2) (E-4), phosphorylated V-akt murine thymoma oncogene homolog 1 (p-Akt) (Ser473), c-Kit (961-976), p-c-Kit (Tyr721), Akt1/2/3 (H136), ERK1/2 (K-23), caspase-3 (H-277), actin, and donkey anti-goat secondary antibodies were purchased from Santa Cruz Biotechnology Inc (Dallas, TX, USA); antibody for p65 (nuclear factor kappa-light-chain-enhance of activated B cells [NF-κB]) was purchased from BD Biosciences (San Jose, CA, USA); poly (ADP-ribose) polymerase (PARP) (recognize total and cleaved PARP [cPARP]), cleaved caspase-3 (cCaspase-3) (D175), secondary anti-rabbit and anti-mouse antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Labeling, Software, Western Blot, Molecular Weight

Journal: Drug Design, Development and Therapy

Article Title: Cyclooxygenase inhibitors potentiate receptor tyrosine kinase therapies in bladder cancer cells in vitro

doi: 10.2147/DDDT.S158518

Figure Lengend Snippet: Co-treatment of AB with Indo inhibited cell viability and AB-induced COX-2 expression in TCC cells. ( A ) h-5637 and K9TCC#1Lillie cells were treated with 5 μM AB, 50 μM Indo, and in combination (AB + Indo). Cell viability of TCC cells was determined by MTS assay. Co-treatment of AB with Indo inhibited cell viability more effectively than either treatment alone in bladder cancer cells. Values represent mean ± SE of four replicates of three independent experiments; paired Student’s t -test was used to compare the treatment to control groups, ** p <0.01 and *** p <0.001. Student’s t -test was used to compare AB + Indo to AB treatment groups, # p <0.05. Student’s t -test was used to compare AB + Indo to Indo treatment groups, ‡‡ p <0.01 and ‡‡‡ p <0.001. ( B ) h-5637 and K9TCC#1Lillie cells were co-treated with 50 μM Indo and 5 μM AB, or each drug alone for 24 hours. The expression of COX-1, COX-2, NF-κB (specific band labeled with arrow), p-Akt (Ser473), Akt1/2/3, and cPARP (specific band labeled with arrow) proteins was determined by WB analysis. Actin was used as a loading control. AB-induced COX-2 expression was inhibited by co-treatment with Indo in h-5637 and K9TCC#1Lillie cells. ( C ) Densitometry analysis of COX-2, p-Akt (Ser473), and cPARP protein bands from WB analysis was performed using VisionWorks analysis software. Values represent the mean ± SE of the measured densitometry of each protein band of three independent experiments. Paired Student’s t -test was used to compare AB, Indo, and AB + Indo treatments to control groups, * p <0.05, ** p <0.01, *** p <0.001. Abbreviations: AB, AB1010; Indo, indomethacin; COX, cyclooxygenase; WB, Western blot; TCC, transitional cell carcinoma; NF-κB, nuclear factor kappa-light-chain-enhance of activated B cells; Akt, V-akt murine thymoma oncogene homolog 1; p-Akt, phosphorylated Akt; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt; MW, molecular weight; cPARP, cleaved poly (ADP-ribose) polymerase; ns, non-specific.

Article Snippet: The antibodies for COX-2 (C-20), COX-1 (H-62), PDGFRα (C-20), phosphorylated PDGFRα (p-PDGFRα) (Tyr720), phosphorylated extracellular signal regulated kinases 1/2 (p-ERK1/2) (E-4), phosphorylated V-akt murine thymoma oncogene homolog 1 (p-Akt) (Ser473), c-Kit (961-976), p-c-Kit (Tyr721), Akt1/2/3 (H136), ERK1/2 (K-23), caspase-3 (H-277), actin, and donkey anti-goat secondary antibodies were purchased from Santa Cruz Biotechnology Inc (Dallas, TX, USA); antibody for p65 (nuclear factor kappa-light-chain-enhance of activated B cells [NF-κB]) was purchased from BD Biosciences (San Jose, CA, USA); poly (ADP-ribose) polymerase (PARP) (recognize total and cleaved PARP [cPARP]), cleaved caspase-3 (cCaspase-3) (D175), secondary anti-rabbit and anti-mouse antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, MTS Assay, Labeling, Software, Western Blot, Molecular Weight