Journal: Cell reports

Article Title: Arachidonic acid inhibition of the NLRP3 inflammasome is a mechanism to explain the anti-inflammatory effects of fasting

doi: 10.1016/j.celrep.2024.113700

Figure Lengend Snippet:

Article Snippet: Immunoblots were probed using the following primary antibodies: caspase-1 p10 (mouse) (sc-514, Santa Cruz) 1 in 500; IL-1β (goat) (AF-401, R&D Systems) 1 in 1000; β-Actin (mouse) (AB3280, ABCAM) 1 in 2500; NLRP3 (rat) (MAB7578-SP, R&D Systems) 1 in 2000; COX2 (goat) (AF4198, R&D Systems).

Techniques: Virus, Recombinant, Modification, Saline, Protease Inhibitor, Marker, Cytotoxicity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Knock-Out, Software

Journal: Regulatory peptides

Article Title: Increased circulating angiotensin-(1-7) protects white adipose tissue against development of a proinflammatory state stimulated by a high-fat diet.

doi: 10.1016/j.regpep.2012.06.009

Figure Lengend Snippet: Fig. 3. Adipose tissue inflammatory markers. Western blot for COX2-epididymal adipose tissue (A), RT-PCR expression of IL-1β‐epididymal adipose tissue (B), SD immunohisto- chemical reaction for IL-1β in epididymal adipose tissue (C), TGR immunohistochemical reaction for IL-1β in epididymal adipose tissue (D), RT-PCR expression of TNF-α‐ epididymal adipose tissue (E), SD immunohistochemical reaction for TNF-α in epididymal adipose tissue (F), TGR immunohistochemical reaction for TNF-α in epididymal adipose tissue (G). Data are presented as mean±SEM; * pb0.05.

Article Snippet: The blot was probed with COX2 enzyme antibody (Cell Signaling, USA; 1:1000), diluted in 1× blocking buffer, washed in 1× TBS with 1% of Tween 20, and then incubated with either IRDye 800 anti-goat secondary antibody (Rockland Immunochemicals®, Gilbertsville, Pa.) diluted to 1:10,000 in blocking buffer followed by further washes with PBS-Tween 20 and one wash in TBS.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemical staining

Journal: International Journal of Clinical and Experimental Pathology

Article Title: MAPK/ERK signal pathway involved expression of COX-2 and VEGF by IL-1? induced in human endometriosis stromal cells in vitro

doi:

Figure Lengend Snippet: Expression of COX-1 by 4 cytokines in endometriosis ESC by Western Blotting in vitro. Expression of COX-1 was expressed in three ESC, the normal was higher than the eutopic or ectopic, and there was statistical difference (P<0.05). The eutopic and ectopic had no statistical difference (P>0.05). And the expression of COX-1 had no statistical differences after 4 kinds CK (IL-1β, TNF-α, IFN-γ and M-CSF) was added (P>0.05).

Article Snippet: Macrophage colony stimulating factor (MCSF) and vascular endothelial growth factor (VEGF) development ELISA Kit were from PeproTech, Inc (Rocky Hill, NJ, USA), protein assay kit was from Bio-RAD (Hercules, CA, USA), antibody against COX-2 was from Cayman Chemicals (Ann arbor, Mi, USA), antibody against COX-1 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), the second antibody, a horseradish peroxidase-conjugated anti-mouse antibody and ECL plus western blotting detection system were obtained from Amersham (Piscataway, NJ, USA).

Techniques: Expressing, Western Blot, In Vitro

Journal: Cell Death & Disease

Article Title: Celecoxib and acetylbritannilactone interact synergistically to suppress breast cancer cell growth via COX-2-dependent and -independent mechanisms

doi: 10.1038/cddis.2011.64

Figure Lengend Snippet: Effects of celecoxib, ABL or their combinations on the apoptosis of breast cancer cells. Cells were treated with celecoxib, ABL or their combination as indicated for 48 h. ( a ) DNA fragmentation of apoptotic cells was evaluated using a Cell Death Detection ELISA Plus kit. * P <0.05, compared with vehicle-treated cells. ( b ) Apoptotic cells were evaluated by ELISA in Control and KD cells after the combination treatment. Control, MDA-MB-231 cells transfected with a general negative sequence of siRNA; KD, COX-2 KD MDA-MB-231 cells. ( c ) MCF-7-mock, MCF-7-COX-2 and TPA (0.1 μ M)-stimulated MCF-7 cells were treated with combination of celecoxib and ABL for 48 h, and apoptotic cells were evaluated. Results were expressed as mean±S.E.M. from at least three independent experiments

Article Snippet: Antibodies against COX-1, COX-2, β -actin, caspase-3, caspase-9, phospho-extracellular signal-regulated kinase1/2 (p-ERK1/2), phospho-Akt (p-Akt), Akt, phospho-GSK-3 β (p-GSK-3 β ), the secondary antibodies, small interfering RNA (siRNA) specific for human COX-2 mRNA and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Sequencing

Journal: Cell Death & Disease

Article Title: Celecoxib and acetylbritannilactone interact synergistically to suppress breast cancer cell growth via COX-2-dependent and -independent mechanisms

doi: 10.1038/cddis.2011.64

Figure Lengend Snippet: Effects of the combination of celecoxib and ABL on COX-2 expression and activity in MDA-MD-231 cells. ( a ) MDA-MD-231 cells were treated with celecoxib, ABL or their combination for 24 h (left) or 48 h (right). COX-2 mRNA and protein expression was examined using RT-PCR (left) and western blot analysis (right). Quantification of COX-2 mRNA and protein expression normalized to actin levels, were provided at the bottom. ( b ) MDA-MB-231 cells were treated with the combination for 48 h, and PGE 2 levels in the culture medium were measured using ELISA. Results were expressed as mean±S.E.M. from at least three independent experiments

Article Snippet: Antibodies against COX-1, COX-2, β -actin, caspase-3, caspase-9, phospho-extracellular signal-regulated kinase1/2 (p-ERK1/2), phospho-Akt (p-Akt), Akt, phospho-GSK-3 β (p-GSK-3 β ), the secondary antibodies, small interfering RNA (siRNA) specific for human COX-2 mRNA and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Celecoxib and acetylbritannilactone interact synergistically to suppress breast cancer cell growth via COX-2-dependent and -independent mechanisms

doi: 10.1038/cddis.2011.64

Figure Lengend Snippet: Effects of celecoxib and ABL individually and in combination on MDA-MB-231 Xenograft model. Female nude mice bearing MDA-MB-231 tumors were treated with vehicle, celecoxib (5 mg/kg) and ABL (15 mg/kg) individually, or in combination. ( a ) Tumors were measured with calipers on alternate days. Points, mean tumor volume in each experimental group containing six mice. * P <0.01 versus vehicle group. ( b ) The expression of COX-2, COX-1, p21, PARP, p-Akt, Akt, p-p38 and p38 were determined by western blot analysis in protein extracted from the tumor specimens. Total protein samples from two mice for each group representing control and experiment were used for western blot. Densitometric analyses of western blot (average of two samples) were provided. Each column represents the mean±S.E.M. ( c ) A strong positive correlation (data shown were obtained from ) between serum PGE 2 level and COX-2 expression in xenograft specimens (Pearson's correlation coefficient, 0.900; P <0.001, two-tailed)

Article Snippet: Antibodies against COX-1, COX-2, β -actin, caspase-3, caspase-9, phospho-extracellular signal-regulated kinase1/2 (p-ERK1/2), phospho-Akt (p-Akt), Akt, phospho-GSK-3 β (p-GSK-3 β ), the secondary antibodies, small interfering RNA (siRNA) specific for human COX-2 mRNA and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: OncoImmunology

Article Title: Stromal-like Wilms tumor cells induce human Natural Killer cell degranulation and display immunomodulatory properties towards NK cells

doi: 10.1080/2162402x.2021.1879530

Figure Lengend Snippet: Figure 5. Str-WT cells up-regulate inhibitory factors on exposure to inflammatory cytokines. (a) Western blot analysis of COX-2 expression in str-WT cells, and in g66 MSC, either unstimulated or stimulated with IL-1β + TNF-α for 48 h. GAPDH expression was analyzed as positive control. Molecular weight (MW) markers (kDa) are indicated on the right. A representative experiment of three is shown. (b) Production of PGE2 by str-WT cells cultured alone (basal), stimulated by IL-1β + TNF-α, or co-cultured with NK cells derived from two different healthy donors (NK1 and NK2). Culture supernatants were collected after 48 h (for cytokine stimulation experiments) or after 72 h (for co-culture experiments) and analyzed by ELISA assay. Data refer to the levels of PGE2 expressed as pg/ml. (c) Western blot analysis of IDO expression in str-WT cells and in g66 MSC, either unstimulated or stimulated with IFN-γ for 48 h. GAPDH expression was analyzed as positive control. Molecular weight (MW) markers (kDa) are indicated on the right. A representative experiment of two is shown. (d) gW1 cells were stimulated with IFN-γ and analyzed by flow cytometry for the expression of HLA-class I and the indicated immune checkpoint molecules. Light and dark gray histograms represent unstimulated and IFN-γ-astimulated cells, respectively. Upper numbers represent MFI of unstimulated cells, while bold lower numbers refer to MFI of stimulated cells.

Article Snippet: The following commercial antibodies were utilized in this study for Western blot analysis: mouse anti-vimentin (clone V9, IgG1; Santa Cruz Biotechnology, sc-6260); mouse anti-pan Cytokeratin mAb (clone PCK-6; IgG1; Abcam, ab6401); rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mAb (clone 14 C10; Cell Signaling, 2118); rabbit anticyclooxygenase-2 (COX-2) mAb (clone D5H5; Cell Signaling, 12282); rabbit anti indoleamine 2,3-dioxygenase (IDO) Ab (ThermoFisher, 711778); goat anti-mouse Ig HRP-conjugated mAb (Southern Biotech, 1031–05); goat anti-rabbit HRPconjugated mAb (Southern Biotech, 4050–05).

Techniques: Western Blot, Expressing, Positive Control, Molecular Weight, Cell Culture, Derivative Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages

doi: 10.1136/jitc-2019-000489

Figure Lengend Snippet: Melanoma cells expressing bcl-2 promote migration and polarization of macrophages to a M2-type phenotype. qRT-PCR analysis of CD206, IL-10, CCL1, CCL22, IL-12, COX-2 mRNA levels in (A) THP-1 cells and (B) human M-DM after 24 hours exposure to serum free medium (M0 macrophages) or to CM from M14 human melanoma control or bcl-2 overexpressing cells. (C) ELISA of IL-10 and IL-12 protein secretion in M-DM stimulated as reported in (B). (D) qRT-PCR analysis of CD206, IL-10, CCL1, CCL22, IL-12, COX-2 mRNA levels in M-DM stimulated with CM derived from M14 melanoma control or bcl-2 silenced cells. (E) qRT-PCR analysis of IL-1β, IL-8 and VEGF mRNA levels in M-DM stimulated as reported in (B). (F) Representative images (left panels) and relative quantification (right panel) of THP-1 cell migration in response to CM from M14 control (CM M14 Control) or bcl-2 overexpressing (CM M14 Bcl-2/6) melanoma cells. The values are reported as number of migrated cells/field. The quantification was performed by counting the number of migrated cells in at least 10 fields for each condition. The results are reported as fold induction relative to (A, B) M0 macrophages or to (C) melanoma control cells. The results are reported as % of mRNA variation in macrophages exposed to CM derived from (D) bcl-2 silenced cells versus control ones and from (E) bcl-2 overexpressing cells versus control ones. The average±SEM (A, B, D, E) or ±SD (C, F) of three independent experiments is reported. *P<0.05. **P<0.01. CM, culture medium; M-DM, monocyte-derived macrophages.

Article Snippet: Immunodetection was performed using antibodies directed to β-actin (Sigma-Aldrich), Cyclooxygenase 2 (COX-2, Cayman Chemical, Ann Arbor, Michigan, USA), histone H3 (Cell Signaling, Danvers, USA), human bcl-2 (100), p65, IKBα and murine bcl-2 (10C4) (Santa Cruz Biotechnology, Dallas, Texas, USA), HSP72/73 (Calbiochem, San Diego, California, USA), HSP90 (BD biosciences, San Diego, California, USA).

Techniques: Expressing, Migration, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages

doi: 10.1136/jitc-2019-000489

Figure Lengend Snippet: Bcl-2 drives expression of COX-2/PGE2 axis and IL-1β, IL-8, IL-17, CCL2 in melanoma cells. Analysis of COX-2 expression by (A) Western blot and (B) qRT-PCR analyses in M14 human melanoma control (M14 Control) and bcl-2 overexpressing (M14 Bcl-2/6) cells. (A) β-actin is shown as loading and transferring control. One representative western blot analysis out of two with similar results is reported. The numbers indicate densitometric analysis relative to control. (C) ELISA of PGE2 levels in CM derived from M14 control and bcl-2 overexpressing cells. PGE2 levels were normalized to the number of adherent cells. (D) qRT-PCR and (E) ELISA analyses of IL-1β, IL-8 and IL-17 expression in M14 control and bcl-2 overexpressing cells. (E) Protein levels were normalized to the number of adherent cells. (F) qRT-PCR analysis of IL-1β (IL-1R1), IL-8 (CXCR1), IL-17 (IL-17RA) receptors in M14 control and bcl-2 overexpressing cells. (G) qRT-PCR analysis of CCL2, CSF-1 and SDF-1 mRNA levels in M14 control and bcl-2 overexpressing cells. (B–G) Fold induction relative to control is reported. The results represent the average±SEM (B, D, F, G) or ±SD (C, E) of three independent experiments. *P<0.05.

Article Snippet: Immunodetection was performed using antibodies directed to β-actin (Sigma-Aldrich), Cyclooxygenase 2 (COX-2, Cayman Chemical, Ann Arbor, Michigan, USA), histone H3 (Cell Signaling, Danvers, USA), human bcl-2 (100), p65, IKBα and murine bcl-2 (10C4) (Santa Cruz Biotechnology, Dallas, Texas, USA), HSP72/73 (Calbiochem, San Diego, California, USA), HSP90 (BD biosciences, San Diego, California, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transferring, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages

doi: 10.1136/jitc-2019-000489

Figure Lengend Snippet: Bcl-2 drives transcriptional activation of IL-1β, IL-17, RORa, RORc and CCL2 in a NF-κB-dependent manner. (A) Western blot analysis of NF-κB subunit p65 in nuclear and cytosol extracts in M14 human melanoma control (M14 Control) and bcl-2 overexpressing clone (M14 Bcl-2/6). HSP90 and histone H3 are shown as loading, transferring and cytoplasmic/nuclear purification control. One representative western blot analysis out of two with similar results is reported. The numbers indicate densitometric analysis relative to control. ChIP analysis of (B) NF-κB subunit p65 recruitment, (C) histone H3 acetylation and (D) Pol II recruitment at IL-1β, IL-8, COX-2 and CCL2 promoters in M14 control and two bcl-2 overexpressing clones (M14 Bcl-2/6, M14 Bcl-2/37). The results are reported as % Input – NoAb. (E) qRT-PCR analysis of IL-1β, IL-8, IL-17, RORa, RORc, COX-2, CCL2 expression in M14 control, Bcl-2/6 and Bcl-2/6 IKBSR cells. (F) qRT-PCR analysis of CD206, IL-10, CCL1, CCL22, IL-12 and COX-2 expression in M-DM after exposure to CM from M14 control, Bcl-2/6 or Bcl-2/6 IKBSR cells. (B–F) Fold induction relative to control cells and the average±SEM of three experiments is reported. P values were calculated between (B–D) control and bcl-2 overexpressing cells or between (E, F) Bcl-2/6 cells and Bcl-2/6 overexpressing IKBSR cells, *P<0.05.

Article Snippet: Immunodetection was performed using antibodies directed to β-actin (Sigma-Aldrich), Cyclooxygenase 2 (COX-2, Cayman Chemical, Ann Arbor, Michigan, USA), histone H3 (Cell Signaling, Danvers, USA), human bcl-2 (100), p65, IKBα and murine bcl-2 (10C4) (Santa Cruz Biotechnology, Dallas, Texas, USA), HSP72/73 (Calbiochem, San Diego, California, USA), HSP90 (BD biosciences, San Diego, California, USA).

Techniques: Activation Assay, Western Blot, Transferring, Purification, Clone Assay, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Mitochondria-Associated Membranes (MAMs) are involved in Bax mitochondrial localization and cytochrome c release

doi: 10.1101/443606

Figure Lengend Snippet: (A) Cytochrome c+c1/Cytochrome b ratios measured on mitochondria isolated from wild-type or Δmdm34 cells expressing Bax-P168A. Each point represents a single mitochondria preparation. The hatched zone corresponds to the typical values found on mitochondria preparations from cells that do not express Bax [ - ]. *: p<0.05 (unpaired Student t-test). (B) Cytochrome c+c1/Cytochrome b ratios measured on mitochondria preparations isolated from strains co-expressing Bax-P168A and Bcl-xL. (C) Separation of whole extracts from cells expressing Bax-P168A on a 15-65% sucrose density gradient. Vertical lines mark the separation between different gels. Data are representative of four independent experiments.

Article Snippet: For western-blots, proteins were separated on 12.5% SDS-PAGE, transferred onto nitrocellulose, saturated in PBST/milk or TBST/BSA (depending on antibodies) and blotted with the following antibodies: anti-human Bax 2D2 (Santa-Cruz, 1/5,000 dilution), anti-yeast porin (Por1) (Novex, 1/50,000 dilution), anti-yeast Phosphoglycerate Kinase (Pgk1) (Novex, 1/10,000 dilution), anti-yeast Dolichol Phosphate Mannose Synthase (Dpm1) (Novex, 1/5,000 dilution), anti-yeast Cytochrome c (Cyc1) (Custom made, Millegen, 1/5,000 dilution), anti-yeast Cytochrome c Oxidase subunit II (Cox2) (Invitrogen, 1/5,000 dilution, anti-yeast carboxypeptidase Y (Cpy1) (Invitrogen, 1/10,000 dilution).

Techniques: Isolation, Expressing

Journal: bioRxiv

Article Title: Mitochondria-Associated Membranes (MAMs) are involved in Bax mitochondrial localization and cytochrome c release

doi: 10.1101/443606

Figure Lengend Snippet: (A) Cytochrome c+c1/Cytochrome b ratios measured on mitochondria from different strains. Values are averages (± s.d.) of 4 to 6 independent experiments. (B) Distribution of Dpm1 (ER), Por1 (OMM) and Bax on a crude mitochondrial fraction separated on sucrose density gradients. Results are representative of 3 independent experiments.

Article Snippet: For western-blots, proteins were separated on 12.5% SDS-PAGE, transferred onto nitrocellulose, saturated in PBST/milk or TBST/BSA (depending on antibodies) and blotted with the following antibodies: anti-human Bax 2D2 (Santa-Cruz, 1/5,000 dilution), anti-yeast porin (Por1) (Novex, 1/50,000 dilution), anti-yeast Phosphoglycerate Kinase (Pgk1) (Novex, 1/10,000 dilution), anti-yeast Dolichol Phosphate Mannose Synthase (Dpm1) (Novex, 1/5,000 dilution), anti-yeast Cytochrome c (Cyc1) (Custom made, Millegen, 1/5,000 dilution), anti-yeast Cytochrome c Oxidase subunit II (Cox2) (Invitrogen, 1/5,000 dilution, anti-yeast carboxypeptidase Y (Cpy1) (Invitrogen, 1/10,000 dilution).

Techniques:

Journal: bioRxiv

Article Title: Mitochondria-Associated Membranes (MAMs) are involved in Bax mitochondrial localization and cytochrome c release

doi: 10.1101/443606

Figure Lengend Snippet: (A) The fraction of NADH-reducible cytochrome c was measured by measuring reduction after adding 1mM NADH, followed by measuring the reduction after adding dithionite on the same sample. Each point is a different mitochondria preparation (**: significantly different p<0.01). (B) The ratio reduction by NADH/reduction by dithionite was measured for cytochrome c and for cytochrome b. The graph shows the ratio (% reduction cytochrome c) / (% reduction of cytochrome b). Each point is a different mitochondria preparation. (*: significantly different p<0.05). (C) Mitochondria were fractionated on a sucrose density gradient. A small pool of cytochrome c was found at a lower density than the main fraction, that may correspond to partly released cytochrome c, not reducible by NADH in experiments (A) and (B). The figure is representative of 3 independent experiments.

Article Snippet: For western-blots, proteins were separated on 12.5% SDS-PAGE, transferred onto nitrocellulose, saturated in PBST/milk or TBST/BSA (depending on antibodies) and blotted with the following antibodies: anti-human Bax 2D2 (Santa-Cruz, 1/5,000 dilution), anti-yeast porin (Por1) (Novex, 1/50,000 dilution), anti-yeast Phosphoglycerate Kinase (Pgk1) (Novex, 1/10,000 dilution), anti-yeast Dolichol Phosphate Mannose Synthase (Dpm1) (Novex, 1/5,000 dilution), anti-yeast Cytochrome c (Cyc1) (Custom made, Millegen, 1/5,000 dilution), anti-yeast Cytochrome c Oxidase subunit II (Cox2) (Invitrogen, 1/5,000 dilution, anti-yeast carboxypeptidase Y (Cpy1) (Invitrogen, 1/10,000 dilution).

Techniques: