Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 7. Validation of compound-target interactions through molecular docking and protein-small molecule binding assays. (A) Chemical structure of EGCG and molecular docking models showing its interactions with NOX2 and p47phox. (B) Chemical structure of quercetin and molecular docking models illustrating its interactions with NOX2 and p47phox. (C) An SPR sensorgram displaying the binding affinity of the NOX2-EGCG interaction. (D) An SPR sensorgram showing the binding affinity of the NOX2-quercetin interaction. (E) A BLI sensorgram illustrating the binding affinity of the p47phox-EGCG interaction. (F)A BLI sensorgram depicting the binding affinity of the p47phox-quercetin interaction. Abbreviations: BLI, Bio-layer interferometry; EGCG, epigallocatechin-3-gallate; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; Que, quercetin; SPR, surface plasmon resonance.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: Biomarker Discovery, Binding Assay, SPR Assay

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 8. QKPC and its components, EGCG and quercetin, reduce NOX2-p47phox/ROS signaling in vivo and in vitro. (A) Protein levels of NOX2 and p47phox in human neutrophils were assessed by Western blotting analysis, with relative density quantification shown as the ratio of NOX2 or p47phox to GAPDH (n = 3). (B) Protein levels of NOX2 and p47phox in lung tissues of mice were determined by Western blotting analysis and presented as the ratio of NOX2 or p47phox to GAPDH (n = 3). (C) Cellular ROS levels were measured using the oxidant-sensing probe DCFH-DA, and fluorescence intensity was recorded for each group. EGCG, quercetin, and the positive drugs NAC (20 μM, a ROS scavenger), DPI (50 μM, a general NADPH oxidase inhibitor), and GSK (50 μM, a NOX2-specific inhibitor) effectively suppressed ROS levels elevated by the combined CSE and LPS exposure (n = 6–8). (D) MDA levels in lung tissues were measured with an MDA assay kit. Data are presented as mean ± SD, with statistical significance set at p < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviations: CS, cigarette smoke; CSE, cigarette smoke extract; DPI, Diphenyleneiodonium chloride; EGCG, epigallocatechin-3-gallate; GSK, GSK2795039; LPS, lipopolysaccharide; MDA, malondialdehyde; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; Que, quercetin; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: In Vivo, In Vitro, Western Blot, Fluorescence, Multiple Displacement Amplification

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 9. Proposed mechanisms of QKPC action against ECOPD. Abbreviations: COPD, chronic obstructive pulmonary disease; EGCG, epigallocatechin-3-gallate; LPS, lipopolysaccharide; MPO, myeloperoxidase; NE, neutrophil Elastase; NET, neutrophil extracellular trap; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: c-Jun NH2-terminal Kinase Targeting and Phosphorylation of Heat Shock Factor-1 Suppress Its Transcriptional Activity

doi: 10.1074/jbc.m000958200

Figure Lengend Snippet: FIG. 2. JNK targets HSF-1 to the D domain. A, in vivo interaction of ERK1 with HSF-1. Human lung carcinoma cells H1299 stably expressing HA-tagged ERK1 (HE-10) were transiently trans- fected with full-length pcDNA3-HSF-1 ex- pression vector. After 48 h, cells were ly- sed, and ERK1 was immunoprecipitated using anti-ERK1 antibodies. Immunopre- cipitates were analyzed by PAGE and im- munoblotted using anti-HSF-1 antibody (23). B, diagram of truncated wild type HSF-1 and deletion mutants. Numbers above indicate amino acid residues. Num- bers below indicate start and end points of deleted region in mutant. C, the con- served sequence (underlined) of the D do- main in HSF-1 and other transcription factors that are the target of MAPKs. D and E, binding of JNK1 and ERK1 to truncated wild type His-tagged-HSF-1 (1–450) and truncated His-tagged HSF-1 mutant D01 (deleted between amino acid residues 203 and 224). Anti-JNK1 or anti- ERK1 antibodies (Ab) were bound to pro- tein A-Sepharose beads and incubated with unheated control (C) or heated (45 °C for 30 min) H1299 cell lysates sta- bly expressing JNK1 (D) or ERK1 (E). Purified truncated wild type His-HSF-1 (1–450) or truncated His-HSF-1 mutant D01 were added to the protein A/JNK or ERK antibody mix. The immunoprecipi- tated materials were analyzed by PAGE, followed by immunoblotting using anti- HSF-1 antibody. The positions of the pu- rified HSF-1 (1–450) and IgG bands are indicated. F, requirement of binding to HSF-1 for efficient JNK or ERK phospho- rylation. Wild type HSF-1 or mutant D01 were phosphorylated for 5–60 min at 30 °C with immunoprecipitated activated JNK1 or purified active JNK2, ERK1, or ERK2. Samples were analyzed by PAGE, and gels were exposed to x-ray film. The gels were quantitated using PhosphorIm- ager. All experiments were performed at least three times, and results were con- sistent. G, deletion of the amino acid res- idues 203–224 in HSF-1 protein prevents its nuclear localization. Representative immunofluorescence photographs (mag- nification, 31000) of HeLa cells trans- fected with HSF-1-GFP (wild type), or HSF-1-GFP D01 (deleted between amino acid residues 203 and 224). HeLa cells were transiently transfected with the ex- pression vectors. 48 h after transfection, cell were heated at 45 °C for 30 min and allowed to recover at 37 °C for 4 h, which is the best recovery time after heat shock to observe HSF-1 granules. GFP fluo- resces green under blue light. Wt, wild type.

Article Snippet: For phosphorylation experiments, 1 mg of each substrate was phosphorylated with [g-32P]ATP for 20 min at 30 °C with immunoprecipitated JNK1 (C17, Santa Cruz) or purified active JNK2 (UBI), ERK1 (United Biotechnology, Inc.), or ERK2 (New England BioLabs).

Techniques: In Vivo, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Sequencing, Binding Assay, Incubation, Control, Purification, Western Blot, Immunofluorescence, Transfection

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

doi: 10.1097/01.lab.0000056999.08437.b2

Figure Lengend Snippet: Figure 3. Activation of extracellular signal-regulated kinase (ERK), p38 mitogen- activated protein kinase (MAPK), and c-Jun NH2-terminal kinase (JNK) in COOH-HBFN-f–stimulated RSF. After incubation with 100 nM COOH-HBFN-f (indicated as HBFN-f) or 2 ng/ml IL-1 for the time periods indicated, RSF were lysed as described. Cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-pospho-ERK1/2 (ph-ERK1/2), anti-ERK1/2, anti-phospho-p38 MAPK (ph-p38), anti-p38, anti-phospho-JNK (ph-JNK), or anti-JNK antibody. RSF derived from four patients with RA were used for experiments with similar results.

Article Snippet: Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Activation Assay, Incubation, SDS Page, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

doi: 10.1097/01.lab.0000056999.08437.b2

Figure Lengend Snippet: Figure 4. Effects of MAPK inhibitors on MMP production by RSF stimulated with COOH-HBFN-f. After preincubation with PD98059 or SB203580 for 1 hour at the concentrations indicated, RSF were incubated for 48 hours with 100 nM COOH-HBFN-f (indicated as HBFN-f). Conditioned media were analyzed by immunoblotting using specific antibodies for MMP-1, MMP-3, and MMP-13. The amount of sample applied was determined on the basis of DNA content of RSF in the well. RSF derived from four patients with RA were used for experiments with similar results.

Article Snippet: Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Incubation, Western Blot, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

doi: 10.1097/01.lab.0000056999.08437.b2

Figure Lengend Snippet: Figure 7. Activation of ERK, p38, and JNK in RSF with 41 integrin ligation with CS-1. After incubation with CS-1 at 10 M for the time periods indicated, RSF were lysed as described. Cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-ph-ERK1/2, anti-ERK1/2, anti-ph- p38, anti-p38, anti-ph-JNK, or anti-JNK antibody. RSF derived from four patients with RA were used for experiments with similar results.

Article Snippet: Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Activation Assay, Ligation, Incubation, SDS Page, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

doi: 10.1097/01.lab.0000056999.08437.b2

Figure Lengend Snippet: Figure 8. Effects of MAPK inhibitors on MMP induction by 41 integrin stimulation with CS-1. RSF were pretreated for 1 hour with the indicated concentrations of PD98059 or SB203580 and thereafter stimulated for 48 hours with 10 M CS-1. Conditioned media were analyzed by SDS-PAGE and immunoblotting using specific antibodies for MMP-1, MMP-3, and MMP-13. The amount of sample applied was determined on the basis of DNA content of RSF in the well. RSF derived from four patients with RA were used for experiments with similar results.

Article Snippet: Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: SDS Page, Western Blot, Derivative Assay

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal weight (A) and umbilical artery serum DHAS (B) and cortisol (C) levels in untreated baboons on days 100 (middle, n = 4) and 165 (late, n = 8) of gestation and on day 165 in animals that were treated daily on days 100–164 with letrozole (0.115 mg/kg body weight per day, n = 8) or letrozole plus estradiol (each at 0.115 mg/kg body weight per day, n = 5). Values indicated by different letter superscripts are different at P < .05 to P < .01 (ANOVA and Newman-Keul's multiple comparison test).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques:

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Western immunoblot of MC2R (A) and MRAP (B) incubated with primary antibody (Ab), with primary Ab preabsorbed with blocking peptide or without primary Ab in the baboon fetal adrenal gland on day 165 of gestation in untreated and letrozole-treated baboons (n = 2 samples each). Panels C and D show Western immunoblots of MC2R and MRAP in the fetal liver and epididymis on day 165 in an untreated baboon.

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Western Blot, Incubation, Blocking Assay

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal MC2R protein expression assessed by PLA in the DZ/TZ (A and B) and FZ (C and D) on day 165 of gestation in baboons untreated (A and C) and treated with letrozole on days 100–164 (B and D). Panel E shows the means (±SE) of fetal adrenal MC2R protein expression, quantified by PLA and image analysis/Metamorph software, on day 100 (midgestation, n = 4) and day 165 (late gestation, n = 8) in untreated baboons and on day 165 in animals treated with letrozole (n = 8) or letrozole plus estradiol (n = 5). Each red PLA signal represents a single molecule of MC2R protein detected by primary MC2R antibody tagged with a secondary antibody conjugated to fluorescently labeled oligonucleotide. Nuclei are labeled blue. P-450C17 immunostaining with Alexa Fluor 488-conjugated IgG within TZ (A and B) and FZ (C and D) cells is shown in green. Final magnification, ×400 (A–D).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Expressing, Software, Labeling, Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal MRAP protein expression assessed by PLA in the DZ/TZ (A and B) and FZ (C and D) on day 165 of gestation in baboons untreated (A and C) and treated with letrozole (B and D). Panel E shows the means (±SE) of fetal adrenal MRAP quantified by PLA and image analysis/Metamorph software in the same baboons in which MC2R is shown in Figure 3. Nuclei are labeled blue. P-450C17 immunostaining within TZ (A and B) and FZ (C and D) are shown in green. Final magnification, ×400 (A–D). *, Significantly different at P < .03 in late vs midgestation.

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Expressing, Software, Labeling, Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal MC2R-MRAP protein interaction assessed by PLA in the DZ/TZ (A and B) and FZ (C and D) on day 165 of gestation in baboons untreated (A and C) and treated with letrozole (B and D). Panel E shows the means (±SE) of fetal adrenal MC2R-MRAP interaction quantified by PLA and image analysis/Metamorph software in the same baboons in which MC2R is shown in Figure 3. For PLA detection of MC2R-MRAP protein interaction, tissue was incubated with a secondary antirabbit PLUS antibody and a secondary antigoat minus antibody conjugated with oligonucleotide. Nuclei are labeled blue. P-450C17 immunostaining within TZ (A and B) and FZ (C and D) are shown in green. Final magnification, ×400 (A–D). *, Significantly different at P < .05 in letrozole-treated animals vs all other groups (ANOVA and Tukey-Kramer multiple comparison test).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Software, Incubation, Labeling, Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal liver hepatocyte (A and B) and fetal epidydimal stroma (D and E) MC2R-MRAP interaction assessed by PLA on day 165 of gestation in untreated (A and D) and letrozole-treated (B and E) baboons. α-Smooth muscle actin immunostaining within fibromuscular cells of epidydimal ducts appears in green in panels D and E. Means ± SE of fetal liver (C) and fetal epidydimal stroma (F) MC2R-MRAP interaction were quantified by PLA in baboons untreated (n = 3) or treated with letrozole (n = 3). Final magnification, ×400 in each panel.

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: A, Fetal pituitary POMC mRNA expression was quantified by in situ hybridization on days 100 (n = 3) and 165 (n = 7) in untreated baboons and on day 165 in animals treated with letrozole (n = 7) or letrozole plus estradiol (n = 7). B, Fetal (ie, umbilical artery) plasma ACTH levels on days 100 (n = 8) and 165 (n = 18) in untreated baboons and on day 165 in animals treated on days 100–164 with letrozole (n = 14) or letrozole plus estradiol (n = 11). Fetal pituitaries for POMC mRNA assay and blood samples for plasma ACTH assay were obtained from animals of the current study and from a contemporaneous group of baboons from our primate colony. Values indicated by different letter superscripts are different at P < .05 (ANOVA and Newman-Keul's multiple comparison test).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Expressing, In Situ Hybridization

Journal:

Article Title: Extracellular Signal-Regulated Kinase Activates Topoisomerase II? through a Mechanism Independent of Phosphorylation

doi:

Figure Lengend Snippet: Coimmunoprecipitation of endogenous topoisomerase IIα and ERK2 from nuclear extracts. ERK2 was immunoprecipitated from nuclear extracts prepared from nocodazole-treated NIH 3T3 cells; this was followed by immunoblotting to visualize both ERK2 and coimmunoprecipitating topoisomerase (Topo) IIα. Lanes 3 and 4 show increasing amounts of immunoprecipitated ERK2 and coimmunoprecipitated topoisomerase IIα. A small amount of topoisomerase IIα nonspecifically bound to the protein A-(Prot. A)-Sepharose resin (lane 2). Ten percent of the extract volume used for the immunoprecipitations served as a loading control (lane 1).

Article Snippet: For coimmunoprecipitations of endogenous ERK2 and topoisomerase IIα, nuclear extracts from nocodazole-treated NIH 3T3 cells were incubated with 0, 0.2, or 2 μg of anti-ERK2 antibody (C-14; Santa Cruz Biotechnology) for 2 h on ice; this was followed by addition of 20 μl of protein A-Sepharose (Pharmacia) that had been pretreated with BSA at 0.5 mg/ml.

Techniques: Immunoprecipitation, Western Blot

Journal: The FASEB Journal

Article Title: Innate immune agonist, dsRNA, induces apoptosis in ovarian cancer cells and enhances the potency of cytotoxic chemotherapeutics

doi: 10.1096/fj.11-202333

Figure Lengend Snippet: Caspase activation is required for dsRNA-induced cell death. A) OVCAR-3 (O), DOV-13 (D), and ascites-derived ovarian cancer cells (M1, M2) were treated with 50 μg/ml pI:pC for 24 h, and whole-cell lysates were collected. Expression of full-length (35-kDa) and cleaved (19- and 17-kDa) caspase 3 was determined by Western blot. Actin was used as a loading control. One representative experiment is shown. B) CAOV-3 (top) and OVCAR-3 (bottom) cells were treated with 25 μM of pan-caspase, caspase 9-, caspase 8-, or caspase 4-specific inhibitor. After 6 h, cells were treated with 50 μg/ml pI:pC for 48 h. Apoptosis was assessed by Hoechst and propidium iodide staining. Data are means ± sd for 3 independent experiments. *P ≤ 0.05. C) Ovarian cancer cell lines were treated with 50 μg/ml pI:pC for indicated times, and cell lysates were collected. Expression of c-IAP2 was determined by Western blot. β-Tubulin was used as a loading control. D) Ovarian cancer cell lines were treated with 50 μg/ml pI:pC for 10 min, and whole-cell lysates were collected. RIP1K was immunoprecipitated, and RIP1K ubiquitination level was determined via Western blot analysis. One representative experiment is shown. E) DOV-13 and SKOV-3 cells were treated with 100 nM SMAC mimetic for 4 h, followed by treatment with 50 μg/ml pI:pC for 48 h. Apoptosis was assessed by Hoechst and propidium iodide staining. Data are means ± sd for 2 independent experiments.

Article Snippet: IFNα/β receptor chain 2 inhibition CAOV-3 and OVCAR-3 cells were treated with 5 μg/ml anti-IgG or anti-IFN-α/βR2 neutralizing antibody, followed 6 h later by stimulation with 50 μg/ml pI:pC for 24 h. Caspase inhibition Cells were treated with 25 μM pan-caspase inhibitor Z-VAD-FMK, caspase 9 inhibitor Z-LEHD-FMK, caspase 8 inhibitor Z-IETD-FMK, or caspase 4 inhibitor Z-YVAD-FMK (R&D Systems), followed 6 h later by 50 μg/ml pI:pC for 24 h. Second mitochondria-derived activator of caspases (SMAC) mimetic DOV-13 and SKOV-3 cells were treated with 100 nM SMAC mimetic ( 22 ) for 4 h and then treated with 50 μg/ml pI:pC for 48 h. RIP1 kinase immunoprecipitation Cell extracts were prepared in 20 mM HEPES, pH 7.4; 150 mM NaCl; 10 mM β-glycerophosphate; 1.5 mM MgCl 2 ; 10 mM NaF; 2 mM dithiothreitol; 1 mM sodium orthovanadate; 2 mM EGTA; 1 mM PMSF; 0.5% Triton X-100; 1:500 protease inhibitor cocktail (Sigma); and 1 mg/ml of N -ethylmaleimide.

Techniques: Activation Assay, Derivative Assay, Expressing, Western Blot, Control, Staining, Immunoprecipitation, Ubiquitin Proteomics

Journal: Oncology Reports

Article Title: Expression level of Bcl-XL critically affects sensitivity of hepatocellular carcinoma cells to LIGHT-enhanced and interferon-γ-induced apoptosis

doi: 10.3892/or.17.5.1067

Figure Lengend Snippet: Figure 3. Expression levels of caspase-3, caspase-8, caspase-9, Bcl-XL, Bak and Bid in Hep3B cells treated with IFN-Á/LIGHT. (A) Hep3B cells were treated with 100 ng/ml IFN-Á and various concentrations of LIGHT for 24 h or 72 h as indicated. Cell lysates containing 30 μg of protein were subjected to 12% Tris-glycine gel electrophoresis followed by Western blotting with rabbit polyclonal antibodies against caspase-3, caspase-8, caspase-9, Bcl- XL, Bak, Bid, respectively. Cells treated with 100 ng/ml IFN-Á or 100 ng/ml LIGHT alone and untreated cells were used as the controls. ß-actin was used as an internal control for total protein loading. (B) Hep3B cells (2x105) treated with 100 ng/ml sLIGHT and 100 ng/ml of IFN-Á were also incubated with 100 μM of caspase-3 inhibitor Z-DEVD-fmk, caspase-9 inhibitor Z- IEHD-fmk, caspase-8 inhibitor Z-IEHD-fmk, or normal saline for 96 h. Cell viability was then measured. The viability of untreated (without any cytokine and inhibitor) cells was set at 100%, and the relative viability of other samples was calculated accordingly. The experiments were performed in triplicate, and in at least two separated experiments. The bar indicates the standard error. **p<0.01, versus group treated with normal saline.

Article Snippet: A recombinant LIGHT soluble protein that only contained the extracellular region of human LIGHT and caspase inhibitors Z-DEVD-fmk, Z-IETD-fmk and Z-LEHD-fmk was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Expressing, Nucleic Acid Electrophoresis, Western Blot, Control, Incubation, Saline

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane