Journal: Frontiers in Immunology

Article Title: CD4+ T Cells of Myasthenia Gravis Patients Are Characterized by Increased IL-21, IL-4, and IL-17A Productions and Higher Presence of PD-1 and ICOS

doi: 10.3389/fimmu.2020.00809

Figure Lengend Snippet: Cytokine production of CD4 + T cells in myasthenia gravis (MG) subgroups. (A) Measurement of intracellular IL-21, IL-4, IL-17A, IL-10, and IFN-γ in CD4 + T cells of a patient and a healthy control (HC) by flow cytometry after 4 h of stimulation with phorbol 12-myristate 13-acetate and ionomycin in cell culture. (B) The AChR-MG ( n = 54) patients had higher IL-21, IL-4, IL-17A, and IL-10 ( p

Article Snippet: Intracellular Staining Freshly isolated PBMCs were seeded in 48-well plates at a final concentration of 2 × 106 cells/ml in complete RPMI1640 medium supplemented with 2 mM L-glutamine, 100 IU/100 mg/ml penicillin/streptomycin (Sigma), and 10% fetal bovine serum (Gibco) and were stimulated by the cell stimulation cocktail (500X) containing phorbol 12-myristate 13-acetate and ionomycin (eBioscience, ThermoFisher) for 4 h at 37°C.

Techniques: Flow Cytometry, Cell Culture

Journal: Oncotarget

Article Title: Comparative proteomic analysis of cat eye syndrome critical region protein 1- function in tumor-associated macrophages and immune response regulation of glial tumors

doi: 10.18632/oncotarget.26063

Figure Lengend Snippet: Schematic illustration of study design and workflow Human monocytic THP-1 cells were differentiated into macrophages (MQs) by PMA stimulation (100 ng/ml) for 48 hours followed by siRNA transfection (CECR1 targeting versus non-targeted scrambled siSham as transfection control, using two different siRNA and scrambled systems). Glioblastoma cell line U87 cells were co-cultured with THP-1 derived MQs with/without siCECR1 in a separate chamber with a 0.4-micron pore size filter to enable paracrine interaction between the two cell types. After 96 hours of incubation, cell lysates of were harvested and analyzed by nano-LC/MS. Raw data was imported into Scaffold 4 for processing and exported to Excel for further analysis.

Article Snippet: THP-1 macrophage differentiation was induced by stimulating with PMA (P8139, Sigma-Aldrich, Zwijndrecht, the Netherlands) for 48 hours at a concentration of 100 ng/ml.

Techniques: Transfection, Cell Culture, Derivative Assay, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: Data in Brief

Article Title: Whole transcriptome sequence data of 5-FU sensitive and 5-FU resistant tumors generated in a mouse model of de novo carcinogenesis

doi: 10.1016/j.dib.2018.08.209

Figure Lengend Snippet: Generation of 5-FU sensitive and resistant tumors in a mouse model of chemically-induced carcinogenesis. Carcinogenesis was initiated with DMBA, followed by bi-weekly treatments with TPA to promote tumor growth. Isolated RNA from 2 pairs of 5-FU sensitive and 5-FU resistant tumors was used to perform whole-transcriptome sequencing using Illumina 2000 , in order to identify differentially expressed genes between sensitive and resistant tumors.

Article Snippet: Two weeks after DMBA administration, we initiated treatment with 2.5 μg TPA diluted in 200 μl acetone twice a week (Sigma-Aldrich, cat. #P8139) until a mouse was sacrificed.

Techniques: Isolation, Sequencing

Journal: Molecules

Article Title: β-Cyclodextrin Inhibits Monocytic Adhesion to Endothelial Cells through Nitric Oxide-Mediated Depletion of Cell Adhesion Molecules

doi: 10.3390/molecules25163575

Figure Lengend Snippet: β-Cyclodextrin inhibits protein kinase Cε through molecular encapsulation of diacylglycerol. T-495 phosphorylation ( A ) and nitric oxide (NO) concentration ( B ) were measured after bovine aortic endothelial cells (BAECs) were pretreated with or without 100 nM phorbol-12-myristate-13-acetate (PMA) for 30 min and subsequently treated with β-cyclodextrin (β-CD) for 30 min. The data are shown as mean ± SE, n = 3. The p value was obtained through ANOVA followed by Tukey’s test. ( C ). BAECs were pretreated with or without 100 μM diacylglycerol (DAG) 30 min before β-CD treatment. Then the cell lysates were immunoblotted with an antibody against T-495 phosphorylation. The data are shown as mean ± SE, n = 3. The p value was obtained through ANOVA followed by Tukey’s test. ( D ). NO was measures as described in panel B. BAECs were pretreated with or without 100 μM DAG with or without β-CD in the presence of 5–20% fetal bovine serum. The data were plotted as bar graphs (means ± S.E., n = 3). The p value was obtained through ANOVA followed by Tukey’s test. ( E ) Purified PKCε (0.89 μM) was incubated with the indicated concentrations of diacylglycerol (DAG) with or without β-CD at a 1:1 molar ratio. The fluorescence spectra of the mixtures were obtained; the average emission wavelengths (

Article Snippet: The activators used were phorbol 12-myristate 13-acetate (PMA, 100 nmol/L, Enzo Life Sciences Inc., Farmingdale, NY, USA) and glycerol-1,3-dipalmitate (diacylglycerol (DAG), 100 μmol/L, Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Concentration Assay, Purification, Incubation, Fluorescence

Journal: Molecules

Article Title: β-Cyclodextrin Inhibits Monocytic Adhesion to Endothelial Cells through Nitric Oxide-Mediated Depletion of Cell Adhesion Molecules

doi: 10.3390/molecules25163575

Figure Lengend Snippet: Protein kinase Cε mediates phosphorylation of endothelial nitric oxide synthetase at threonine-495 in bovine aortic endothelial cells. ( A ). Bovine aortic endothelial cells (BAECs) were incubated with 400 μg/mL β-cyclodextrin (β-CD) for the time-course experiment. Then the cell lysates were immunoblotted with anti-caveolin and p-caveolin antibodies. The quantified data are shown in the bottom panel (mean ± SE, n = 3). ( B ). BAECs were pretreated with 400 μg/mL β-CD for the indicated times and then incubated with 1 μM A23187 for 20 min. Subsequently, intracellular [Ca 2+ ] was measured by fluorescence spectroscopy (Ex: 490 nm, Em: 525 nm) 30 min after incubation with 5 μM Fluo-8AM (mean ± SE, n = 3). ( C ). BAECs were incubated with 100 nM phorbol-12-myristate-13-acetate (PMA) in the time-course experiment. Cell lysates were immunoblotted with anti-eNOS and various anti-p-T495 eNOS antibodies. The data were quantified using densitometry (bottom panel, mean ± SE, n = 3). * p

Article Snippet: The activators used were phorbol 12-myristate 13-acetate (PMA, 100 nmol/L, Enzo Life Sciences Inc., Farmingdale, NY, USA) and glycerol-1,3-dipalmitate (diacylglycerol (DAG), 100 μmol/L, Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Incubation, Fluorescence, Spectroscopy

Journal: Journal of Bone Metabolism

Article Title: Bone Morphogenetic Protein-2 Desensitizes MC3T3-E1 Osteoblastic Cells to Estrogen Through Transcriptional Downregulation of Estrogen Receptor 1

doi: 10.11005/jbm.2013.20.2.83

Figure Lengend Snippet: Bone morphogenetic protein-2 downregulates a reporter activity driven by mouse estrogen receptor1 gene promoter in MC3T3-E1 cells. (A) MC3T3-E1-MERAluc1 cells (clone#1-4) were treated with 1alpha,25-dihydroxy-vitamin D 3 (1,25-[OH] 2 D 3 ; 10 nM), estradiol (E2; 100 nM), 4-hydroxy-tamoxifen (4-OH-Tam; 100 nM), phorbol-12-myristate-13-acetate (PMA; 10 nM), dibutyryl cyclic adenosine monophosphate (dbcAMP; 1 mM), recombinant mouse epidermal growth factor (rmEGF; 100 ng/mL) and recombinant human bone morphogenetic protein-2 (rhBMP-2; 250 ng/mL). After a 48-hour incubation, these cells were harvested and lysed then, luciferase activities in the lysates were measured. Luciferase activities in the vehicle-treated cells were set at 1. (B) MC3T3-E1 cells were transiently transfected with pMERAluc1 (0.5 µg) along with pSV-β-gal (0.3 µg) for normalization of transfection efficiency, incubated for 24 hours and exposed by rhBMP-2 (250 ng/mL) or vehicle. Forty-eight hours later, the cells were harvested and lysed in LCβ lysis buffer. Luciferase activities were measured as described in the Materials and Methods section. The results are indicated as relative values when the normalized luciferase activity of the vehicle-treated cells is set at 1. * P

Article Snippet: Phorbol-12-myristate-13-acetate (PMA) and dibutyryl cyclic adenosine monophosphate (dbcAMP) were purchased from Wako (Osaka, Japan).

Techniques: Activity Assay, Recombinant, Incubation, Luciferase, Transfection, Lysis

Journal: Viruses

Article Title: Mucosal Immune Response to Feline Enteric Coronavirus Infection

doi: 10.3390/v11100906

Figure Lengend Snippet: Infection by feline enteric coronavirus (FECV) did not result in an increase in total IFNγ-producing cells or FCoV-specific IFNγ-producing cells. The total number of IFNγ-secreting cells after phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation (spot forming units, SFU) ( a ) and the number of FCoV-specific IFNγ-secreting cells ( b ) were determined by ELISPOT. Each symbol represents a single cat. Bars show the mean and 95% confidence interval. Linear models were used to compare group means, and p values are shown when ≤0.05. One was added to the number of FCoV-specific IFNγ-secreting cells prior to log transformation.

Article Snippet: IFNγ ELISPOT Capture and detection antibodies from the feline IFNγ Development Module (R & D Systems) were used with MultiScreen-IP 96-well plates (MAIPSWU10; MilliporeSigma) to quantify IFNγ-producing mucosal lymphocytes after stimulation with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL; LC Laboratories, Woburn, MA) and ionomycin (300 ng/mL; LC Laboratories) or with FIPV antigen (60 µg/mL; IVD Technologies, Santa Ana, CA, USA) as previously described [ ].

Techniques: Infection, Enzyme-linked Immunospot, Transformation Assay

Journal: Scientific Reports

Article Title: Quantitative proteomics screen identifies a substrate repertoire of rhomboid protease RHBDL2 in human cells and implicates it in epithelial homeostasis

doi: 10.1038/s41598-017-07556-3

Figure Lengend Snippet: Identification of substrates specific for RHBDL2 ( A ). Strep tagged substrates were co-expressed with HA tagged forms of mouse RHBDL2, the four human rhomboids (R1/R2/R3/R4) and their corresponding inactivated forms where the catalytic serine residue was mutated to an alanine (SA). Twenty four hours after transfection, the medium was replaced by serum-free medium containing 10 µM broad spectrum matrix metalloprotease inhibitor BB94 to exclude shedding by these extracellular proteases. Forty eight hours after transfection, the media and cell lysates were harvested and analysed by immunoblotting. Cells were co-transfected with FLAG tagged prolactin as a secretion control and to confirm recovery of TCA precipitated proteins from the media. ( B ) Twenty four hours after transfection with plasmids encoding Strep tagged substrates, the medium was replaced with serum-free medium containing 1 µM ionomycin, 1 µM ionomycin and 2 mM EGTA, or 1 µM ionomycin and 10 µM BB94. One hour after media replacement the media (upper panels) and cell lysate (lower panels) were harvested as previously described and analysed by immunoblotting. ( C ) Twenty four hours after transfection with plasmids encoding Strep tagged substrates, the medium was replaced with serum-free medium containing 0.5 µM phorbol 12-myristate 13-acetate (PMA), the inactive analogue 4α-phorbol 12,13-didecanoate (4αPDD), or both 0.5 µM PMA and 10 µM BB94. One hour after media replacement the media and cell lysate were harvested as previously described and analysed by immunoblotting. Asterisks indicate samples which required treatment with PNGase F prior to SDS-PAGE to reduce smearing and improve resolution of bands on the gel.

Article Snippet: Phorbol-12-myristate-13-acetate (PMA), 4α-phorbol 12,13-didecanoate (4αPDD) and ionomycin were purchased from Merck Millipore.

Techniques: Transfection, SDS Page

Journal: International Journal of Molecular Sciences

Article Title: Azithromycin and Chloramphenicol Diminish Neutrophil Extracellular Traps (NETs) Release

doi: 10.3390/ijms18122666

Figure Lengend Snippet: Effect of azithromycin and chloramphenicol on NET release. 100 nM phorbol 12-myristate 13-acetate (PMA) was added to stimulate the release of NETs, neutrophils treated with antibiotics without stimulation served as the negative control ( n = 6).

Article Snippet: Positive control with 100 nM phorbol 12-myristate 13-acetate (PMA, Merck Millipore, Burlington, MA, USA) was used, whereas granulocytes with RPMI alone constituted negative control.

Techniques: Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Azithromycin and Chloramphenicol Diminish Neutrophil Extracellular Traps (NETs) Release

doi: 10.3390/ijms18122666

Figure Lengend Snippet: Effect of azithromycin and chloramphenicol on respiratory burst ( n = 6), $$ p ≤ 0.01 vs. unstimulated cells (Un), * p ≤ 0.05 vs. phorbol 12-myristate 13-acetate (PMA).

Article Snippet: Positive control with 100 nM phorbol 12-myristate 13-acetate (PMA, Merck Millipore, Burlington, MA, USA) was used, whereas granulocytes with RPMI alone constituted negative control.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Azithromycin and Chloramphenicol Diminish Neutrophil Extracellular Traps (NETs) Release

doi: 10.3390/ijms18122666

Figure Lengend Snippet: Effect of azithromycin and chloramphenicol on neutrophil degranulation assessed by measuring the granularity degree of neutrophils in the side scatter channel (SSC). 100 nM phorbol 12-myristate 13-acetate (PMA) was used as a positive control of degranulation ( n ≥ 3) (* p ≤ 0.05 vs. unstimulated (Un) cells).

Article Snippet: Positive control with 100 nM phorbol 12-myristate 13-acetate (PMA, Merck Millipore, Burlington, MA, USA) was used, whereas granulocytes with RPMI alone constituted negative control.

Techniques: Positive Control