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: Molecular Medicine Reports

Article Title: CD8+ T cells with high TGF-β1 expression cause lymph node fibrosis following HIV infection

doi: 10.3892/mmr.2018.8964

Figure Lengend Snippet: CD8 + T cells with high TGF-β1 expression induced collagen I secretion from human lymphatic fibroblasts. (A) CD8 + T cells from LNs of non-HIV infected subjects were stimulated for 6 h with 50 µg/ml phorbol-12-myristate 13-acetate and the proportion of TGF-β1-highly expressing CD8 + T cells was determined. (B) Detection of type I collagen expression by western blot analysis in different groups. (C) Relative expression levels of collagen I based on western blot analysis. The data indicate the mean ± standard deviation (n=3). (D) Immunofluorescence staining of type I collagen secreted by human lymphatic fibroblasts in each group. Green (fluorescein isothiocyanate) indicates the type I collagen-positive cells. Magnification, ×400. *P

Article Snippet: Lymphocytes were stimulated with phorbol-12-myristate 13-acetate (PMA; cat. no. ab120297, Abcam) at 50 µg/ml at 37°C for 6 h. The above two LNs were fixed and embedded as described in the LN processing section.

Techniques: Expressing, Infection, Western Blot, Standard Deviation, Immunofluorescence, Staining

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: 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: Pflugers Archiv : European journal of physiology

Article Title: Extracellular Cl− regulates human SO42−/anion exchanger SLC26A1 by altering pH-sensitivity of anion transport

doi: 10.1007/s00424-016-1823-8

Figure Lengend Snippet: Inhibition of SLC26A1 by phorbol 12-myristate 13-acetate (PMA)

Article Snippet: Phorbol-12-myristate-13-acetate (PMA) was from LC Laboratories (Woburn, MA).

Techniques: Inhibition