Journal: Kidney international

Article Title: Beta 2 adrenergic receptor agonists are novel regulators of macrophage activation in diabetic renal and cardiovascular complications

doi: 10.1016/j.kint.2017.02.013

Figure Lengend Snippet: High glucose (HG) and diabetes increase TNF-α production and PKC activity in BM-derived macrophages (A–C). A: Rat BM-derived macrophages were cultured under control glucose (CG, 5.6 mM) or HG (25 mM) for 72 h. TNF-α production was measured in conditioned medium (n=17). B: PKC activity in rat BM-derived macrophages stimulated with HG for 72 h or PMA (100 nM) for 30 min (n=3). C: PKC activity in BM-derived macrophages isolated from control and diabetic mice at 12 weeks of diabetes (n=4, also indicates the number of mice studied individually). Diabetes was induced in 6-week-old male C57Bl6/J mice fasted for 12 h, with intraperitoneal injections of STZ in citrate buffer (90 mg/kg) for 2 consecutive days. D: β2AR agonists decreased LPS-induced TNF-α production in rat BM-derived macrophages. Cells were preincubated with metaproterenol or terbutaline hemisulfate for 1 h and then stimulated with LPS (50 ng/mL) for 6 or 48 h (n=3). E: β2AR agonists decreased diabetes-induced TNF-α production in rat PBMCs isolated from control and diabetic rats at 4 weeks of diabetes. Cells were incubated in RPMI medium with 10% FBS for 1 h, and then treated with 500 nM metaproterenol or terbutaline hemisulfate for an additional 16 h with or without 1 h pretreatment with ICI118551 (100 nM), a selective β2AR blocker (n=9~11, also indicates the number of rats studied individually). * p

Article Snippet: Cells were cultured in RPMI containing 10% fetal bovine serum (FBS) and 20% L929 cell (American Type Culture Collection [ATCC], Manassas, VA) conditioned media as a source of macrophage colony stimulating factor .

Techniques: Activity Assay, Derivative Assay, Cell Culture, Isolation, Mouse Assay, Incubation

Journal: Molecular Neurobiology

Article Title: Metabolomic Analysis Reveals Increased Aerobic Glycolysis and Amino Acid Deficit in a Cellular Model of Amyotrophic Lateral Sclerosis

doi: 10.1007/s12035-015-9165-7

Figure Lengend Snippet: Serum deprivation causes selective cytotoxicity, caspase 3/7 activation, and increased lactate formation in G93ASOD1 cells. a The viability of the NSC-34, WT-NSC, and G93A-NSC cell lines was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay after culture with or without 5 % fetal bovine serum (FBS) for 22 h. Percentages of the MTT conversion after culture with serum (100 %; n = 12) are shown. b Activation of caspase 3/7 of the NSC-34, WT-NSC, and G93A-NSC cell lines cultured without 5 % serum for 17 h. Values are percentages of the NSC-34 activity (100 %; n = 15). c Levels of lactate in the medium (μmol/mg of protein; n = 8) of the NSC-34, WT-NSC, and G93A-NSC cell lines cultured with or without 5 % serum for 22 h. All values are mean ± s.e.m. For each parameter, statistical significance of differences was assessed by one-way ANOVA and Tukey’s post hoc test comparing the levels of the different cell lines with or without 5 % serum (respectively ### p

Article Snippet: Materials Flasks and plates were obtained from Corning Inc. High-glucose D-MEM and fetal bovine serum (FBS) were from Lonza, and high-glucose D-MEM without phenol red, geneticin (G418 sulfate), pyruvate, l -glutamine and penicillin/streptomycin were from GIBCO, Invitrogen.

Techniques: Activation Assay, MTT Assay, Cell Culture, Activity Assay

Journal: PLoS ONE

Article Title: Alpha-tocopherol attenuates the anti-tumor activity of crizotinib against cells transformed by NPM-ALK

doi: 10.1371/journal.pone.0183003

Figure Lengend Snippet: Crizotinib-induced apoptosis in Ba/F3 cells transformed by NPM-ALK. Ba/F3 cells were infected with an empty virus (-) and retrovirus expressing NPM-ALK or TEL-JAK2. (A) Whole cell lysates were immunoblotted with an anti-ALK antibody, anti-Flag antibody, anti-JAK2 antibody, anti-phospho-STAT3 antibody, anti-STAT3 antibody, anti-phospho-STAT5 antibody, anti-STAT5 antibody or anti-β-actin antibody. (B) Transduced Ba/F3 cells were incubated with RPMI containing 10% FBS in the absence of IL-3 for 2 days. Viable cell numbers at 24 hr and 48 hr were counted by the Trypan blue exclusion method. Values are the mean ± SD of three independent experiments. (C-F) Transduced Ba/F3 cells were incubated with RPMI containing 10% FBS in the presence of crizotinib (0.25, 0.5, 1 μM) and MMC (0.1, 1, 10 μg/mL) for 24 hr. (C) Cell viability was measured by the WST assay. Values are the mean ± S.D. of four independent experiments. (D) Cells were fixed, treated with propidium iodide, and subjected to a flow cytometric analysis. The percentages of cells in the sub-G1 phase were graphed. Values are the mean ± S.D. of four independent experiments. (E) To evaluate the apoptotic cell death, the chromatin DNA was isolated from cells and subjected to agarose gel electrophoresis. (F) Whole cell lysates were prepared and the phosphorylation of STAT3 and STAT5 was analyzed by immunoblotting. The relative phosphorylation level of STAT3 and STAT5 is shown in the graphs. Values are given as the mean ± SD of three independent experiments. *** P

Article Snippet: These cells were cultured in RPMI-1640 medium (Nacalai Tesque, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) (BioWest, Nuaillé, France), 100 units/ml penicillin (Nacalai Tesque), 100 μg/ml streptomycin (Nacalai Tesque), 2 ng/mL IL-3 (PEPROTECH), and 5 μg/mL puromycin (InVivoGen).

Techniques: Transformation Assay, Infection, Expressing, Incubation, WST Assay, Flow Cytometry, Isolation, Agarose Gel Electrophoresis

Journal: Nutrition Research and Practice

Article Title: Ethanol extract of Innotus obliquus (Chaga mushroom) induces G1 cell cycle arrest in HT-29 human colon cancer cells

doi: 10.4162/nrp.2015.9.2.111

Figure Lengend Snippet: Effect of EEIO on HT-29 cell growth. HT-29 cells were plated in 24-well plates at a density of 50,000 cells/well in DMEM/F12 supplemented with 10% FBS. One day later, the monolayers were serum-starved with serum-free DMEM/F12 supplemented with 5 mg/L transferrin, 0.1 g/L BSA, and 5 µg/mL selenium for 24 h. After serum starvation, cells were incubated in serum-free medium in the absence or presence of various concentrations of EEIO. Cell numbers were estimated by MTT assay. Each bar represents the mean ± SEM (n = 6). Bars with different letters are significantly different at P

Article Snippet: Materials The reagents used in this study were purchased from the following suppliers: Dulbecco's modified Eagle's medium/Ham's F12 nutrient mixture (DMEM/F12) and selenium from Gibco BRL (Gaithersburg, MD, USA); fetal bovine serum (FBS), trypsin-EDTA, and penicillin/streptomycin from Cambrex Bio Technology (Walkersville, MD, USA); 3-[4,5-dimetjylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT), anti-β-actin, RIA-grade bovine serum albumin (BSA), and transferrin from Sigma-Aldrich Co. (St. Louis, MO, USA); antibodies against cyclin D1 and phospho-Rb (Ser807/811) from Cell Signaling Technology (Beverly, MA, USA); antibodies against p21CIP1/WAF1 (c-19), p27KIP1 , p53, CDK2 (M-2), CDK4 (c-22), E2F-1 (C-20), and Rb (c-15) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Incubation, MTT Assay

Journal: Arthritis Research & Therapy

Article Title: Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair

doi: 10.1186/ar2703

Figure Lengend Snippet: The internalization of chitosan by PMNs. Freshly isolated polymorphonuclear neutrophils (PMNs) were resuspended in RPMI 1640 supplemented with 0.1% decomplemented fetal bovine serum, pre-stained with 1 μg/ml calcein-AM for 30 minutes at 37°C and incubated with 100 μg/1 × 10 6 cells rhodamine B isothiocyanate (RITC)-zymosan for 1.5 hours (a positive control), 15 μg/ml RITC-80% deacetylated (80 M) or RITC-95% deacetylated (95 M) chitosan for 3 hours at 37°C. PMNs were then centrifuged and plated on a slide coated with 100% decomplemented autologous serum and visualized by live confocal microscopy. The index of internalization of chitosan by PMNs was calculated as the percentage of cells that internalized RITC-chitosan. Results are presented as mean ± standard error. This figure represents the results of three independent experiments.

Article Snippet: Ficoll-Paque and dextran used for the isolation of PMN were obtained from Pharmacia (Kirkland, Québec, Canada) and fetal bovine serum (FBS) as well as RPMI 1640 were purchased from Wisent (St-Bruno, Québec, Canada).

Techniques: Isolation, Staining, Incubation, Positive Control, Confocal Microscopy

Journal: Arthritis Research & Therapy

Article Title: Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair

doi: 10.1186/ar2703

Figure Lengend Snippet: The effect of inhibitors on PMN chemotaxis towards chitosan. Freshly isolated polymorphonuclear neutrophils (PMNs) were resuspended in RPMI 1640 supplemented with 0.1% decomplemented fetal bovine serum, pre-stained with 1 μg/ml calcein-AM for 30 minutes at 37°C and incubated with 0.5 μg/ml pertussis toxin for 90 minutes and seeded on a polycarbonate filter above a well containing 50 μg/ml 80% deacetylated (80 M) chitosan. Alternatively, PMNs were incubated with 10 -7 mol/l pyrrolidine for the last 10 minutes of the incubation with calcein-AM. Chemotaxis was performed as described in Figure 1. The percentage inhibition of migration corresponds to the fluorescence of PMNs incubated with the inhibitors that migrated toward 80 M chitosan versus fluorescence of PMN incubated in media that migrated toward 80 M chitosan. This figure represents the results of at least three independent experiments. * P = 0.02 and *** P = 0.0001.

Article Snippet: Ficoll-Paque and dextran used for the isolation of PMN were obtained from Pharmacia (Kirkland, Québec, Canada) and fetal bovine serum (FBS) as well as RPMI 1640 were purchased from Wisent (St-Bruno, Québec, Canada).

Techniques: Chemotaxis Assay, Isolation, Staining, Incubation, Inhibition, Migration, Fluorescence

Journal: Arthritis Research & Therapy

Article Title: Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair

doi: 10.1186/ar2703

Figure Lengend Snippet: Production of superoxide anions by PMN in response to chitosan. Superoxide anion production was determined using the cytochrome c reduction assay. Freshly isolated polymorphonuclear neutrophils (PMNs) resuspended in RPMI 1640 supplemented with 0.1% decomplemented fetal bovine serum were incubated with the indicated concentrations of 80% deacetylated (80 M) or 95% deacetylated (95 M) chitosan for 10 minutes at 37°C. Results are presented as mean ± standard error. The difference from the negative control is statistically significant: * P

Article Snippet: Ficoll-Paque and dextran used for the isolation of PMN were obtained from Pharmacia (Kirkland, Québec, Canada) and fetal bovine serum (FBS) as well as RPMI 1640 were purchased from Wisent (St-Bruno, Québec, Canada).

Techniques: Isolation, Incubation, Negative Control

Journal: Arthritis Research & Therapy

Article Title: Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair

doi: 10.1186/ar2703

Figure Lengend Snippet: Release of myeloperoxidase and lactoferrin by PMNs in response of chitosan. Degranulation was determined using the (a, b) myeloperoxidase (MPO) and (c, d) lactoferrin assay, as described in 'Materials and Methods'. Freshly isolated polymorphonuclear neutrophils (PMNs) resuspended in RPMI 1640 supplemented with 0.1% decomplemented fetal bovine serum were treated with cytochalasin B and further incubated with the indicated concentrations of 80% deacetylated (80 M; panels a and c) or 95% deacetylated (95 M; panels b and d) chitosan for 30 minutes at 37°C. The quantity of MPO released is expressed as '% MPO', which corresponds to the ratio of the amount of MPO released/total amount of cellular MPO. The amount of lactoferrin released is expressed in ng/ml. Results are presented as mean ± standard error. The difference from the negative control is statistically significant: * P

Article Snippet: Ficoll-Paque and dextran used for the isolation of PMN were obtained from Pharmacia (Kirkland, Québec, Canada) and fetal bovine serum (FBS) as well as RPMI 1640 were purchased from Wisent (St-Bruno, Québec, Canada).

Techniques: Isolation, Incubation, Negative Control

Journal: Molecular Medicine

Article Title: Nonproteolytic Properties of Murine Alternatively Spliced Tissue Factor: Implications for Integrin-Mediated Signaling in Murine Models

doi: 10.2119/molmed.2011.00416

Figure Lengend Snippet: Effects of masTF and hasTF in the scratch assay. bEnd.3 cells (A) and HRECs (B) were grown to confluence in six-well plates and serum-starved overnight in DMEM containing 2% FBS. Eight radial scratches per well were introduced (see Materials and Methods).

Article Snippet: Murine endothelial cells (ECs) (bEnd.3 from the American Type Culture Collection [ATCC], Manassas, VA, USA) and murine embryonic endothelial cells [MEECs], provided by MJ Goumans, Leiden University Medical Center [LUMC], Leiden, the Netherlands), primary human retinal endothelial cells (HRECs, Cell Systems) and murine monocytes/ macrophages (J774A.1, ATCC) were grown in filter-cap tissue culture flasks containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/ streptomycin (all from Hyclone/ Thermo Scientific, Rockford, IL, USA).

Techniques: Wound Healing Assay

Journal: The Journal of Biological Chemistry

Article Title: BCR-ABL but Not JAK2 V617F Inhibits Erythropoiesis through the Ras Signal by Inducing p21CIP1/WAF1 *

doi: 10.1074/jbc.M110.118653

Figure Lengend Snippet: Inhibition of Ras-dependent cell proliferation and survival by GATA-1. A , Ba/F3/N-RasE12/G1ERT cells were seeded at a density of 100/μl and cultured in RPMI supplemented with 1% FBS without IL-3 in the presence or absence of 1 μ m 4-HT.

Article Snippet: A murine IL-3-dependent hematopoietic cell line, Ba/F3, was maintained in RPMI (nacalai tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS) (Equitech-Bio, Kerrville, TX) and 0.3 ng/ml rmIL-3.

Techniques: Inhibition, Cell Culture

Journal: International Journal of Nanomedicine

Article Title: Cellular effects of paclitaxel-loaded iron oxide nanoparticles on breast cancer using different 2D and 3D cell culture models

doi: 10.2147/IJN.S187886

Figure Lengend Snippet: Physicochemical properties of Ptx-loaded SPIONs. Notes: ( A ) Hydrodynamic diameter of Ptx-loaded and unloaded particles in water and different cell culture media. ( B ) Dialysis-based release kinetics of SPION-adsorbed Ptx compared with free Ptx. ( C , D ) ζ potential as a function of pH in SPION LA-HSA and SPION LA-HSA-Ptx during ( C ) forward titration and ( D ) backward titration. ( E ) Magnetization curves showing the M(H) data of SPION LA-HSA and SPION LA-HSA-Ptx . ( F ) AC susceptibility spectra of SPION LA-HSA and SPION LA-HSA-Ptx . ( G , H ) Fourier transform infrared spectroscopy spectra of ( G ) SPION LA-HSA , SPION LA-HSA-Ptx , and ( H ) free Ptx. ( I ) Stability of SPION LA-HSA and SPION LA-HSA-Ptx in human blood. SPIONs coated with lauric acid and aminated human serum albumin (SPION LA-HSA-NH2 ) were used as a positive control for nonstable particles. Negative control = corresponding amount of H 2 O instead of water-based ferrofluid. Representative images were recorded using optical bright-field microscopy. Abbreviations: DMEM, Dulbecco’s Modified Eagle’s Medium; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; H, applied magnetic field; M φ, magnetization; Ptx, paclitaxel; RPMI, Roswell Park Memorial Institute; SPION, superparamagnetic iron oxide nanoparticles; SPION LA-HSA , lauric acid- and human serum albumin-coated SPIONs; SPION LA-HSA-NH2 , lauric acid- and aminated human serum albumin-coated SPIONs; SPION LA-HSA-Ptx , SPION LA-HSA functionalized with Ptx; Xi, real part of the magnetic susceptibility; Xii, imaginary part of the magnetic susceptibility.

Article Snippet: Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were provided by Biochrom (Berlin, Germany).

Techniques: Cell Culture, Titration, Spectroscopy, Positive Control, Negative Control, Microscopy, Modification

Journal: Arthritis Research & Therapy

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

doi: 10.1186/s13075-018-1699-4

Figure Lengend Snippet: Arhalofenate acid activated AMPK downstream targets involved in regulation of mitochondrial function and maintained mitochondrial cristae area. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulation with monosodium urate (MSU) crystals (0.2 mg/mL) for 1 h or 18 h in RPMI containing 1% FBS. Cell lysates prepared from 18-h treatment samples were subjected to Western blot analysis of phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α, expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM), and expression of thioredoxin (TRX)1, TRX2, and thioredoxin-interacting protein (TXNIP) ( a ). Cells from 1-h treatment were then stained with MitoSOX Red and MitoTracker Green and visualized by fluorescence microscopy ( b , magnification 20×). TEM analysis was performed to examine mitochondrial cristae (the folds of inner mitochondrial membrane indicated by white arrows in c ), and the cristae volume density are presented ( d ). Data in a and b are representative of three individual experiments. Data in c are representative of 30 cells examined for each condition. Data in d are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid

Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

Techniques: Western Blot, Expressing, Pyrolysis Gas Chromatography, Staining, Fluorescence, Microscopy, Transmission Electron Microscopy

Journal: Arthritis Research & Therapy

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

doi: 10.1186/s13075-018-1699-4

Figure Lengend Snippet: Arhalofenate acid prevented prolonged accumulation of p62 by promoting autophagy flux in response to MSU crystals. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 2 h in the presence or absence of bafilomycin (Baf; 100 nM) and for 6 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis of LC3 and p62 ( a ). TEM was performed to examine autophagosomes (indicated by black arrows in b ), and the numbers of autophagosomes containing electron dense material per μm 2 are presented ( c ). Data in a and b are representative of three individual experiments. Data in c are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid

Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

Techniques: Western Blot, Transmission Electron Microscopy

Journal: Arthritis Research & Therapy

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

doi: 10.1186/s13075-018-1699-4

Figure Lengend Snippet: Arhalofenate acid attenuates MSU crystal-induced IL-1β release by inhibiting NLRP3 inflammasome activation in BMDMs in vitro. BMDMs were pretreated with arhalofenate acid at a concentration of 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) in RPMI containing 1% FBS for 18 h. The conditioned media was used for ELISA for interleukin (IL)-1β ( a ), and the cell lysates were subjected to Western blot analysis ( b ) for expression of NLRP3, pro-caspase 1, and cleaved caspase 1 (p10). Data in a are the mean ± SD of three individual experiments, and p values represent comparisons between none and MSU crystals alone, or between MSU crystal alone and MSU crystals plus arhalofenate acid. Data in b are representative of three individual experiments

Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

Techniques: Activation Assay, In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Arthritis Research & Therapy

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

doi: 10.1186/s13075-018-1699-4

Figure Lengend Snippet: Arhalofenate acid inhibited MSU crystal-induced IL-1β via AMPK in BMDMs in vitro. BMDMs were treated with arhalofenate acid at 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 18 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α ( a ). The conditioned medium was used for ELISA analysis of interleukin (IL)-1β release ( b ). Data in a are representative of three individual experiments. Data in b are the mean ± SD of three individual experiments. The p values in b represent comparisons between none and MSU crystals alone in the presence or absence of arhalofenate acid in either wild-type (WT) or AMPKα1 knockout (KO) BMDMs. ns not significant

Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

Techniques: In Vitro, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Arthritis Research & Therapy

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

doi: 10.1186/s13075-018-1699-4

Figure Lengend Snippet: Arhalofenate acid induced phosphorylation of AMPKα and expression of SIRT1 in BMDMs in vitro. BMDMs prepared from wild-type (WT) and AMPKα1 knockout (KO) mice were pretreated with arhalofenate acid at the concentrations indicated for 18 h ( a ) or at 100 μM for 1 h ( b ) in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α and sirtuin 1 (SIRT1). Data in both a and b are representative of three individual experiments

Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

Techniques: Expressing, In Vitro, Knock-Out, Mouse Assay, Western Blot

Journal: Neurochemical research

Article Title: Cocaine Induces Alterations in Mitochondrial Membrane Potential and Dual Cell Cycle Arrest in Rat C6 Astroglioma Cells

doi: 10.1007/s11064-009-0053-2

Figure Lengend Snippet: Effect of cocaine on cell cycle after 24 h. The glial cells with starting density of 1.3 × 10 6 per dish in complete RPMI 1640 medium containing 10% FBS were treated with 0, 3, 4 and 5 mM of cocaine for 24 h. Cells were harvested and stained by propidium iodide staining solution for 1 h in dark and analyzed by flow cytometry. Data were represented as mean ± SEM ( n = 5, * P

Article Snippet: RPMI 1640 (modified), fetal bovine serum (FBS), penicillin/streptomycin sulfate, amphotericin B, phosphate-buffered saline (PBS) and l -glutamine were purchased from Media Tech (Herndon, VA, US).

Techniques: Staining, Flow Cytometry, Cytometry

Journal: Neurochemical research

Article Title: Cocaine Induces Alterations in Mitochondrial Membrane Potential and Dual Cell Cycle Arrest in Rat C6 Astroglioma Cells

doi: 10.1007/s11064-009-0053-2

Figure Lengend Snippet: Population doubling period of rat glial cells. Cells with starting density of 5 × 10 3 per well in 96-well plates ( n = 4, and each value is the average absorbance of eight wells, so final n at each time point = 32) with complete RPMI 1640 medium containing 10% FBS were incubated for designated time points. The cells were stained with crystal violet and the absorbance at 540 nm was read in a plate reader. The absorbance values at any time point were so close to each other that error bars were not seen on the plot

Article Snippet: RPMI 1640 (modified), fetal bovine serum (FBS), penicillin/streptomycin sulfate, amphotericin B, phosphate-buffered saline (PBS) and l -glutamine were purchased from Media Tech (Herndon, VA, US).

Techniques: Incubation, Staining

Journal: Neurochemical research

Article Title: Cocaine Induces Alterations in Mitochondrial Membrane Potential and Dual Cell Cycle Arrest in Rat C6 Astroglioma Cells

doi: 10.1007/s11064-009-0053-2

Figure Lengend Snippet: Effect of cocaine on glial cell viability. The cells (2 × 10 4 per well) were seeded in 96 well plates with complete RPMI 1640 medium containing 10% FBS and were treated with various concentrations of cocaine for 24 or 48 h. Data were represented as mean ± SEM ( n = 36, * P

Article Snippet: RPMI 1640 (modified), fetal bovine serum (FBS), penicillin/streptomycin sulfate, amphotericin B, phosphate-buffered saline (PBS) and l -glutamine were purchased from Media Tech (Herndon, VA, US).

Techniques:

Journal: Neurochemical research

Article Title: Cocaine Induces Alterations in Mitochondrial Membrane Potential and Dual Cell Cycle Arrest in Rat C6 Astroglioma Cells

doi: 10.1007/s11064-009-0053-2

Figure Lengend Snippet: Effect of cocaine on glial cell cycle after 48 h. The glial cells with starting density of 0.7 × 10 6 per dish in complete RPMI 1640 medium containing 10% FBS were treated with 0, 3, 4 and 5 mM of cocaine for 48 h. Cells were harvested and stained by propidium iodide staining solution for 1 h in dark and analyzed by flow cytometry. Data were represented as mean ± SEM ( n = 9, * P

Article Snippet: RPMI 1640 (modified), fetal bovine serum (FBS), penicillin/streptomycin sulfate, amphotericin B, phosphate-buffered saline (PBS) and l -glutamine were purchased from Media Tech (Herndon, VA, US).

Techniques: Staining, Flow Cytometry, Cytometry

Journal: Neurochemical research

Article Title: Cocaine Induces Alterations in Mitochondrial Membrane Potential and Dual Cell Cycle Arrest in Rat C6 Astroglioma Cells

doi: 10.1007/s11064-009-0053-2

Figure Lengend Snippet: Effect of cocaine on mitochondrial membrane potential. The glial cells (2 × 10 4 per well) were seeded in 96 well plates with complete RPMI 1640 media containing 10% FBS and were treated with various concentrations of cocaine (2–6 mM) for 24 h. Membrane potential was evaluated fluorometrically as a measure of rhodamine- 123 uptake. Data were represented as mean ± SEM ( n = 12, * P

Article Snippet: RPMI 1640 (modified), fetal bovine serum (FBS), penicillin/streptomycin sulfate, amphotericin B, phosphate-buffered saline (PBS) and l -glutamine were purchased from Media Tech (Herndon, VA, US).

Techniques:

Journal: PLoS ONE

Article Title: Ligand-activated PPARδ inhibits angiotensin II-stimulated hypertrophy of vascular smooth muscle cells by targeting ROS

doi: 10.1371/journal.pone.0210482

Figure Lengend Snippet: GW501516-activated PPARδ suppresses Ang II-induced ROS generation and [ 3 H]-leucine incorporation in a PI3K/Akt-dependent manner. (A) VSMCs were treated with Ang II for the indicated durations. (B and C) VSMCs pretreated with LY294002 for 30 min (B) or transfected with PPARδ-targeting siRNA for 48 h (C) were incubated in the presence or absence of GW501516 for 24 h and then exposed to Ang II for 24 h. Levels of phosphorylated and total Akt were determined by western blotting. Representative blots from three independent experiments and densitometric measurements are shown. (D and E) VSMCs pretreated with LY294002 for 30 min were incubated in the presence or absence of GW501516 for 24 h and then exposed to Ang II for 24 h. Intracellular ROS were detected by confocal laser fluorescence microscopy using CM-H 2 DCF-DA (D), and the fluorescence intensity was quantified (E). Bars, 200 μm. (F) VSMCs pretreated with LY294002 for 30 min were incubated in the presence or absence of GW501516 for 24 h in DMEM containing 0.1% FBS. Thereafter, cells were exposed to Ang II for 24 h and pulsed with 1 μCi/mL [ 3 H]-leucine for the final 6 h. Data represent means ± SE (n = 3 or 4). ** p

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and WelFect-si transfection reagent were obtained from WelGENE Inc. (Gyeongsan-si, Gyeongsangbuk-do, Republic of Korea).

Techniques: Transfection, Incubation, Western Blot, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Ligand-activated PPARδ inhibits angiotensin II-stimulated hypertrophy of vascular smooth muscle cells by targeting ROS

doi: 10.1371/journal.pone.0210482

Figure Lengend Snippet: NOX is involved in the effects of GW501516 on VSMCs. (A) VSMCs transfected with siRNA against NOX1 or NOX4 for 48 h were pretreated with GW501516 for 24 h in DMEM containing 0.1% FBS. Thereafter, cells were treated with Ang II for 24 h and pulsed with 1 μCi/mL [ 3 H]-leucine for the final 6 h. (B) VSMCs transfected with siRNA against NOX1 or NOX4 for 48 h were incubated in the presence or absence of GW501516 for 24 h and then exposed to Ang II for 24 h. Intracellular ROS were detected by confocal laser fluorescence microscopy using the peroxide-sensitive dye CM-H 2 DCF-DA (10 μM, B), and the fluorescence intensity was quantified (C). Data represent means ± SE (n = 5). Bars, 200 μm. ** p

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and WelFect-si transfection reagent were obtained from WelGENE Inc. (Gyeongsan-si, Gyeongsangbuk-do, Republic of Korea).

Techniques: Transfection, Incubation, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Ligand-activated PPARδ inhibits angiotensin II-stimulated hypertrophy of vascular smooth muscle cells by targeting ROS

doi: 10.1371/journal.pone.0210482

Figure Lengend Snippet: GW501516-activated PPARδ inhibits Ang II-induced incorporation of [ 3 H]-leucine in VSMCs. (A) VSMCs were treated with increasing concentrations of Ang II for 24 h in DMEM containing 0.1% FBS. (B) VSMCs were pretreated with increasing concentrations of GW501516 for 24 h and then treated with Ang II for 24 h in DMEM containing 0.1% FBS. (C) VSMCs transfected with PPARδ-targeting siRNA for 48 h were pretreated with GW501516 for 24 h in DMEM containing 0.1% FBS and then exposed to Ang II for 24 h. (D) VSMCs pretreated with GW501516 (for 24 h), DPI (for 30 min), or NAC (for 30 min) were treated with Ang II for 24 h in DMEM containing 0.1% FBS and pulsed with 1 μCi/mL [ 3 H]-leucine for the final 6 h. Data represent means ± SE (n = 4). ** p

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and WelFect-si transfection reagent were obtained from WelGENE Inc. (Gyeongsan-si, Gyeongsangbuk-do, Republic of Korea).

Techniques: Transfection

Journal: Oncotarget

Article Title: Tumor cells derived exosomes contain hTERT mRNA and transform nonmalignant fibroblasts into telomerase positive cells

doi: 10.18632/oncotarget.10384

Figure Lengend Snippet: Validation of exosome isolation Exosomes were isolated from Jurkat, K562, MCF7 and HCT116 cells growth medium depleted from FBS exosomes as described in the Methods section following 72 hours of growth. A . Exosomal markers from all cells lines analyzed by Western blotting; B. Capture imaging of Jurkat exosomes obtained by using the NanoSight device; C. amount of these exosomes obtained specified by the NanoSight device.

Article Snippet: Experimental system Jurkat and K562 cell lines were cultured in RPMI-1640 supplemented with 20% and 10% fetal bovine serum (FBS), respectively, containing 100 units/ml L-glutamine and 1% penicillin/streptomycin (Biological Industries Beit Haemek, Israel).

Techniques: Isolation, Western Blot, Imaging

Journal: BioMed Research International

Article Title: Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages

doi: 10.1155/2014/901617

Figure Lengend Snippet: Stability of natural and modified siRNAs in 100% fetal bovine serum at 37°C. After incubation, siRNAs were analyzed by PAGE and ethidium bromide staining. Gel images were captured by ChemiDoc XRS (Bio-Rad) and RNA bands were quantified by Image Lab software (Bio-Rad). Signal intensity values at t 0 were set at 1.

Article Snippet: Therefore, the nuclease resistance of 2–19 was investigated through incubation in 100% fetal bovine serum (FBS) at 37°C using unmodified siRNA 1 as control ( ).

Techniques: Modification, Incubation, Polyacrylamide Gel Electrophoresis, Staining, Software

Journal: Applied and Environmental Microbiology

Article Title: Corexit 9500 Inactivates Two Enveloped Viruses of Aquatic Animals but Enhances the Infectivity of a Nonenveloped Fish Virus

doi: 10.1128/AEM.03569-13

Figure Lengend Snippet: Cytotoxic effect of Corexit 9500 on EPC and CHSE-214. EPC (a) or CHSE-214 (b) cells were exposed to 10-fold serial TCID 50 dilutions, up to 10 −6 , of Corexit 9500 (10%, initial concentration) in L15 with 2% FBS before cell viability was determined

Article Snippet: Both cell lines were grown in 75-cm2 flasks (BD Biosciences, Fisher Scientific) using Leibovitz's L15 medium (HyClone; Fisher Scientific, Mississauga, ON, Canada) supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories, VWR International, Mississauga, ON, Canada) and 1% penicillin-streptomycin (PS) (HyClone; Fisher Scientific, Mississauga, ON, Canada).

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Epithelial-Mesenchymal Transition Stimulates Human Cancer Cells to Extend Microtubule-based Invasive Protrusions and Suppresses Cell Growth in Collagen Gel

doi: 10.1371/journal.pone.0053209

Figure Lengend Snippet: Migration activity of EMT-induced cancer cells. (A and B) To measure cell migration activity, A549 (left panels) and Panc-1 (right panels) cells were subjected to the in vitro wound healing assay in the presence or absence (Control) of TGF-ß, as described in the text. Phase-contrast micrographs of the cultures were taken after 16-h incubation. Black broken lines indicate initial wound edges. Scale bar, 50 µm. B) Cell migration area was estimated by analyzing the pixel with Image J. Each bar indicates the mean ± SD for the areas of migrated cells in 3 different fields (right panels). (C) Cells that had been pretreated without or with TGF-ß for 24 h were incubated in 1% FBS-containing medium without or with TGF-ß onto a 24-well plate for 6 h at 37°C. The cell migration was monitored with a time-lapse video system for 12 h. Cell migration distance was measured for 15 randomly selected cells. Each bar indicates the mean ± SD for cell migration velocity of 15 cells. Other experimental conditions are described in Materials and Methods .

Article Snippet: These cell lines were cultured in DMEM/F12 medium (Invitrogen, Carlsbed, CA) supplemented with 10% fetal bovine serum (FBS) (Nichirei Biosciences, Tokyo) at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

Techniques: Migration, Activity Assay, In Vitro, Wound Healing Assay, Incubation

Journal: PLoS ONE

Article Title: Constitutive Expression of Insulin Receptor Substrate (IRS)-1 Inhibits Myogenic Differentiation through Nuclear Exclusion of Foxo1 in L6 Myoblasts

doi: 10.1371/journal.pone.0025655

Figure Lengend Snippet: Foxo1 localization in L6 and satellite cells. A: L6-mock or L6-mIRS1 was incubated in DMEM containing 2% FBS for 18 h and 6 days. Cells were immunostained with anti-Foxo1 antibody. Foxo1 localization is shown. B: Satellite cells were separated from the rat soleus muscle and incubated in DMEM containing 20% FBS. Plasmid expressing myc-tagged Foxo1 H215R mutant was transfected into satellite cells. One day after transfection, muscle differentiation was induced by changing medium to 2% FBS. One day after induction of differentiation, cells were fixed, permeabilized and immunostained with anti-myc antibody. DAPI staining was shown in blue. Myc staining (Foxo1) is shown in green.

Article Snippet: Materials Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Nissui Pharmaceutical CO., LTD. (Ibaraki, Japan).

Techniques: Incubation, Plasmid Preparation, Expressing, Mutagenesis, Transfection, Staining

Journal: PLoS ONE

Article Title: Constitutive Expression of Insulin Receptor Substrate (IRS)-1 Inhibits Myogenic Differentiation through Nuclear Exclusion of Foxo1 in L6 Myoblasts

doi: 10.1371/journal.pone.0025655

Figure Lengend Snippet: Effects of IRS-1 constitutive expression on cell growth. A: [Methyl- 3 H] thymidine incorporation into DNA was measured during the last 4 h of IGF-I treatment time. The mean ± SEM of three replicate dishes is shown. B: 3×10 3 cells of L6-GFP control or L6-mIRS1 cells were inoculated in 35 mm dishes. Cells were grown in DMEM containing 10% FBS and cell number was counted in each day. *, difference between L6-GFP control cells and L6-mIRS1 cells is significant with p

Article Snippet: Materials Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Nissui Pharmaceutical CO., LTD. (Ibaraki, Japan).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Constitutive Expression of Insulin Receptor Substrate (IRS)-1 Inhibits Myogenic Differentiation through Nuclear Exclusion of Foxo1 in L6 Myoblasts

doi: 10.1371/journal.pone.0025655

Figure Lengend Snippet: Effects of Foxo1 (Δ256) expression on myogenic differentiation. A: Differentiation of L6 myoblast cells were induced by changing medium from 10% FBS-DMEM to 2% FBS-DMEM. During induction of differentiation, various concentrations of SB216763 (a specific inhibitor to GSK3) or LiCl were added to the medium. Cells were harvested at the indicated day after induction of differentiation. Immunoblotting analyses were carried out using indicated antibodies ( IB ). These are representative immunoblots independently performed three times. B: Differentiation of L6 myoblast cells stably expressing dominant interfering form of Foxo1 (L6- Δ 256Foxo1) and cells infected with mock retrovirus vector (L6-mock) were induced by exchanging medium containing 2% FBS from 10% FBS. Cells were lysed on the indicated day (0, 1, 2, 4 or 6: days after induction of differentiation). Ten µg of total cell lysates were separated by SDS-PAGE, and subjected to immunoblotting analyses with indicated antibodies ( IB ). These are representative immunoblots independently performed three times. C: At 6 days after induction of differentiation, cell morphology was shown.

Article Snippet: Materials Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Nissui Pharmaceutical CO., LTD. (Ibaraki, Japan).

Techniques: Expressing, Western Blot, Stable Transfection, Infection, Plasmid Preparation, SDS Page

Journal: PLoS ONE

Article Title: Constitutive Expression of Insulin Receptor Substrate (IRS)-1 Inhibits Myogenic Differentiation through Nuclear Exclusion of Foxo1 in L6 Myoblasts

doi: 10.1371/journal.pone.0025655

Figure Lengend Snippet: Effects of IRS-1 constitutive expression on myogenic differentiation in L6 myoblasts. A: Differentiation of L6 myoblasts stably expressing GFP (L6-GFP) or myc-IRS1 (L6-mIRS1) was induced by changing medium from 10% FBS-DMEM to 2% FBS-DMEM. At 0 or 6 days after induction of differentiation, cell morphology was shown. B: Differentiation of L6-GFP or L6-mIRS1 cells was induced. Cells were fixed on 6 days after induction of differentiation and stained with DAPI (blue) or phalloidine (red). C: Differentiation of L6-GFP cells or L6-mIRS1 cells was induced. Cells were lysed on the indicated day (0, 1, 2, 3, 4 or 6: days after induction of differentiation). Ten µg of total cell lysates was separated by SDS-PAGE, and subjected to immunoblotting analyses with indicated antibodies ( IB ). These are representative immunoblots independently performed three times.

Article Snippet: Materials Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Nissui Pharmaceutical CO., LTD. (Ibaraki, Japan).

Techniques: Expressing, Stable Transfection, Staining, SDS Page, Western Blot

Journal: Redox Report

Article Title: Nuclear receptor subfamily 4, group A, member 1 inhibits extrinsic apoptosis and reduces caspase-8 activity in H2O2-induced human HUC-F2 fibroblasts

doi: 10.1179/1351000214Y.0000000109

Figure Lengend Snippet: Effect of si-NR4A1 on mRNA expression and cell growth. (A) HUC-F2 cells were cultured with 10 nM siRNA and 0.2% Lipofectamine 2000 in α-MEM containing 10% fetal bovine serum and 150 IU/ml to 100 μg/ml penicillin to streptomycin medium for 24, 48, and 72 hours in 5% CO 2 . Real-time PCR analysis showed that NR4A1 expression is reduced by 70.4, 87.2, and 83.7%, respectively. (B) HUC-F2 cells were transfected with si-NR4A1 for 48 hours and treated with 200μM H 2 O 2 for 2–8 hours. Expression levels of NR4A1 and NR4A3 were determined using real-time PCR. Normalization of data was achieved by quantitating the cycle time at an arbitrary fluorescence intensity in the linear exponential phase using StepOnePlus Real-Time system software by calculating the ratio of the concentration of each enzyme relative to that of β-actin and GAPDH cDNA, respectively. (C) Effects of the H 2 O 2 concentration on the HUC-F2 cells treated with si-NR4A1 were determined with the MTT assay. Specifically, HUC-F2 cells were transfected with si-NR4A1 or si-NC, incubated in 5% CO 2 for 48 hours, and treated with H 2 O 2 . After 24 hours, cell viability was assessed with the MTT assay. Results are presented as mean ± standard deviation of three samples in each treatment group. The differences between the groups were analyzed by ANOVA with Fisher's PLSD post hoc method. * P

Article Snippet: Cells were cultured in Alpha-Modified Eagle's Medium (α-MEM) (Cellgro, Manassas, VA, USA) containing 10% foetal bovine serum (FBS) (MP Biomedicals Inc., Solon, OH, USA), 150 IU/ml penicillin (Meiji Seika Kaisha, Tokyo, Japan), and 100 mg/ml streptomycin (Meiji Seika Kaisha) in humidified air at 37°C with 5% CO2 .

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Fluorescence, Software, Concentration Assay, MTT Assay, Incubation, Standard Deviation