fatal bovine serum fbs Search Results


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  • fbs  (ATCC)
    99
    ATCC fbs
    LPA-induced FOXM1 in other EOC cell lines and FOXM1C was the dominant form in <t>CAOV3</t> cells A. LPA (10 μM and 6 hr) induced FOXM1 up-regulation in CAOV3 and OVCAR5 cells. CAOV3 and OVCAR5 cells expressed the FOXM1B with apparent MW of 80 KDa (Sigma, Cat. Log # AV39518). B. OVCA433 cells did not express FOXM1C, while CAOV3 cells expressed the FOXM1C with apparent MW of 100 KDa (Cell Signaling, Cat. Log # D12D5). C. CAOV3-FOXM1-KD cell line was established using shRNA against FOXM1 (detailed in Methods). The parental or control-shRNA transfected CAOV3 cells responded to LPA (10 μM, 6 hr) for FOXM1 up-regulation, which was blocked by KD-FOXM1. D. The dominant splicing form of FOXM1 in CAOV3 was FOXM1C. E. Comparison of the relative mRNA expression levels FOXM1 isoform in three EOC cell lines. F. KD-YAP and PTX inhibited LPA-induced FOXM1 up-regulation in CAOV3 cells. G. KD-FOXM1 inhibited LPA-induced cell migration and invasion. H. KD-FOXM1 inhibited <t>FBS</t> (2%)-induced cell proliferation. * P
    Fbs, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 915 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher heat inactivated fetal bovine serum fbs
    Effect of EPA on EPA on PC3 ( a ) cell migration and ( b ) invasion. Double-chambered cell culture dishes with a transwell insert separating the two chambers were used to evaluate PC3 cell migration and invasion. Cells were seeded in the upper chamber, which was uncoated (migration) or coated (invasion) with Matrigel, while the lower chamber was filled with <t>DMEM</t> containing 10% <t>FBS.</t> Data represent mean + SEM ( n = 3). * P
    Heat Inactivated Fetal Bovine Serum Fbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7928 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher fetal bovine serum fbs
    CRISPR/Cas9 mediated deletion of 191bp region flanking rs7098889 confers significant increase of MSMB expression. (A)Sanger sequencing showed both LNCaP and <t>AGS</t> cells are heterozygous (C/T) at rs7098889 site.(B)CRISPR/Cas9 mediated rs7098889 deletion was created by paired guide RNAs (rs7098889-g1 and -g4) transfection followed by puromycin selection. The deletion was confirmed by PCR amplification with primer pair (rs7098889-For1 and -Rev1) flanking the deleted region, PCR product runs at 178bp on agarose gel with deletion, and at 369bp without deletion. (C) Real time qPCR showed 9.5 folds MSMB over-expression in prostate cancer LNCaP cells with bulk transfection but not the gastric cancer AGS cells. The expression of downstream NCOA4 gene is barely affected. (D) Western blot showed that the MSMB protein product β-MSP is significantly up-regulated in LNCaP cells with deletion, but not in AGS cells. (E) ELISA assay showed that the secreting β-MSP level significantly up-regulated in LNCaP cells with deletion either in the presence (28.0 ng/ml) or absence (2.8ng/ml) of <t>FBS</t> in cell culture.
    Fetal Bovine Serum Fbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 104651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare fbs
    Dcp1a is phosphorylated during early differentiation of 3T3-L1 preadipocytes. (A) The expression profile of endogenous Dcp1a during the early differentiation of 3T3-L1 preadipocytes. Two days after reaching confluency, cultures of 3T3-L1 preadipocytes were induced to differentiate with an induction cocktail for 0, 1, 2, 4, 8, and 16 h. Cell extracts were isolated, and western blot analysis for detection of Dcp1a protein was conducted using 30 µg of each sample. Tubulin served as the protein input control. (B) CIP treatment. Confluent cultures of 3T3-L1 preadipocytes were induced to differentiate for 0–16 h, and cell extracts were isolated and treated with CIP. Western blot analysis was performed as in panel A. (C) Two days after reaching confluency, 3T3-L1 preadipocytes were induced to differentiate with individual components of the induction cocktail <t>(FBS,</t> MIX, DEX, insulin) or with the induction cocktail <t>(FMDI)</t> or non-induced control (NI). After incubating for 2 h, cell extracts were analyzed as in panel A. (D) Two days after reaching confluency, 3T3-L1 preadipocytes were pretreated with either DMSO (vehicle control) or 20 µM U0126 for 30 min before induction and then were induced with FMDI for 0, 1, 4, and 8 h in DMSO or U0126. Cell extracts were isolated for western blot analysis using anti-Dcp1a, anti-phospho-ERK (p-ERK), and anti-total ERK (t-ERK). All experiments were independently repeated three to five times with similar results, one of which is shown here.
    Fbs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 28650 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore fetal bovine serum fbs
    Expression of pancreatic endocrine genes in islet-derived cells cultured with different media. Analysis of gene expression (mRNA) using qRT-PCR was performed on cells isolated from different pigs and representative results are presented. (a) Islet-derived cells were cultured in CMRL media containing 10% <t>FBS</t> (CF) or 20% PS (CP) and in <t>DMEM</t> media containing 10% FBS (DF) or 20% PS (DP) and analyzed for the expression of insulin mRNA. (b–d) Islet-derived cells were cultured in DMEM media containing 10% FBS and 1 g/L (L) or 4.5 g/L (H) glucose and analyzed for the expression of insulin (b), glucagon (c), and somatostatin (d) mRNA. The results are the average fold increase for each indicated mRNA compared with the level of this mRNA in adipose-derived stem cells (ADSC) and normalized for the expression of GAPDH.
    Fetal Bovine Serum Fbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16849 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare fetal bovine serum fbs
    Knockdown of IC53 blocked cell proliferation, migration and adhesion. (A) Stealth siRNA against IC53 eliminated IC53 protein expression. IC53 expression was analyzed in serum-starved HCT-116 cells. The cells were transfected with stealth siRNA against IC53 (siRNA) or a negative control (siRNA negative). The IC53 proteins were detected by using Western blot analysis with an anti-IC53 monoclonal antibody (1:1,000). An anti-GAPDH antibody was included in the analysis as a loading control. (B) Knockdown of IC53 inhibited HCT-116 cell proliferation. IC53-mediated cell proliferation was analyzed by using the MTT assay. The cells were maintained in <t>DMEM</t> containing 10% <t>FBS</t> and cultured in an incubator with 5% CO 2 at 37°C. The culture media were replaced every 48 h. HCT-116 cells transfected with stealth siRNA against IC53 showed a 64% decrease in proliferation compared with cells transfected with the negative control. (C) Knockdown of IC53 inhibited HCT-116 cell adhesion. HCT-116 cells were suspended in serum-free medium and seeded into collagen I (20 μg/mL)–coated 24-well plates. After incubating for 60 min at 37°C, the percentage of adhered cells was determined by using the colorimetric crystal violet assay. HCT-116 cells transfection with stealth siRNA against IC53 showed a 74% decrease in cell adhesion compared with cells transfected with the negative control. (D) Knockdown of IC53 inhibited HCT-116 cell migration. IC53-mediated cell migration was analyzed by the transwell assay. The cells were maintained in DMEM containing 10% FBS at 37°C. HCT-116 cells transfected with stealth siRNA against IC53 showed an 85% decrease in migration compared with cells transfected with the negative control. This figure represents 1 of 3 independent experiments, with quadruplicate samples in each experiment. The results represent the means ± SD in triplicate. ** P
    Fetal Bovine Serum Fbs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 17568 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher heat inactivated fbs
    (A) Representative TEM image of IDE-NLCs. (B) Size measurement of IDE-NLCs performed by DLS. (C) ζ-potential measurement of IDE-NLCs. (D) Average hydrodynamic diameter measurement of IDE-NLCs performed by DLS. (E) PDI measurement of IDE-NLCs performed by DLS. Stability measurements were performed in water, PBS, <t>DMEM,</t> and DMEM supplemented with 10% <t>FBS</t> at 37 °C.
    Heat Inactivated Fbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 10705 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Atlanta Biologicals fbs
    l -C14 carnitine does not induce NFκB in TLR 2/1 or 2/6 in transfected HEK-293T cells. HEK-293T cells were cultured in 10% <t>FBS/DMEM</t> medium and cotransfected with TLR2 and TLR1, TLR2 and TLR6 in addition to NF-κB-luciferase reporter and
    Fbs, supplied by Atlanta Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 5766 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Gemini Bio fbs
    p53 Inhibits SREBP-2 Maturation under Low-Sterol Conditions (A) HCT116 cells were cultured for 24 hr in medium plus fetal bovine serum <t>(FBS;</t> gray bars), <t>delipidated</t> FBS (DL-FBS; black bars), or delipidated FBS plus 25-hydroxycholesterol (DL-FBS+25-HC; white bars). Expression of HMGCR , HMGCS1 , and SREBF-2 was assessed by qRT-PCR (n = 3; *p
    Fbs, supplied by Gemini Bio, used in various techniques. Bioz Stars score: 92/100, based on 2611 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dialyzed fbs
    Phosphorylation of Girdin is required for its interaction with 4F2hc. (A) Amino acid sequences of Girdins from different species. Conserved serines at positions 233 (red) and 237 (green) that conform to the MAPK substrate motif are highlighted. (B) Endogenous interaction between MAPK1/2 and Girdin, as detected by co-IP in 293FT cells. (C) In vitro kinase assay showing that MAPK phosphorylates recombinant Girdin NT but not its mutants. (D) Lysates from 293FT cells transfected with the indicated plasmids were separated on Phos-tag gel. Arrowheads indicate multiple bands that may represent phosphorylated Girdin NT. (E) Lysates from 293FT cells stimulated with <t>FBS</t> in the presence or absence of U0126 were subjected to IP to enrich for endogenous Girdin, followed by the enrichment of phosphopeptides using a Titansphere Phos-TiO kit. The phosphopeptides were analyzed by mass spectrometry. The mass spectrum of a tryptic fragment at m/z 843.3431 (mass error, 0.46 ppm) that matched to the peptide 219-DGLHFLPHASSSAQpSPCGpSPGMK-241 containing phosphorylated S233 and S237 is shown. This phosphopeptide was detected with a high peptide confidence (false discovery rate of less than 1%) in cells stimulated with FBS (Mascot score, 28; expectation value, 0.431) but not in cells pretreated with U0126. (F) 293FT cells were transfected with the indicated plasmids, followed by co-IP, showing that mutation of MAPK phosphorylation sites in Girdin disrupts 4F2hc/Girdin interaction. (G) 293FT cells starved for serum were pretreated with DMSO or U0126 for 30 min and stimulated with <t>DMEM</t> containing 10% FBS for 30 min, followed by co-IP (left panel). 293FT cells were transfected with indicated plasmids, followed by co-IP to test endogenous 4F2hc/Girdin interaction (right panel). co-IP, co-immunoprecipitation; CT, carboxyl terminal; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethyl sulphoxide; FBS, fetal bovine serum; Flag-NT, Flag-tagged Girdin NT domain; GFP, green fluorescent protein; Girdin, girders of actin filaments; GST, glutathione S-transferase; IgG, immunoglobulin G; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKK CA, constitutively active mutant of MAPKK; Mr , molecular marker; NT, amino terminal; Rf , relative mobility; S233, serine at position 233; S237, serine at position 237; WT, wild-type; 4F2hc, 4F2 heavy chain.
    Dialyzed Fbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2312 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare heat inactivated fbs
    Protein coronas modulate sulphide formation. Proposed mechanism of protein corona-modulated nano-Ag 2 S formation at Ag <t>NPs,</t> with hard corona proteins trapping Ag + released from the nanoparticle surface and soft corona proteins transporting said ions away from the sulphide-formation centres in the long-lived corona ( a ); TEM images of silver nanocubes after 24 h in RPMI-1640 cell culture medium supplemented with 1% <t>FBS</t> ( b ), followed by 6 days incubation in RPMI-1640 with 0% FBS (inset cartoon showing only hard corona around Ag NPs) ( c ), 1% FBS ( d ) or 10% FBS ( e ; common inset cartoon showing hard and soft coronas, as well as free bulk proteins around Ag NPs); TEM images of silver nanocubes after 7 days incubation in RPMI-1640 with 0.4 mg ml −1 BSA ( f ) or 4 mg ml −1 BSA ( g ) (common inset cartoon showing hard corona and free bulk proteins around Ag NPs); Ultraviolet–visible spectra of cubic ( h ) and quasi-spherical ( i ) Ag NPs after 24 h incubation in RPMI-1640 cell culture medium supplemented with 1, 10 or 50% FBS; TEM images of silver nanocubes after 24 h in RPMI-1640 supplemented with 1% FBS ( j ), 10% FBS ( k ) and 50% FBS ( l ). Scale bars are 100 nm ( b , j – l ) or 50 nm ( c – g ).
    Heat Inactivated Fbs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 2732 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Biological Industries Inc fetal bovine serum fbs
    Validation of exosome isolation Exosomes were isolated from Jurkat, <t>K562,</t> MCF7 and HCT116 cells growth medium depleted from <t>FBS</t> 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.
    Fetal Bovine Serum Fbs, supplied by Biological Industries Inc, used in various techniques. Bioz Stars score: 94/100, based on 1561 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biowest SAS fbs
    (A,B) Time of addition assay for analysis of mechanism of CAM and SCM against influenza A/shanghai/37T (2009H1N1) infection. MDCK cells cultured in <t>DMEM</t> supplemented with 10% <t>FBS</t> (Biowest, France), penicillin (100 U/ml), and streptomycin (10 μg/ml) at 37°C/5% CO 2 were infected with influenza A/shanghai/37T (2009H1N1) at 0.1 MOI in DMEM containing 2 μg/mL TPCK-Trypsin. CAM and SCM diluted in DMEM to give a final concentration of 20 μM were added to mixture of virus and cells at 0, 0.5, 1, 2, 4, and 12 h post infection. Inhibition activity of CAM and SCM was measured by CPE reduction assay using CCK-8, according to the manufacturer’s instruction. The data were presented as means ± SE of triplicate assays and the experiment was repeated at least twice.
    Fbs, supplied by Biowest SAS, used in various techniques. Bioz Stars score: 92/100, based on 1540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biochrom fbs
    BCR ablation leads to reduced metabolic flexibility. (A) Pictures show Mito Tracker Red-CMXRos staining alone (upper panels) and overlaid with transmitted light (lower panels) of WT and KO cells (magnification 63×, scale bar = 5 μm). (B) Cells were plated on d0 with or without oligomycin and cell numbers were assessed on d1 and d2 using the CCK8-kit. Values were normalized to the measurement obtained on d1. Statistical significance was determines using the ANOVA test. N = 7; * P = 0.0213 and 0.0341, ** P = 0.0012. (C) Cells were left untreated or incubated with oligomycin overnight. Cells were resupsended in medium containing pyruvate, glutamine, and glucose, and OCR was measured using Seahorse flux technology. One of three independent experiments is shown. a, oligomycin, b, FCCP, c, rotenone + antimycin. (D) Cells were cultured in glucose free <t>RPMI+</t> dialyzed <t>FBS</t> in the presence of 10 mM glucose, 10 mM galactose, or 10 mM GlutaMAX with or without oligomycin. Cell numbers were assessed using the CCK8 kit after overnight incubation. Results are normalized to the values obtained from the untreated sample cultured with glucose. Differences between the indicated pairs of samples were tested for significance using the unpaired t test. N = 3; * P = 0.0242, ** P = 0.0033 and 0.0059, *** P = 0.0005 and 0.0002. KO, BCR-KO Ramos cells; WT, Ramos wild-type cells.
    Fbs, supplied by Biochrom, used in various techniques. Bioz Stars score: 92/100, based on 1846 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    PAA Laboratories fetal bovine serum fbs
    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 <t>L15</t> with 2% <t>FBS</t> before cell viability was determined
    Fetal Bovine Serum Fbs, supplied by PAA Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 1248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    LPA-induced FOXM1 in other EOC cell lines and FOXM1C was the dominant form in CAOV3 cells A. LPA (10 μM and 6 hr) induced FOXM1 up-regulation in CAOV3 and OVCAR5 cells. CAOV3 and OVCAR5 cells expressed the FOXM1B with apparent MW of 80 KDa (Sigma, Cat. Log # AV39518). B. OVCA433 cells did not express FOXM1C, while CAOV3 cells expressed the FOXM1C with apparent MW of 100 KDa (Cell Signaling, Cat. Log # D12D5). C. CAOV3-FOXM1-KD cell line was established using shRNA against FOXM1 (detailed in Methods). The parental or control-shRNA transfected CAOV3 cells responded to LPA (10 μM, 6 hr) for FOXM1 up-regulation, which was blocked by KD-FOXM1. D. The dominant splicing form of FOXM1 in CAOV3 was FOXM1C. E. Comparison of the relative mRNA expression levels FOXM1 isoform in three EOC cell lines. F. KD-YAP and PTX inhibited LPA-induced FOXM1 up-regulation in CAOV3 cells. G. KD-FOXM1 inhibited LPA-induced cell migration and invasion. H. KD-FOXM1 inhibited FBS (2%)-induced cell proliferation. * P

    Journal: Oncotarget

    Article Title: FOXM1 is a downstream target of LPA and YAP oncogenic signaling pathways in high grade serous ovarian cancer

    doi:

    Figure Lengend Snippet: LPA-induced FOXM1 in other EOC cell lines and FOXM1C was the dominant form in CAOV3 cells A. LPA (10 μM and 6 hr) induced FOXM1 up-regulation in CAOV3 and OVCAR5 cells. CAOV3 and OVCAR5 cells expressed the FOXM1B with apparent MW of 80 KDa (Sigma, Cat. Log # AV39518). B. OVCA433 cells did not express FOXM1C, while CAOV3 cells expressed the FOXM1C with apparent MW of 100 KDa (Cell Signaling, Cat. Log # D12D5). C. CAOV3-FOXM1-KD cell line was established using shRNA against FOXM1 (detailed in Methods). The parental or control-shRNA transfected CAOV3 cells responded to LPA (10 μM, 6 hr) for FOXM1 up-regulation, which was blocked by KD-FOXM1. D. The dominant splicing form of FOXM1 in CAOV3 was FOXM1C. E. Comparison of the relative mRNA expression levels FOXM1 isoform in three EOC cell lines. F. KD-YAP and PTX inhibited LPA-induced FOXM1 up-regulation in CAOV3 cells. G. KD-FOXM1 inhibited LPA-induced cell migration and invasion. H. KD-FOXM1 inhibited FBS (2%)-induced cell proliferation. * P

    Article Snippet: CAOV3 were cultured in DMEM with glutamine, 10% FBS (ATCC, Manassas, VA), and 100 μg/mL P/S.

    Techniques: shRNA, Transfection, Expressing, Migration

    Mtb and BCG bacilli cannot spread between BMECs in a monolayer Cells were infected with fluorescent Mtb or BCG bacilli (MOI = 0.1) for 24 h, then washed three times with PBS to remove unbound bacteria. The cells were overlaid with 1% agarose/DMEM with 1% FBS, with or without amikacin (50 μg/ml) and cultured for up to 7 days. Extracellular bacterial replication was observed in amikacin-free infections with Mtb and BCG. However, plaque formation, indicating bacterial spread and cytotoxicity, was only found in Mtb -infected cells in the absence of amikacin. Infections were performed in triplicate and experiments repeated two times.

    Journal: Oncotarget

    Article Title: Lack of intracellular replication of M. tuberculosis and M. bovis BCG caused by delivering bacilli to lysosomes in murine brain microvascular endothelial cells

    doi:

    Figure Lengend Snippet: Mtb and BCG bacilli cannot spread between BMECs in a monolayer Cells were infected with fluorescent Mtb or BCG bacilli (MOI = 0.1) for 24 h, then washed three times with PBS to remove unbound bacteria. The cells were overlaid with 1% agarose/DMEM with 1% FBS, with or without amikacin (50 μg/ml) and cultured for up to 7 days. Extracellular bacterial replication was observed in amikacin-free infections with Mtb and BCG. However, plaque formation, indicating bacterial spread and cytotoxicity, was only found in Mtb -infected cells in the absence of amikacin. Infections were performed in triplicate and experiments repeated two times.

    Article Snippet: Cell culture Mouse BMECs (bEnd.3, ATCC® CRL-2299) and macrophages (J774A.1, ATCC® TIB-67) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (ATCC).

    Techniques: Infection, Cell Culture

    Effect of EPA on EPA on PC3 ( a ) cell migration and ( b ) invasion. Double-chambered cell culture dishes with a transwell insert separating the two chambers were used to evaluate PC3 cell migration and invasion. Cells were seeded in the upper chamber, which was uncoated (migration) or coated (invasion) with Matrigel, while the lower chamber was filled with DMEM containing 10% FBS. Data represent mean + SEM ( n = 3). * P

    Journal: Lipids in Health and Disease

    Article Title: Contribution of Pyk2 pathway and reactive oxygen species (ROS) to the anti-cancer effects of eicosapentaenoic acid (EPA) in PC3 prostate cancer cells

    doi: 10.1186/s12944-019-1122-4

    Figure Lengend Snippet: Effect of EPA on EPA on PC3 ( a ) cell migration and ( b ) invasion. Double-chambered cell culture dishes with a transwell insert separating the two chambers were used to evaluate PC3 cell migration and invasion. Cells were seeded in the upper chamber, which was uncoated (migration) or coated (invasion) with Matrigel, while the lower chamber was filled with DMEM containing 10% FBS. Data represent mean + SEM ( n = 3). * P

    Article Snippet: Cell culture PC3 cells (Riken BRC Cell Bank, Tsukuba, Japan) were grown in DMEM (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Gibco).

    Techniques: Migration, Cell Culture

    CRISPR/Cas9 mediated deletion of 191bp region flanking rs7098889 confers significant increase of MSMB expression. (A)Sanger sequencing showed both LNCaP and AGS cells are heterozygous (C/T) at rs7098889 site.(B)CRISPR/Cas9 mediated rs7098889 deletion was created by paired guide RNAs (rs7098889-g1 and -g4) transfection followed by puromycin selection. The deletion was confirmed by PCR amplification with primer pair (rs7098889-For1 and -Rev1) flanking the deleted region, PCR product runs at 178bp on agarose gel with deletion, and at 369bp without deletion. (C) Real time qPCR showed 9.5 folds MSMB over-expression in prostate cancer LNCaP cells with bulk transfection but not the gastric cancer AGS cells. The expression of downstream NCOA4 gene is barely affected. (D) Western blot showed that the MSMB protein product β-MSP is significantly up-regulated in LNCaP cells with deletion, but not in AGS cells. (E) ELISA assay showed that the secreting β-MSP level significantly up-regulated in LNCaP cells with deletion either in the presence (28.0 ng/ml) or absence (2.8ng/ml) of FBS in cell culture.

    Journal: bioRxiv

    Article Title: Validation of prostate cancer risk variants by CRISPR/Cas9 mediated genome editing

    doi: 10.1101/337022

    Figure Lengend Snippet: CRISPR/Cas9 mediated deletion of 191bp region flanking rs7098889 confers significant increase of MSMB expression. (A)Sanger sequencing showed both LNCaP and AGS cells are heterozygous (C/T) at rs7098889 site.(B)CRISPR/Cas9 mediated rs7098889 deletion was created by paired guide RNAs (rs7098889-g1 and -g4) transfection followed by puromycin selection. The deletion was confirmed by PCR amplification with primer pair (rs7098889-For1 and -Rev1) flanking the deleted region, PCR product runs at 178bp on agarose gel with deletion, and at 369bp without deletion. (C) Real time qPCR showed 9.5 folds MSMB over-expression in prostate cancer LNCaP cells with bulk transfection but not the gastric cancer AGS cells. The expression of downstream NCOA4 gene is barely affected. (D) Western blot showed that the MSMB protein product β-MSP is significantly up-regulated in LNCaP cells with deletion, but not in AGS cells. (E) ELISA assay showed that the secreting β-MSP level significantly up-regulated in LNCaP cells with deletion either in the presence (28.0 ng/ml) or absence (2.8ng/ml) of FBS in cell culture.

    Article Snippet: The LNCaP cells were cultured in RPMI 1640 medium (Gibco 11875-093, Life Technologies) supplemented with 15% fetal bovine serum (FBS) and the AGS cells were cultured in F12K medium (Gibco 21127-022, Life Technologies) with 10% FBS, both with the presence of 1% penicillin/streptomycin (Gibco 15140-122, Life Technologies).

    Techniques: CRISPR, Expressing, Sequencing, Transfection, Selection, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Over Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture

    CXCR4 surface expression in PANC-1 adherent cells and spheres CXCR4 cell surface expression in PANC-1 adherently growing cells ( A ; 6.4%) and spheres ( B ; 19.4%) using FACS analysis. All results are expressed as means of three independent experiments. ( C ) Transwell migration assay of PANC-1 spheres compared with adherently growing cells. Average percentage of cells (normalized to DMEM serum-free medium as the control) that migrated after 48 h from the upper to the lower chamber is shown. The medium in the lower chamber contained the following chemoattractants: DMEM serum-free medium (as the control, ctrl); complete CSM (CSC); HGF at 200 ng/ml; DMEM serum-free medium supplemented with SDF-1 (200 ng/ml; SDF) and DMEM supplemented with 10% FBS (10% FBS). Statistically significant (Student's t test; * P

    Journal: Bioscience Reports

    Article Title: Pancreatic cancer spheres are more than just aggregates of stem marker-positive cells

    doi: 10.1042/BSR20100018

    Figure Lengend Snippet: CXCR4 surface expression in PANC-1 adherent cells and spheres CXCR4 cell surface expression in PANC-1 adherently growing cells ( A ; 6.4%) and spheres ( B ; 19.4%) using FACS analysis. All results are expressed as means of three independent experiments. ( C ) Transwell migration assay of PANC-1 spheres compared with adherently growing cells. Average percentage of cells (normalized to DMEM serum-free medium as the control) that migrated after 48 h from the upper to the lower chamber is shown. The medium in the lower chamber contained the following chemoattractants: DMEM serum-free medium (as the control, ctrl); complete CSM (CSC); HGF at 200 ng/ml; DMEM serum-free medium supplemented with SDF-1 (200 ng/ml; SDF) and DMEM supplemented with 10% FBS (10% FBS). Statistically significant (Student's t test; * P

    Article Snippet: Subsequently, both cell suspensions were washed twice with Hanks balanced salt solution (Gibco, Invitrogen) containing 2% FBS (FACS buffer) and resuspended in FACS buffer at a density of 100000 cells/100 μl.

    Techniques: Expressing, FACS, Transwell Migration Assay

    Binding studies of Z mab25 or protein G with fetal bovine serum and species selective affinity recovery of two monoclonal antibodies. a 10% FBS was flowed over a Z mab25 ( gray solid trace ) or protein G C2–C3 fragment (SPG) ( black dashed trace ) BIAcore biosensor surface. For comparison, a sample containing 10% FBS spiked with mouse IgG 1 mAb3 was also flowed over the Z mab25 surface ( black solid trace ). b : SDS-PAGE analysis (reducing conditions) of samples, flow-through (FT), and eluate (E) fractions from affinity chromatography experiments using either a protein G (SPG) column or a Z mab25 column, and samples of 10% FBS only ( lanes 1 – 6 ) or 10% FBS with in-spiked mouse IgG 1 mAb3 ( lanes 7 – 12 ) or 10% FBS with in-spiked mouse IgG 1 8F11 ( lanes 13 – 15 ). M LMW-SDS molecular weight marker; lane 1 : 10% FBS sample; lane 2 : SPG column, FT; lane 3 : SPG column, E; lane 4 : 10% FBS sample; lane 5 : Z mab25 column, FT; lane 6 : Z mab25 column, E; lane 7 : 10% FBS + mAb3 sample; lane 8 : SPG column, FT; lane 9 : SPG column, E; lane 10 : 10% FBS + mAb3 sample; lane 11 : Z mab25 column, FT; lane 12 : Z mab25 column, E; lane 13 : 10% FBS + mAb 8F11 sample; lane 14 : Z mab25 column, FT; lane 15 : Z mab25 column, E. The black arrows to the right indicate nominal molecular weights of antibody heavy and light chains, respectively. The numbers to the left indicate molecular weights in kilo Dalton (kDa). c Results from a biosensor analysis of pH neutralized eluates from the SPG and Z mab25 columns, originating from the use of the 10% FBS sample containing in-spiked mAb3 ( 1 – 4 ) or mAb 8F11 ( 5 , 6 ). On separate flow cell surfaces, anti-cow IgG and anti-mouse Ig antibodies were immobilized, and samples were injected. (1) black solid trace SPG column eluate, anti-cow IgG surface; (2) black dashed trace SPG column eluate, anti-mouse Ig surface; (3) gray solid trace Z mab25 column eluate, anti-cow IgG surface; (4) gray dashed trace Z mab25 column eluate, anti-mouse Ig surface; (5) black dotted trace Z mab25 column eluate, anti-cow IgG surface; (6) gray dotted trace Z mab25 column eluate, anti-mouse Ig surface

    Journal: Molecular Biotechnology

    Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies

    doi: 10.1007/s12033-010-9367-1

    Figure Lengend Snippet: Binding studies of Z mab25 or protein G with fetal bovine serum and species selective affinity recovery of two monoclonal antibodies. a 10% FBS was flowed over a Z mab25 ( gray solid trace ) or protein G C2–C3 fragment (SPG) ( black dashed trace ) BIAcore biosensor surface. For comparison, a sample containing 10% FBS spiked with mouse IgG 1 mAb3 was also flowed over the Z mab25 surface ( black solid trace ). b : SDS-PAGE analysis (reducing conditions) of samples, flow-through (FT), and eluate (E) fractions from affinity chromatography experiments using either a protein G (SPG) column or a Z mab25 column, and samples of 10% FBS only ( lanes 1 – 6 ) or 10% FBS with in-spiked mouse IgG 1 mAb3 ( lanes 7 – 12 ) or 10% FBS with in-spiked mouse IgG 1 8F11 ( lanes 13 – 15 ). M LMW-SDS molecular weight marker; lane 1 : 10% FBS sample; lane 2 : SPG column, FT; lane 3 : SPG column, E; lane 4 : 10% FBS sample; lane 5 : Z mab25 column, FT; lane 6 : Z mab25 column, E; lane 7 : 10% FBS + mAb3 sample; lane 8 : SPG column, FT; lane 9 : SPG column, E; lane 10 : 10% FBS + mAb3 sample; lane 11 : Z mab25 column, FT; lane 12 : Z mab25 column, E; lane 13 : 10% FBS + mAb 8F11 sample; lane 14 : Z mab25 column, FT; lane 15 : Z mab25 column, E. The black arrows to the right indicate nominal molecular weights of antibody heavy and light chains, respectively. The numbers to the left indicate molecular weights in kilo Dalton (kDa). c Results from a biosensor analysis of pH neutralized eluates from the SPG and Z mab25 columns, originating from the use of the 10% FBS sample containing in-spiked mAb3 ( 1 – 4 ) or mAb 8F11 ( 5 , 6 ). On separate flow cell surfaces, anti-cow IgG and anti-mouse Ig antibodies were immobilized, and samples were injected. (1) black solid trace SPG column eluate, anti-cow IgG surface; (2) black dashed trace SPG column eluate, anti-mouse Ig surface; (3) gray solid trace Z mab25 column eluate, anti-cow IgG surface; (4) gray dashed trace Z mab25 column eluate, anti-mouse Ig surface; (5) black dotted trace Z mab25 column eluate, anti-cow IgG surface; (6) gray dotted trace Z mab25 column eluate, anti-mouse Ig surface

    Article Snippet: For analysis of Zmab25 or protein G C2–C3 domain interaction with fetal bovine serum (FBS), a third CM5 sensor chip was prepared onto which 488 RU of protein G C2–C3 and 1817 RU of Zmab25 were immobilized on separate surfaces, and 100 μl of 10% FBS (Gibco) or 10% FBS and 133 nM mAb3 were flowed over both surfaces.

    Techniques: Binding Assay, SDS Page, Flow Cytometry, Affinity Chromatography, Molecular Weight, Marker, Injection

    Dcp1a is phosphorylated during early differentiation of 3T3-L1 preadipocytes. (A) The expression profile of endogenous Dcp1a during the early differentiation of 3T3-L1 preadipocytes. Two days after reaching confluency, cultures of 3T3-L1 preadipocytes were induced to differentiate with an induction cocktail for 0, 1, 2, 4, 8, and 16 h. Cell extracts were isolated, and western blot analysis for detection of Dcp1a protein was conducted using 30 µg of each sample. Tubulin served as the protein input control. (B) CIP treatment. Confluent cultures of 3T3-L1 preadipocytes were induced to differentiate for 0–16 h, and cell extracts were isolated and treated with CIP. Western blot analysis was performed as in panel A. (C) Two days after reaching confluency, 3T3-L1 preadipocytes were induced to differentiate with individual components of the induction cocktail (FBS, MIX, DEX, insulin) or with the induction cocktail (FMDI) or non-induced control (NI). After incubating for 2 h, cell extracts were analyzed as in panel A. (D) Two days after reaching confluency, 3T3-L1 preadipocytes were pretreated with either DMSO (vehicle control) or 20 µM U0126 for 30 min before induction and then were induced with FMDI for 0, 1, 4, and 8 h in DMSO or U0126. Cell extracts were isolated for western blot analysis using anti-Dcp1a, anti-phospho-ERK (p-ERK), and anti-total ERK (t-ERK). All experiments were independently repeated three to five times with similar results, one of which is shown here.

    Journal: PLoS ONE

    Article Title: Phosphorylation of mRNA Decapping Protein Dcp1a by the ERK Signaling Pathway during Early Differentiation of 3T3-L1 Preadipocytes

    doi: 10.1371/journal.pone.0061697

    Figure Lengend Snippet: Dcp1a is phosphorylated during early differentiation of 3T3-L1 preadipocytes. (A) The expression profile of endogenous Dcp1a during the early differentiation of 3T3-L1 preadipocytes. Two days after reaching confluency, cultures of 3T3-L1 preadipocytes were induced to differentiate with an induction cocktail for 0, 1, 2, 4, 8, and 16 h. Cell extracts were isolated, and western blot analysis for detection of Dcp1a protein was conducted using 30 µg of each sample. Tubulin served as the protein input control. (B) CIP treatment. Confluent cultures of 3T3-L1 preadipocytes were induced to differentiate for 0–16 h, and cell extracts were isolated and treated with CIP. Western blot analysis was performed as in panel A. (C) Two days after reaching confluency, 3T3-L1 preadipocytes were induced to differentiate with individual components of the induction cocktail (FBS, MIX, DEX, insulin) or with the induction cocktail (FMDI) or non-induced control (NI). After incubating for 2 h, cell extracts were analyzed as in panel A. (D) Two days after reaching confluency, 3T3-L1 preadipocytes were pretreated with either DMSO (vehicle control) or 20 µM U0126 for 30 min before induction and then were induced with FMDI for 0, 1, 4, and 8 h in DMSO or U0126. Cell extracts were isolated for western blot analysis using anti-Dcp1a, anti-phospho-ERK (p-ERK), and anti-total ERK (t-ERK). All experiments were independently repeated three to five times with similar results, one of which is shown here.

    Article Snippet: Two days after reaching confluency, 3T3-L1 cells were stimulated to undergo differentiation into adipocytes by replacing fresh medium with an induction cocktail, FMDI, that was comprised of 10% FBS (HyClone-Characterized), 0.5 mM MIX (Sigma-Aldrich), 5 µM DEX (Sigma-Aldrich), and 1.7 µM bovine insulin.

    Techniques: Expressing, Isolation, Western Blot

    Expression of pancreatic endocrine genes in islet-derived cells cultured with different media. Analysis of gene expression (mRNA) using qRT-PCR was performed on cells isolated from different pigs and representative results are presented. (a) Islet-derived cells were cultured in CMRL media containing 10% FBS (CF) or 20% PS (CP) and in DMEM media containing 10% FBS (DF) or 20% PS (DP) and analyzed for the expression of insulin mRNA. (b–d) Islet-derived cells were cultured in DMEM media containing 10% FBS and 1 g/L (L) or 4.5 g/L (H) glucose and analyzed for the expression of insulin (b), glucagon (c), and somatostatin (d) mRNA. The results are the average fold increase for each indicated mRNA compared with the level of this mRNA in adipose-derived stem cells (ADSC) and normalized for the expression of GAPDH.

    Journal: Journal of Diabetes Research

    Article Title: In Vitro Proliferation of Porcine Pancreatic Islet Cells for β-Cell Therapy Applications

    doi: 10.1155/2016/5807876

    Figure Lengend Snippet: Expression of pancreatic endocrine genes in islet-derived cells cultured with different media. Analysis of gene expression (mRNA) using qRT-PCR was performed on cells isolated from different pigs and representative results are presented. (a) Islet-derived cells were cultured in CMRL media containing 10% FBS (CF) or 20% PS (CP) and in DMEM media containing 10% FBS (DF) or 20% PS (DP) and analyzed for the expression of insulin mRNA. (b–d) Islet-derived cells were cultured in DMEM media containing 10% FBS and 1 g/L (L) or 4.5 g/L (H) glucose and analyzed for the expression of insulin (b), glucagon (c), and somatostatin (d) mRNA. The results are the average fold increase for each indicated mRNA compared with the level of this mRNA in adipose-derived stem cells (ADSC) and normalized for the expression of GAPDH.

    Article Snippet: In this study, two types of basal media were applied, namely, Connaught Medical Research Laboratories Medium (CMRL-1066) (Sigma) and Dulbecco's Modified Eagle Medium (DMEM) (HyClone Laboratories), supplied with 10% fetal bovine serum (FBS) (Sigma) or 20% porcine serum (PS) (Sigma), 1% penicillin-streptomycin (HyClone Laboratories), and 4.0 μ M L-glutamine (as shown in ).

    Techniques: Expressing, Derivative Assay, Cell Culture, Quantitative RT-PCR, Isolation

    Islet-derived cell proliferation in culture. The doubling time (hours) of islet-derived cells was measured in CMRL and DMEM media supplemented with 10% fetal bovine serum (FBS) and 20% porcine serum (PS) at the indicated passages.

    Journal: Journal of Diabetes Research

    Article Title: In Vitro Proliferation of Porcine Pancreatic Islet Cells for β-Cell Therapy Applications

    doi: 10.1155/2016/5807876

    Figure Lengend Snippet: Islet-derived cell proliferation in culture. The doubling time (hours) of islet-derived cells was measured in CMRL and DMEM media supplemented with 10% fetal bovine serum (FBS) and 20% porcine serum (PS) at the indicated passages.

    Article Snippet: In this study, two types of basal media were applied, namely, Connaught Medical Research Laboratories Medium (CMRL-1066) (Sigma) and Dulbecco's Modified Eagle Medium (DMEM) (HyClone Laboratories), supplied with 10% fetal bovine serum (FBS) (Sigma) or 20% porcine serum (PS) (Sigma), 1% penicillin-streptomycin (HyClone Laboratories), and 4.0 μ M L-glutamine (as shown in ).

    Techniques: Derivative Assay

    Human dermal fibroblasts (HDF; 106-05a) obtained from ECACC grown to 80 % confluence using the DMEM medium and supplemented with 10 % ( v / v ) FBS and 1 % ( v / v ) Gibco® GlutaMAX™ prior to treatment with test compounds. ×40 objective

    Journal: Applied Microbiology and Biotechnology

    Article Title: Antimicrobial activity, cytotoxicity and chemical analysis of lemongrass essential oil (Cymbopogon flexuosus) and pure citral

    doi: 10.1007/s00253-016-7807-y

    Figure Lengend Snippet: Human dermal fibroblasts (HDF; 106-05a) obtained from ECACC grown to 80 % confluence using the DMEM medium and supplemented with 10 % ( v / v ) FBS and 1 % ( v / v ) Gibco® GlutaMAX™ prior to treatment with test compounds. ×40 objective

    Article Snippet: HDFs were cultured in DMEM (Sigma, Poole, UK, D6546) supplemented with 10 % (v /v ) FBS (Sigma) and 1 % (v /v ) GIBCO® GlutaMAX™ (ThermoFisher Scientific, Paisley, UK).

    Techniques:

    Micro- and ultrastructure of human retinal pigment epithelial cells cultured with or without sericin. Photomicrographs ( a–f ) show human retinal pigment epithelial cells cultured for 12 days with or without 1% sericin or 10% fetal bovine serum (FBS) using two different basal media. ( a ) DMEM/FBS; ( b ) DMEM/sericin; ( c ) DMEM; ( d ) MEM-α/FBS; ( e ) MEM-α/sericin; ( f ) MEM-α. Cellular pigmentation is indicated by white arrowheads and cobblestone morphology is indicated with black arrowheads. Magnification: 200×. Photomicrographs were captured by a black-and-white camera and are representative of eight samples. Electron microscopy analyses ( g,h ) were performed on human retinal pigment epithelial (hRPE) cells cultured in DMEM with 1% sericin for 12 days. ( g ) Scanning electron microscopy image showing the apical surface of a confluent layer of hexagonal hRPE cells with extensive microvilli (arrow). Image is representative of three samples. ( h ) Transmission electron microscopy image showing a polarized hRPE cell with apical tight junctions (black arrows), a basal nuclei and numerous melanosomes between stage I (non-melanized) and stage IV (melanized). Image is representative of four samples.

    Journal: Scientific Reports

    Article Title: The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

    doi: 10.1038/srep22671

    Figure Lengend Snippet: Micro- and ultrastructure of human retinal pigment epithelial cells cultured with or without sericin. Photomicrographs ( a–f ) show human retinal pigment epithelial cells cultured for 12 days with or without 1% sericin or 10% fetal bovine serum (FBS) using two different basal media. ( a ) DMEM/FBS; ( b ) DMEM/sericin; ( c ) DMEM; ( d ) MEM-α/FBS; ( e ) MEM-α/sericin; ( f ) MEM-α. Cellular pigmentation is indicated by white arrowheads and cobblestone morphology is indicated with black arrowheads. Magnification: 200×. Photomicrographs were captured by a black-and-white camera and are representative of eight samples. Electron microscopy analyses ( g,h ) were performed on human retinal pigment epithelial (hRPE) cells cultured in DMEM with 1% sericin for 12 days. ( g ) Scanning electron microscopy image showing the apical surface of a confluent layer of hexagonal hRPE cells with extensive microvilli (arrow). Image is representative of three samples. ( h ) Transmission electron microscopy image showing a polarized hRPE cell with apical tight junctions (black arrows), a basal nuclei and numerous melanosomes between stage I (non-melanized) and stage IV (melanized). Image is representative of four samples.

    Article Snippet: Dulbecco’s modified eagle’s medium (high glucose, with pyruvate; hereafter named DMEM), Minimal Essential Medium (α-modification; MEM-α), heat-inactivated fetal bovine serum (FBS), N1 growth supplement, taurine, triiodo-thyronine, non-essential amino acids, glutamine-penicillin-streptomycin, hydrocortisone, propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) and 4-methyl-N1-(3-phenylpropyl)-1,2-benzenediamine (JSH-23) were obtained from Sigma Aldrich (St Louis, MO).

    Techniques: Cell Culture, Electron Microscopy, Transmission Assay

    Effect of Sericin on the alternate NF-κB pathway. The effects of sericin, the NF-κB activator inhibitor 4-methyl-N1-(3-phenylpropyl)-1,2-benzenediamine (JSH-23), the NF-κB agonist phorbol-12-myristate-13-acetate (PMA) and fetal bovine serum (FBS) on the protein level of NF-κB p52 in the alternate NF-κB pathway was assessed by western blot on primary human retinal pigment epithelial (hRPE) cells cultured for 12 days. Bar chart shows amount of protein relative to the DMEM group. N = 3. * P = 0.04 compared to DMEM alone.

    Journal: Scientific Reports

    Article Title: The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

    doi: 10.1038/srep22671

    Figure Lengend Snippet: Effect of Sericin on the alternate NF-κB pathway. The effects of sericin, the NF-κB activator inhibitor 4-methyl-N1-(3-phenylpropyl)-1,2-benzenediamine (JSH-23), the NF-κB agonist phorbol-12-myristate-13-acetate (PMA) and fetal bovine serum (FBS) on the protein level of NF-κB p52 in the alternate NF-κB pathway was assessed by western blot on primary human retinal pigment epithelial (hRPE) cells cultured for 12 days. Bar chart shows amount of protein relative to the DMEM group. N = 3. * P = 0.04 compared to DMEM alone.

    Article Snippet: Dulbecco’s modified eagle’s medium (high glucose, with pyruvate; hereafter named DMEM), Minimal Essential Medium (α-modification; MEM-α), heat-inactivated fetal bovine serum (FBS), N1 growth supplement, taurine, triiodo-thyronine, non-essential amino acids, glutamine-penicillin-streptomycin, hydrocortisone, propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) and 4-methyl-N1-(3-phenylpropyl)-1,2-benzenediamine (JSH-23) were obtained from Sigma Aldrich (St Louis, MO).

    Techniques: Western Blot, Cell Culture

    Assessment of CRALBP levels by immunofluorescence. The expression of the cellular retinaldehyde-binding protein (CRALBP) was measured by quantitative immunofluorescence in human retinal pigment epithelial cells cultured for 12 days in DMEM with or without 1% sericin and/or fetal bovine serum (FBS). ( a ) Photomicrographs show that CRALBP expression (red) was lowest in the DMEM/FBS group and highest in the DMEM/sericin group. Cell nuclei were counterstained with DAPI (blue). Magnification: 200×. Photomicrographs are representative of four to eight samples. ( b ) Bar chart showing CRALBP expression in fold change relative to the DMEM group. N = 4 to 8. Error bars: standard deviation. * P

    Journal: Scientific Reports

    Article Title: The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

    doi: 10.1038/srep22671

    Figure Lengend Snippet: Assessment of CRALBP levels by immunofluorescence. The expression of the cellular retinaldehyde-binding protein (CRALBP) was measured by quantitative immunofluorescence in human retinal pigment epithelial cells cultured for 12 days in DMEM with or without 1% sericin and/or fetal bovine serum (FBS). ( a ) Photomicrographs show that CRALBP expression (red) was lowest in the DMEM/FBS group and highest in the DMEM/sericin group. Cell nuclei were counterstained with DAPI (blue). Magnification: 200×. Photomicrographs are representative of four to eight samples. ( b ) Bar chart showing CRALBP expression in fold change relative to the DMEM group. N = 4 to 8. Error bars: standard deviation. * P

    Article Snippet: Dulbecco’s modified eagle’s medium (high glucose, with pyruvate; hereafter named DMEM), Minimal Essential Medium (α-modification; MEM-α), heat-inactivated fetal bovine serum (FBS), N1 growth supplement, taurine, triiodo-thyronine, non-essential amino acids, glutamine-penicillin-streptomycin, hydrocortisone, propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) and 4-methyl-N1-(3-phenylpropyl)-1,2-benzenediamine (JSH-23) were obtained from Sigma Aldrich (St Louis, MO).

    Techniques: Immunofluorescence, Expressing, Binding Assay, Cell Culture, Standard Deviation

    Effect of sericin on cell survival. To assess the effect of sericin on cell survival confluent layers of human retinal pigment epithelial cells were cultured in DMEM with or without sericin or fetal bovine serum (FBS) for 12 days. ( a ) Bar chart showing cell density in fold change relative to the DMEM-group. Cell density was measured by counting DAPI-stained nuclei using ImageJ. N = 4 to 8. Error bars: standard deviation. * P

    Journal: Scientific Reports

    Article Title: The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

    doi: 10.1038/srep22671

    Figure Lengend Snippet: Effect of sericin on cell survival. To assess the effect of sericin on cell survival confluent layers of human retinal pigment epithelial cells were cultured in DMEM with or without sericin or fetal bovine serum (FBS) for 12 days. ( a ) Bar chart showing cell density in fold change relative to the DMEM-group. Cell density was measured by counting DAPI-stained nuclei using ImageJ. N = 4 to 8. Error bars: standard deviation. * P

    Article Snippet: Dulbecco’s modified eagle’s medium (high glucose, with pyruvate; hereafter named DMEM), Minimal Essential Medium (α-modification; MEM-α), heat-inactivated fetal bovine serum (FBS), N1 growth supplement, taurine, triiodo-thyronine, non-essential amino acids, glutamine-penicillin-streptomycin, hydrocortisone, propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) and 4-methyl-N1-(3-phenylpropyl)-1,2-benzenediamine (JSH-23) were obtained from Sigma Aldrich (St Louis, MO).

    Techniques: Cell Culture, Staining, Standard Deviation

    F4/80 + CD11b + Gr1 + (Subset 1) cells accumulated at the mucosal damaged area in early phase of healing. 1 × 10 7 BMC were labeled using CFDA-SE. These cells were injected into AA + 5-FU mice via tail vein. After 24 hours, colon was dissected and incubated in calcium and magnesium-free HBSS containing 2.5% heat-inactivated FBS and 1 mM DTT to remove mucus. After the tissues were treated with collagenase and DNase I for 60 min at 37°C, cells were washed and stained with 7-AAD, F4/80-APC, and C11b-PE or 7-AAD, F4/80-APC, and Gr1-PE, for the flow cytometric analysis. 7-AAD was used for exclusion of dead cells. (a) CFDA-SE positive cells indicated bone marrow-derived cells. (b) CFDA-SE positive population was CD11b + F4/80 + monocytes. (c) F4/80 positive population was almost all Gr1 + cells, indicating the F4/80 + Gr1 + Subset 1 monocytes. (d) The population had typical monocytes morphology.

    Journal: Mediators of Inflammation

    Article Title: Role of Bone Marrow-Derived Monocytes/Macrophages in the Repair of Mucosal Damage Caused by Irradiation and/or Anticancer Drugs in Colitis Model

    doi: 10.1155/2010/634145

    Figure Lengend Snippet: F4/80 + CD11b + Gr1 + (Subset 1) cells accumulated at the mucosal damaged area in early phase of healing. 1 × 10 7 BMC were labeled using CFDA-SE. These cells were injected into AA + 5-FU mice via tail vein. After 24 hours, colon was dissected and incubated in calcium and magnesium-free HBSS containing 2.5% heat-inactivated FBS and 1 mM DTT to remove mucus. After the tissues were treated with collagenase and DNase I for 60 min at 37°C, cells were washed and stained with 7-AAD, F4/80-APC, and C11b-PE or 7-AAD, F4/80-APC, and Gr1-PE, for the flow cytometric analysis. 7-AAD was used for exclusion of dead cells. (a) CFDA-SE positive cells indicated bone marrow-derived cells. (b) CFDA-SE positive population was CD11b + F4/80 + monocytes. (c) F4/80 positive population was almost all Gr1 + cells, indicating the F4/80 + Gr1 + Subset 1 monocytes. (d) The population had typical monocytes morphology.

    Article Snippet: Preparation of Damaged Mucosa and Identification of Injected BMCs Dissected mucosa was incubated in calcium and magnesium-free HBSS (GIBCO) containing 2.5% heat-inactivated FBS and 1 mM DTT (Sigma-Aldrich) to remove mucus.

    Techniques: Labeling, Injection, Mouse Assay, Incubation, Staining, Flow Cytometry, Derivative Assay

    Knockdown of IC53 blocked cell proliferation, migration and adhesion. (A) Stealth siRNA against IC53 eliminated IC53 protein expression. IC53 expression was analyzed in serum-starved HCT-116 cells. The cells were transfected with stealth siRNA against IC53 (siRNA) or a negative control (siRNA negative). The IC53 proteins were detected by using Western blot analysis with an anti-IC53 monoclonal antibody (1:1,000). An anti-GAPDH antibody was included in the analysis as a loading control. (B) Knockdown of IC53 inhibited HCT-116 cell proliferation. IC53-mediated cell proliferation was analyzed by using the MTT assay. The cells were maintained in DMEM containing 10% FBS and cultured in an incubator with 5% CO 2 at 37°C. The culture media were replaced every 48 h. HCT-116 cells transfected with stealth siRNA against IC53 showed a 64% decrease in proliferation compared with cells transfected with the negative control. (C) Knockdown of IC53 inhibited HCT-116 cell adhesion. HCT-116 cells were suspended in serum-free medium and seeded into collagen I (20 μg/mL)–coated 24-well plates. After incubating for 60 min at 37°C, the percentage of adhered cells was determined by using the colorimetric crystal violet assay. HCT-116 cells transfection with stealth siRNA against IC53 showed a 74% decrease in cell adhesion compared with cells transfected with the negative control. (D) Knockdown of IC53 inhibited HCT-116 cell migration. IC53-mediated cell migration was analyzed by the transwell assay. The cells were maintained in DMEM containing 10% FBS at 37°C. HCT-116 cells transfected with stealth siRNA against IC53 showed an 85% decrease in migration compared with cells transfected with the negative control. This figure represents 1 of 3 independent experiments, with quadruplicate samples in each experiment. The results represent the means ± SD in triplicate. ** P

    Journal: Molecular Medicine

    Article Title: A Functional Variant of IC53 Correlates with the Late Onset of Colorectal Cancer

    doi: 10.2119/molmed.2010.00192

    Figure Lengend Snippet: Knockdown of IC53 blocked cell proliferation, migration and adhesion. (A) Stealth siRNA against IC53 eliminated IC53 protein expression. IC53 expression was analyzed in serum-starved HCT-116 cells. The cells were transfected with stealth siRNA against IC53 (siRNA) or a negative control (siRNA negative). The IC53 proteins were detected by using Western blot analysis with an anti-IC53 monoclonal antibody (1:1,000). An anti-GAPDH antibody was included in the analysis as a loading control. (B) Knockdown of IC53 inhibited HCT-116 cell proliferation. IC53-mediated cell proliferation was analyzed by using the MTT assay. The cells were maintained in DMEM containing 10% FBS and cultured in an incubator with 5% CO 2 at 37°C. The culture media were replaced every 48 h. HCT-116 cells transfected with stealth siRNA against IC53 showed a 64% decrease in proliferation compared with cells transfected with the negative control. (C) Knockdown of IC53 inhibited HCT-116 cell adhesion. HCT-116 cells were suspended in serum-free medium and seeded into collagen I (20 μg/mL)–coated 24-well plates. After incubating for 60 min at 37°C, the percentage of adhered cells was determined by using the colorimetric crystal violet assay. HCT-116 cells transfection with stealth siRNA against IC53 showed a 74% decrease in cell adhesion compared with cells transfected with the negative control. (D) Knockdown of IC53 inhibited HCT-116 cell migration. IC53-mediated cell migration was analyzed by the transwell assay. The cells were maintained in DMEM containing 10% FBS at 37°C. HCT-116 cells transfected with stealth siRNA against IC53 showed an 85% decrease in migration compared with cells transfected with the negative control. This figure represents 1 of 3 independent experiments, with quadruplicate samples in each experiment. The results represent the means ± SD in triplicate. ** P

    Article Snippet: The colon cancer adenocarcinoma cell lines HCT-116, HT-29 and mouse embryonic fibroblast cell line NIH3T3 were obtained from the Institute of Cell Biology, Academic Sinica, and propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% penicillin/ streptomycin.

    Techniques: Migration, Expressing, Transfection, Negative Control, Western Blot, MTT Assay, Cell Culture, Crystal Violet Assay, Transwell Assay

    Overexpression of IC53 promoted proliferation, migration and adhesion of HCT-116 cells. (A) Stable transfection of IC53 plasmids increased IC53 expression. IC53 expression was analyzed in serum-starved HCT-116 cells by Western blot analysis with an anti-IC53 monoclonal antibody (1:1,000). The expression level of IC53 in the cells transfected with IC53 plasmids (HCT-116-IC53) was four-fold greater than that of the cells transfected with empty plasmid (HCT-116-A) or the untransfected control (HCT-116). An anti-GAPDH antibody was included in the analysis as a loading control. (B) Overexpression of IC53 induced HCT-116 cell proliferation. Cell proliferation was assayed by using the MTT assay. The cells were maintained in DMEM containing 10% FBS and 200 μg/mL G418 and cultured in 5% CO 2 at 37°C. The media were replaced every 48 h. Stable transfection of IC53 (HCT-116-IC53) markedly promoted HCT-116 cell proliferation 2.1-fold on day 3, 2.6-fold on day 5 and 1.97-fold on day 7 after plating, compared with either the untransfected (HCT-116) control or the empty vector control (HCT-116-A), as determined by the optical density (OD) measurements. (C) Overexpression of IC53 induced HCT-116 cell adhesion. HCT-116 cells were suspended in serum-free medium and seeded into collagen I (20 μg/mL)–coated 24-well plates. After incubation for 60 min at 37°C, the percentage of adhered cells was determined by using the colorimetric crystal violet assay. Cells stably transfected with the IC53 expression construct (HCT-116-IC53) dramatically promoted HCT-116 cell adhesion by 183% after 60 min compared with the untransfected control (HCT-116) or cells transfected with empty vector (HCT-116-A). (D) Overexpression of IC53 induced HCT-116 cell migration. Cell migration was analyzed by using the transwell assay. The cells were maintained in DMEM containing 10% FBS and 200 μg/mL G418 and cultured in 5% CO 2 at 37°C. Cells stably transfected with IC53 expression construct (HCT-116-IC53) migrated 180% or 300% more than their parental (HCT-116) or vector transfected cells (HCT-116-A), respectively. This figure represents 1 of 3 independent experiments, with quadruplicate samples in each experiment. The results represent the mean ± SD in triplicate. ** P

    Journal: Molecular Medicine

    Article Title: A Functional Variant of IC53 Correlates with the Late Onset of Colorectal Cancer

    doi: 10.2119/molmed.2010.00192

    Figure Lengend Snippet: Overexpression of IC53 promoted proliferation, migration and adhesion of HCT-116 cells. (A) Stable transfection of IC53 plasmids increased IC53 expression. IC53 expression was analyzed in serum-starved HCT-116 cells by Western blot analysis with an anti-IC53 monoclonal antibody (1:1,000). The expression level of IC53 in the cells transfected with IC53 plasmids (HCT-116-IC53) was four-fold greater than that of the cells transfected with empty plasmid (HCT-116-A) or the untransfected control (HCT-116). An anti-GAPDH antibody was included in the analysis as a loading control. (B) Overexpression of IC53 induced HCT-116 cell proliferation. Cell proliferation was assayed by using the MTT assay. The cells were maintained in DMEM containing 10% FBS and 200 μg/mL G418 and cultured in 5% CO 2 at 37°C. The media were replaced every 48 h. Stable transfection of IC53 (HCT-116-IC53) markedly promoted HCT-116 cell proliferation 2.1-fold on day 3, 2.6-fold on day 5 and 1.97-fold on day 7 after plating, compared with either the untransfected (HCT-116) control or the empty vector control (HCT-116-A), as determined by the optical density (OD) measurements. (C) Overexpression of IC53 induced HCT-116 cell adhesion. HCT-116 cells were suspended in serum-free medium and seeded into collagen I (20 μg/mL)–coated 24-well plates. After incubation for 60 min at 37°C, the percentage of adhered cells was determined by using the colorimetric crystal violet assay. Cells stably transfected with the IC53 expression construct (HCT-116-IC53) dramatically promoted HCT-116 cell adhesion by 183% after 60 min compared with the untransfected control (HCT-116) or cells transfected with empty vector (HCT-116-A). (D) Overexpression of IC53 induced HCT-116 cell migration. Cell migration was analyzed by using the transwell assay. The cells were maintained in DMEM containing 10% FBS and 200 μg/mL G418 and cultured in 5% CO 2 at 37°C. Cells stably transfected with IC53 expression construct (HCT-116-IC53) migrated 180% or 300% more than their parental (HCT-116) or vector transfected cells (HCT-116-A), respectively. This figure represents 1 of 3 independent experiments, with quadruplicate samples in each experiment. The results represent the mean ± SD in triplicate. ** P

    Article Snippet: The colon cancer adenocarcinoma cell lines HCT-116, HT-29 and mouse embryonic fibroblast cell line NIH3T3 were obtained from the Institute of Cell Biology, Academic Sinica, and propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% penicillin/ streptomycin.

    Techniques: Over Expression, Migration, Stable Transfection, Expressing, Western Blot, Transfection, Plasmid Preparation, MTT Assay, Cell Culture, Incubation, Crystal Violet Assay, Construct, Transwell Assay

    (A) Representative TEM image of IDE-NLCs. (B) Size measurement of IDE-NLCs performed by DLS. (C) ζ-potential measurement of IDE-NLCs. (D) Average hydrodynamic diameter measurement of IDE-NLCs performed by DLS. (E) PDI measurement of IDE-NLCs performed by DLS. Stability measurements were performed in water, PBS, DMEM, and DMEM supplemented with 10% FBS at 37 °C.

    Journal: ACS Omega

    Article Title: Development of Nanostructured Lipid Carriers for the Delivery of Idebenone in Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay

    doi: 10.1021/acsomega.0c01282

    Figure Lengend Snippet: (A) Representative TEM image of IDE-NLCs. (B) Size measurement of IDE-NLCs performed by DLS. (C) ζ-potential measurement of IDE-NLCs. (D) Average hydrodynamic diameter measurement of IDE-NLCs performed by DLS. (E) PDI measurement of IDE-NLCs performed by DLS. Stability measurements were performed in water, PBS, DMEM, and DMEM supplemented with 10% FBS at 37 °C.

    Article Snippet: Cell Cultures Mouse astrocyte cells C8-D1A (ATCC CRL-2541), mouse brain endothelial cells bEnd.3 (ATCC CRL-2299), and human healthy and ARSACS patient’s primary fibroblasts derived from skin punch biopsies were cultured in DMEM high glucose (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS (Gibco), 2 mM l-glutamine (Gibco), 1 mM sodium pyruvate (Gibco), 100 IU/mL of penicillin, and 100 μg/mL of streptomycin (all from Gibco).

    Techniques: Transmission Electron Microscopy

    l -C14 carnitine does not induce NFκB in TLR 2/1 or 2/6 in transfected HEK-293T cells. HEK-293T cells were cultured in 10% FBS/DMEM medium and cotransfected with TLR2 and TLR1, TLR2 and TLR6 in addition to NF-κB-luciferase reporter and

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Acylcarnitines activate proinflammatory signaling pathways

    doi: 10.1152/ajpendo.00656.2013

    Figure Lengend Snippet: l -C14 carnitine does not induce NFκB in TLR 2/1 or 2/6 in transfected HEK-293T cells. HEK-293T cells were cultured in 10% FBS/DMEM medium and cotransfected with TLR2 and TLR1, TLR2 and TLR6 in addition to NF-κB-luciferase reporter and

    Article Snippet: These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) FBS (premium select FBS, cat. no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

    Techniques: Transfection, Cell Culture, Luciferase

    l -C14 carnitine induced reactive oxygen species (ROS) but not mitochondrial ROS in murine monocyte/macrophages. RAW 264.7 cells were serum-starved for 6 h (0.25% FBS/DMEM) then treated with LPS (100 ng/mL), 25 μM l -C14 acylcarnitine ( l -C14 carn),

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Acylcarnitines activate proinflammatory signaling pathways

    doi: 10.1152/ajpendo.00656.2013

    Figure Lengend Snippet: l -C14 carnitine induced reactive oxygen species (ROS) but not mitochondrial ROS in murine monocyte/macrophages. RAW 264.7 cells were serum-starved for 6 h (0.25% FBS/DMEM) then treated with LPS (100 ng/mL), 25 μM l -C14 acylcarnitine ( l -C14 carn),

    Article Snippet: These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) FBS (premium select FBS, cat. no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

    Techniques:

    Induction of COX-2 and proinflammatory cytokines are reduced in MyD88 knock-down RAW 264.7 cells. Stable MyD88 KD cells were generated by lentiviral mediated shRNA in RAW 264.7 cells. Cells were then serum-starved for 4–6 h (0.25% FBS/DMEM) then

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Acylcarnitines activate proinflammatory signaling pathways

    doi: 10.1152/ajpendo.00656.2013

    Figure Lengend Snippet: Induction of COX-2 and proinflammatory cytokines are reduced in MyD88 knock-down RAW 264.7 cells. Stable MyD88 KD cells were generated by lentiviral mediated shRNA in RAW 264.7 cells. Cells were then serum-starved for 4–6 h (0.25% FBS/DMEM) then

    Article Snippet: These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) FBS (premium select FBS, cat. no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

    Techniques: Generated, shRNA

    l -C14 carnitine increases phosphorylation of ERK and JNK in a time-dependent manner: MAP kinases and NF-κB contribution to l -C14 carnitine induced cytokine production. RAW 264.7 cells were serum-starved for 6 h (0.25% FBS/DMEM) then treated with

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Acylcarnitines activate proinflammatory signaling pathways

    doi: 10.1152/ajpendo.00656.2013

    Figure Lengend Snippet: l -C14 carnitine increases phosphorylation of ERK and JNK in a time-dependent manner: MAP kinases and NF-κB contribution to l -C14 carnitine induced cytokine production. RAW 264.7 cells were serum-starved for 6 h (0.25% FBS/DMEM) then treated with

    Article Snippet: These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) FBS (premium select FBS, cat. no. S11595, lot no. K0109; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin.

    Techniques:

    MQ-induced changes in cellular ATP and cytosolic lactate. HT29 cells (1 × 10 4 ) were seeded in 96-well plates and grown overnight at 37°C. The next day, the culture media were replaced by fresh phenol-red and fetal bovine serum-free Dulbecco's modified essential medium containing 1.2, 4.6, or 18.3 m M glucose, and the cells were exposed to 50 or 200 μ M MQ. (A) In time course experiments, cells were treated with MQ for 0–60 min. ATP was measured with a luciferin/luciferase bioluminescence Promega kit according to manufacturer's protocol. Each experiment was conducted in quadruplicates and ATP contents are expressed as percent relative to untreated controls (100%). Results are mean ± SEM for six cell preparations. (B) The contribution of mitochondria-derived ( upper panel ) and glycolysis-derived ( bottom panel ) ATP to total ATP levels was determined in cells treated with antimycin A (2 μ M ) or 2-deoxyglucose (2DG), respectively. Concentrations of 2DG, namely, 3, 11.5, and 45.8 m M were 2.5 × higher than the respective glucose concentrations. Results are mean ± SEM for n = 6 for 18.3 m M glucose, and n = 4 each for 4.6 and 1.2 m M glucose. * p

    Journal: Antioxidants & Redox Signaling

    Article Title: Disruption of Pyridine Nucleotide Redox Status During Oxidative Challenge at Normal and Low-Glucose States: Implications for Cellular Adenosine Triphosphate, Mitochondrial Respiratory Activity, and Reducing Capacity in Colon Epithelial Cells

    doi: 10.1089/ars.2010.3489

    Figure Lengend Snippet: MQ-induced changes in cellular ATP and cytosolic lactate. HT29 cells (1 × 10 4 ) were seeded in 96-well plates and grown overnight at 37°C. The next day, the culture media were replaced by fresh phenol-red and fetal bovine serum-free Dulbecco's modified essential medium containing 1.2, 4.6, or 18.3 m M glucose, and the cells were exposed to 50 or 200 μ M MQ. (A) In time course experiments, cells were treated with MQ for 0–60 min. ATP was measured with a luciferin/luciferase bioluminescence Promega kit according to manufacturer's protocol. Each experiment was conducted in quadruplicates and ATP contents are expressed as percent relative to untreated controls (100%). Results are mean ± SEM for six cell preparations. (B) The contribution of mitochondria-derived ( upper panel ) and glycolysis-derived ( bottom panel ) ATP to total ATP levels was determined in cells treated with antimycin A (2 μ M ) or 2-deoxyglucose (2DG), respectively. Concentrations of 2DG, namely, 3, 11.5, and 45.8 m M were 2.5 × higher than the respective glucose concentrations. Results are mean ± SEM for n = 6 for 18.3 m M glucose, and n = 4 each for 4.6 and 1.2 m M glucose. * p

    Article Snippet: Antibiotic/antimycotic, trypsin, L-glutamine, McCoys' medium, and Dulbecco's modified essential medium (DMEM) were from Gibco Corporation, and fetal bovine serum (FBS) was from Atlanta Biologicals.

    Techniques: Modification, Luciferase, Derivative Assay

    p53 Inhibits SREBP-2 Maturation under Low-Sterol Conditions (A) HCT116 cells were cultured for 24 hr in medium plus fetal bovine serum (FBS; gray bars), delipidated FBS (DL-FBS; black bars), or delipidated FBS plus 25-hydroxycholesterol (DL-FBS+25-HC; white bars). Expression of HMGCR , HMGCS1 , and SREBF-2 was assessed by qRT-PCR (n = 3; *p

    Journal: Cell

    Article Title: p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression

    doi: 10.1016/j.cell.2018.11.011

    Figure Lengend Snippet: p53 Inhibits SREBP-2 Maturation under Low-Sterol Conditions (A) HCT116 cells were cultured for 24 hr in medium plus fetal bovine serum (FBS; gray bars), delipidated FBS (DL-FBS; black bars), or delipidated FBS plus 25-hydroxycholesterol (DL-FBS+25-HC; white bars). Expression of HMGCR , HMGCS1 , and SREBF-2 was assessed by qRT-PCR (n = 3; *p

    Article Snippet: For analysis of cell cycle changes during sterol starvation, cells were rinsed once with serum-free medium and then placed in medium with 10% delipidated FBS (Gemini Bio-products, catalog #900–123).

    Techniques: Cell Culture, Expressing, Quantitative RT-PCR

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    Phosphorylation of Girdin is required for its interaction with 4F2hc. (A) Amino acid sequences of Girdins from different species. Conserved serines at positions 233 (red) and 237 (green) that conform to the MAPK substrate motif are highlighted. (B) Endogenous interaction between MAPK1/2 and Girdin, as detected by co-IP in 293FT cells. (C) In vitro kinase assay showing that MAPK phosphorylates recombinant Girdin NT but not its mutants. (D) Lysates from 293FT cells transfected with the indicated plasmids were separated on Phos-tag gel. Arrowheads indicate multiple bands that may represent phosphorylated Girdin NT. (E) Lysates from 293FT cells stimulated with FBS in the presence or absence of U0126 were subjected to IP to enrich for endogenous Girdin, followed by the enrichment of phosphopeptides using a Titansphere Phos-TiO kit. The phosphopeptides were analyzed by mass spectrometry. The mass spectrum of a tryptic fragment at m/z 843.3431 (mass error, 0.46 ppm) that matched to the peptide 219-DGLHFLPHASSSAQpSPCGpSPGMK-241 containing phosphorylated S233 and S237 is shown. This phosphopeptide was detected with a high peptide confidence (false discovery rate of less than 1%) in cells stimulated with FBS (Mascot score, 28; expectation value, 0.431) but not in cells pretreated with U0126. (F) 293FT cells were transfected with the indicated plasmids, followed by co-IP, showing that mutation of MAPK phosphorylation sites in Girdin disrupts 4F2hc/Girdin interaction. (G) 293FT cells starved for serum were pretreated with DMSO or U0126 for 30 min and stimulated with DMEM containing 10% FBS for 30 min, followed by co-IP (left panel). 293FT cells were transfected with indicated plasmids, followed by co-IP to test endogenous 4F2hc/Girdin interaction (right panel). co-IP, co-immunoprecipitation; CT, carboxyl terminal; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethyl sulphoxide; FBS, fetal bovine serum; Flag-NT, Flag-tagged Girdin NT domain; GFP, green fluorescent protein; Girdin, girders of actin filaments; GST, glutathione S-transferase; IgG, immunoglobulin G; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKK CA, constitutively active mutant of MAPKK; Mr , molecular marker; NT, amino terminal; Rf , relative mobility; S233, serine at position 233; S237, serine at position 237; WT, wild-type; 4F2hc, 4F2 heavy chain.

    Journal: PLoS Biology

    Article Title: Negative regulation of amino acid signaling by MAPK-regulated 4F2hc/Girdin complex

    doi: 10.1371/journal.pbio.2005090

    Figure Lengend Snippet: Phosphorylation of Girdin is required for its interaction with 4F2hc. (A) Amino acid sequences of Girdins from different species. Conserved serines at positions 233 (red) and 237 (green) that conform to the MAPK substrate motif are highlighted. (B) Endogenous interaction between MAPK1/2 and Girdin, as detected by co-IP in 293FT cells. (C) In vitro kinase assay showing that MAPK phosphorylates recombinant Girdin NT but not its mutants. (D) Lysates from 293FT cells transfected with the indicated plasmids were separated on Phos-tag gel. Arrowheads indicate multiple bands that may represent phosphorylated Girdin NT. (E) Lysates from 293FT cells stimulated with FBS in the presence or absence of U0126 were subjected to IP to enrich for endogenous Girdin, followed by the enrichment of phosphopeptides using a Titansphere Phos-TiO kit. The phosphopeptides were analyzed by mass spectrometry. The mass spectrum of a tryptic fragment at m/z 843.3431 (mass error, 0.46 ppm) that matched to the peptide 219-DGLHFLPHASSSAQpSPCGpSPGMK-241 containing phosphorylated S233 and S237 is shown. This phosphopeptide was detected with a high peptide confidence (false discovery rate of less than 1%) in cells stimulated with FBS (Mascot score, 28; expectation value, 0.431) but not in cells pretreated with U0126. (F) 293FT cells were transfected with the indicated plasmids, followed by co-IP, showing that mutation of MAPK phosphorylation sites in Girdin disrupts 4F2hc/Girdin interaction. (G) 293FT cells starved for serum were pretreated with DMSO or U0126 for 30 min and stimulated with DMEM containing 10% FBS for 30 min, followed by co-IP (left panel). 293FT cells were transfected with indicated plasmids, followed by co-IP to test endogenous 4F2hc/Girdin interaction (right panel). co-IP, co-immunoprecipitation; CT, carboxyl terminal; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethyl sulphoxide; FBS, fetal bovine serum; Flag-NT, Flag-tagged Girdin NT domain; GFP, green fluorescent protein; Girdin, girders of actin filaments; GST, glutathione S-transferase; IgG, immunoglobulin G; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKK CA, constitutively active mutant of MAPKK; Mr , molecular marker; NT, amino terminal; Rf , relative mobility; S233, serine at position 233; S237, serine at position 237; WT, wild-type; 4F2hc, 4F2 heavy chain.

    Article Snippet: Amino acid–free DMEM (Cell Science and Technology Institute, Inc., Sendai, Japan) supplemented with 10% dialyzed FBS (Thermo Fisher Scientific) was used to starve cells. siRNAs or plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Co-Immunoprecipitation Assay, In Vitro, Kinase Assay, Recombinant, Transfection, Mass Spectrometry, Mutagenesis, Immunoprecipitation, Modification, Marker

    Protein coronas modulate sulphide formation. Proposed mechanism of protein corona-modulated nano-Ag 2 S formation at Ag NPs, with hard corona proteins trapping Ag + released from the nanoparticle surface and soft corona proteins transporting said ions away from the sulphide-formation centres in the long-lived corona ( a ); TEM images of silver nanocubes after 24 h in RPMI-1640 cell culture medium supplemented with 1% FBS ( b ), followed by 6 days incubation in RPMI-1640 with 0% FBS (inset cartoon showing only hard corona around Ag NPs) ( c ), 1% FBS ( d ) or 10% FBS ( e ; common inset cartoon showing hard and soft coronas, as well as free bulk proteins around Ag NPs); TEM images of silver nanocubes after 7 days incubation in RPMI-1640 with 0.4 mg ml −1 BSA ( f ) or 4 mg ml −1 BSA ( g ) (common inset cartoon showing hard corona and free bulk proteins around Ag NPs); Ultraviolet–visible spectra of cubic ( h ) and quasi-spherical ( i ) Ag NPs after 24 h incubation in RPMI-1640 cell culture medium supplemented with 1, 10 or 50% FBS; TEM images of silver nanocubes after 24 h in RPMI-1640 supplemented with 1% FBS ( j ), 10% FBS ( k ) and 50% FBS ( l ). Scale bars are 100 nm ( b , j – l ) or 50 nm ( c – g ).

    Journal: Nature Communications

    Article Title: Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

    doi: 10.1038/ncomms11770

    Figure Lengend Snippet: Protein coronas modulate sulphide formation. Proposed mechanism of protein corona-modulated nano-Ag 2 S formation at Ag NPs, with hard corona proteins trapping Ag + released from the nanoparticle surface and soft corona proteins transporting said ions away from the sulphide-formation centres in the long-lived corona ( a ); TEM images of silver nanocubes after 24 h in RPMI-1640 cell culture medium supplemented with 1% FBS ( b ), followed by 6 days incubation in RPMI-1640 with 0% FBS (inset cartoon showing only hard corona around Ag NPs) ( c ), 1% FBS ( d ) or 10% FBS ( e ; common inset cartoon showing hard and soft coronas, as well as free bulk proteins around Ag NPs); TEM images of silver nanocubes after 7 days incubation in RPMI-1640 with 0.4 mg ml −1 BSA ( f ) or 4 mg ml −1 BSA ( g ) (common inset cartoon showing hard corona and free bulk proteins around Ag NPs); Ultraviolet–visible spectra of cubic ( h ) and quasi-spherical ( i ) Ag NPs after 24 h incubation in RPMI-1640 cell culture medium supplemented with 1, 10 or 50% FBS; TEM images of silver nanocubes after 24 h in RPMI-1640 supplemented with 1% FBS ( j ), 10% FBS ( k ) and 50% FBS ( l ). Scale bars are 100 nm ( b , j – l ) or 50 nm ( c – g ).

    Article Snippet: We incubated Ag NPs (cubic or quasi-spherical) in RPMI-1640 with or without added supplements of heat-inactivated FBS (HyClone; 1–50% by volume), for 1 or 7 days.

    Techniques: Transmission Electron Microscopy, Cell Culture, Incubation

    Silver sulphide forms close to the surface of Ag NPs. TEM image with arrows highlighting nano-Ag 2 S ( a , scale bar 50 nm), X-rays elemental mapping of Ag (red), S (blue, with white rings marking the approximate contour of the Ag NPs) and overlaid Ag and S ( b ), EDS spectrum—with arrows pointing at the peaks corresponding to each element—( c ) and diffraction pattern—arrow pointing at the diffraction line corresponding to monoclinic Ag 2 S—( d ) of silver nanocubes after 7 days incubation in RPMI-1640 supplemented with 1% FBS and formation of Ag 2 S at the surface of the Ag NPs.

    Journal: Nature Communications

    Article Title: Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

    doi: 10.1038/ncomms11770

    Figure Lengend Snippet: Silver sulphide forms close to the surface of Ag NPs. TEM image with arrows highlighting nano-Ag 2 S ( a , scale bar 50 nm), X-rays elemental mapping of Ag (red), S (blue, with white rings marking the approximate contour of the Ag NPs) and overlaid Ag and S ( b ), EDS spectrum—with arrows pointing at the peaks corresponding to each element—( c ) and diffraction pattern—arrow pointing at the diffraction line corresponding to monoclinic Ag 2 S—( d ) of silver nanocubes after 7 days incubation in RPMI-1640 supplemented with 1% FBS and formation of Ag 2 S at the surface of the Ag NPs.

    Article Snippet: We incubated Ag NPs (cubic or quasi-spherical) in RPMI-1640 with or without added supplements of heat-inactivated FBS (HyClone; 1–50% by volume), for 1 or 7 days.

    Techniques: Transmission Electron Microscopy, Incubation

    Silver nanoparticle dissolution is involved in nano-Ag 2 S formation. Ultraviolet–visible full spectra of quasi-spherical Ag NPs ( a ) and quadrupole peak detail of nanocubes ( b ) incubated (1 day: blue or 7 days: pink) in RPMI-1640 cell culture medium supplemented with 1% FBS, with or without added extra 10% (by mass) Ag + ions from AgNO 3 ; EDS spectra of the supernatant obtained after centrifugation of Ag NPs incubated for 7 days in RPMI-1640 with 1% FBS, before ( c ) and after ( d ) spiking with 5 nm PVP-coated Ag NPs, with dotted red line highlighting the presence of a silver signal only in the spiked sample.

    Journal: Nature Communications

    Article Title: Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

    doi: 10.1038/ncomms11770

    Figure Lengend Snippet: Silver nanoparticle dissolution is involved in nano-Ag 2 S formation. Ultraviolet–visible full spectra of quasi-spherical Ag NPs ( a ) and quadrupole peak detail of nanocubes ( b ) incubated (1 day: blue or 7 days: pink) in RPMI-1640 cell culture medium supplemented with 1% FBS, with or without added extra 10% (by mass) Ag + ions from AgNO 3 ; EDS spectra of the supernatant obtained after centrifugation of Ag NPs incubated for 7 days in RPMI-1640 with 1% FBS, before ( c ) and after ( d ) spiking with 5 nm PVP-coated Ag NPs, with dotted red line highlighting the presence of a silver signal only in the spiked sample.

    Article Snippet: We incubated Ag NPs (cubic or quasi-spherical) in RPMI-1640 with or without added supplements of heat-inactivated FBS (HyClone; 1–50% by volume), for 1 or 7 days.

    Techniques: Incubation, Cell Culture, Centrifugation

    Sulphur sources and the Ag:S ratio influence Ag 2 S formation. TEM images of Ag NPs after 7 days incubation in PBS ( a ), PBS supplemented with 1% FBS ( b ) and PBS supplemented with L -cysteine and L -methionine at the same concentrations of amino acids as those found in RPMI-1640 ( c ) and corresponding EDS spectra ( d ); TEM images and corresponding EDS spectra (insets) of Ag NPs after 7 days incubation in RPMI-1640 supplemented with 1% FBS, with initial silver concentrations of 2 μg ml −1 ( e ), 10 μg ml −1 (f) and 100 μg ml −1 ( g ), with elemental mapping images provided in Supplementary Fig. 26 . Scale bars are 100 nm ( a – c ) or 50 nm ( e – g ).

    Journal: Nature Communications

    Article Title: Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

    doi: 10.1038/ncomms11770

    Figure Lengend Snippet: Sulphur sources and the Ag:S ratio influence Ag 2 S formation. TEM images of Ag NPs after 7 days incubation in PBS ( a ), PBS supplemented with 1% FBS ( b ) and PBS supplemented with L -cysteine and L -methionine at the same concentrations of amino acids as those found in RPMI-1640 ( c ) and corresponding EDS spectra ( d ); TEM images and corresponding EDS spectra (insets) of Ag NPs after 7 days incubation in RPMI-1640 supplemented with 1% FBS, with initial silver concentrations of 2 μg ml −1 ( e ), 10 μg ml −1 (f) and 100 μg ml −1 ( g ), with elemental mapping images provided in Supplementary Fig. 26 . Scale bars are 100 nm ( a – c ) or 50 nm ( e – g ).

    Article Snippet: We incubated Ag NPs (cubic or quasi-spherical) in RPMI-1640 with or without added supplements of heat-inactivated FBS (HyClone; 1–50% by volume), for 1 or 7 days.

    Techniques: Transmission Electron Microscopy, Incubation

    Corona-mediated sulphidation of Ag NPs impacts particle toxicity. TEM images of partially sulphidated Ag NPs after pre-incubation in RPMI-1640 with 10% FBS ( a ) and completely sulphidated Ag NPs after pre-incubation in RPMI-1640 with 1% FBS ( b ); scale bars are 50 nm; Viability of J774 murine macrophages (as measured with MTT assays) after 24 h exposure to various concentrations (2, 5, 10, 15, 25, 50 and 100 μg ml −1 ) of Ag + ions (black diamonds), pristine Ag NPs (red triangles), partially sulphidated Ag NPs (blue squares) and completely sulphidated Ag NPs (orange circles); error bars are provided as standard deviation; statistically significant differences (two-tailed t -test, with all data sets showing normal distribution and similar variance values) as compared with the control are marked with ** P

    Journal: Nature Communications

    Article Title: Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

    doi: 10.1038/ncomms11770

    Figure Lengend Snippet: Corona-mediated sulphidation of Ag NPs impacts particle toxicity. TEM images of partially sulphidated Ag NPs after pre-incubation in RPMI-1640 with 10% FBS ( a ) and completely sulphidated Ag NPs after pre-incubation in RPMI-1640 with 1% FBS ( b ); scale bars are 50 nm; Viability of J774 murine macrophages (as measured with MTT assays) after 24 h exposure to various concentrations (2, 5, 10, 15, 25, 50 and 100 μg ml −1 ) of Ag + ions (black diamonds), pristine Ag NPs (red triangles), partially sulphidated Ag NPs (blue squares) and completely sulphidated Ag NPs (orange circles); error bars are provided as standard deviation; statistically significant differences (two-tailed t -test, with all data sets showing normal distribution and similar variance values) as compared with the control are marked with ** P

    Article Snippet: We incubated Ag NPs (cubic or quasi-spherical) in RPMI-1640 with or without added supplements of heat-inactivated FBS (HyClone; 1–50% by volume), for 1 or 7 days.

    Techniques: Transmission Electron Microscopy, Incubation, MTT Assay, Standard Deviation, Two Tailed Test

    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.

    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

    Effect of OA-GL12 on the repair rate of HaCaT cells scratches HaCaT cells (2.5 × 10 5 /well) were cultured in 24-well plates for 12–24 h with serum starvation and then made to the mechanical scratch wound by a sterile pipette tip. After washed, cells were cultured for next periods (from 0 to 24 h) in a serum-free basal medium with the continued presence of OA-GL12. The same volume of DMEM (serum free) was added as vehicle and OM-LV20 (50 nM) as positive control. ( A ). OA-GL12 (10 pM) showed prominent wound-healing capacity on HaCaT cells. ( B ). OA-GL12 illustrated time- and concentration- dependent wound-healing capacity on HaCaT cells. Contain cells were added into 96-well plates with basic DMEM (90 μl) without 10% FBS and incubated for 2-4 h. Afterward, OA-GL12 (10 μl) was added to each well and incubated for 24 h at various final concentrations then tested using a CellTiter 96® AQueous One Solution Assay. ( C ). OM-LV20 (50 nM) showed proliferative effect, but OA-GL12 did not show on HaCaT cells from 2 to 10 pM. Data were presented as mean ± S.D., n =9. * P

    Journal: Bioscience Reports

    Article Title: A short peptide potentially promotes the healing of skin wound

    doi: 10.1042/BSR20181734

    Figure Lengend Snippet: Effect of OA-GL12 on the repair rate of HaCaT cells scratches HaCaT cells (2.5 × 10 5 /well) were cultured in 24-well plates for 12–24 h with serum starvation and then made to the mechanical scratch wound by a sterile pipette tip. After washed, cells were cultured for next periods (from 0 to 24 h) in a serum-free basal medium with the continued presence of OA-GL12. The same volume of DMEM (serum free) was added as vehicle and OM-LV20 (50 nM) as positive control. ( A ). OA-GL12 (10 pM) showed prominent wound-healing capacity on HaCaT cells. ( B ). OA-GL12 illustrated time- and concentration- dependent wound-healing capacity on HaCaT cells. Contain cells were added into 96-well plates with basic DMEM (90 μl) without 10% FBS and incubated for 2-4 h. Afterward, OA-GL12 (10 μl) was added to each well and incubated for 24 h at various final concentrations then tested using a CellTiter 96® AQueous One Solution Assay. ( C ). OM-LV20 (50 nM) showed proliferative effect, but OA-GL12 did not show on HaCaT cells from 2 to 10 pM. Data were presented as mean ± S.D., n =9. * P

    Article Snippet: Briefly, human keratinocytes (HaCaT) (KCB 200442YJ) and human skin fibroblasts (HSF) (KCB 200537) were provided by Conservation Genetics CAS Kunming Cell Bank of Kunming Institute of Zoology, the Chinese Academy of Sciences, and cultured in Dulbecco’s modified Eagle’s medium (DMEM, BI, Israel) with 10% FBS (BI, Israel) and penicillin (100 units/ml)–streptomycin (100 units/ml) in a humidified atmosphere of 5% CO2 (37°C).

    Techniques: Cell Culture, Transferring, Positive Control, Concentration Assay, Incubation

    Effect of OA-GL12 on the repair rate of HSF cell scratches HSF cells (2.5 × 10 5 /well) were cultured in 24-well plates for 12–24 h and then made to the mechanical scratch wound by a sterile pipette tip. After washing, cells were cultured for next periods in a serum-free basal medium (500 μl) with the continued presence of OA-GL12. The same volume of DMEM (serum free) was added as vehicle. ( A ) OA-GL12 (10 pM) illustrated distinct HSF cells scratch-healing activity. ( B ) OA-GL12 illustrated time- and concentration-dependent HSF cells scratch-healing activity. Contain cells were added into 96-well plates with basic DMEM (90 μl) without 10% FBS and incubated for 2–4 h. Afterward, OA-GL12 (10 μl) was added to each well and incubated for 24 h at various final concentrations then tested using a CellTiter 96® AQueous One Solution Assay. ( C , D ). OA-GL12 showed no impact on the proliferation of HSF and HUVEC cells from 2 pM to 10 nM. Data were presented as mean ± S.D., n =9. * P

    Journal: Bioscience Reports

    Article Title: A short peptide potentially promotes the healing of skin wound

    doi: 10.1042/BSR20181734

    Figure Lengend Snippet: Effect of OA-GL12 on the repair rate of HSF cell scratches HSF cells (2.5 × 10 5 /well) were cultured in 24-well plates for 12–24 h and then made to the mechanical scratch wound by a sterile pipette tip. After washing, cells were cultured for next periods in a serum-free basal medium (500 μl) with the continued presence of OA-GL12. The same volume of DMEM (serum free) was added as vehicle. ( A ) OA-GL12 (10 pM) illustrated distinct HSF cells scratch-healing activity. ( B ) OA-GL12 illustrated time- and concentration-dependent HSF cells scratch-healing activity. Contain cells were added into 96-well plates with basic DMEM (90 μl) without 10% FBS and incubated for 2–4 h. Afterward, OA-GL12 (10 μl) was added to each well and incubated for 24 h at various final concentrations then tested using a CellTiter 96® AQueous One Solution Assay. ( C , D ). OA-GL12 showed no impact on the proliferation of HSF and HUVEC cells from 2 pM to 10 nM. Data were presented as mean ± S.D., n =9. * P

    Article Snippet: Briefly, human keratinocytes (HaCaT) (KCB 200442YJ) and human skin fibroblasts (HSF) (KCB 200537) were provided by Conservation Genetics CAS Kunming Cell Bank of Kunming Institute of Zoology, the Chinese Academy of Sciences, and cultured in Dulbecco’s modified Eagle’s medium (DMEM, BI, Israel) with 10% FBS (BI, Israel) and penicillin (100 units/ml)–streptomycin (100 units/ml) in a humidified atmosphere of 5% CO2 (37°C).

    Techniques: Cell Culture, Transferring, Activity Assay, Concentration Assay, Incubation

    (A,B) Time of addition assay for analysis of mechanism of CAM and SCM against influenza A/shanghai/37T (2009H1N1) infection. MDCK cells cultured in DMEM supplemented with 10% FBS (Biowest, France), penicillin (100 U/ml), and streptomycin (10 μg/ml) at 37°C/5% CO 2 were infected with influenza A/shanghai/37T (2009H1N1) at 0.1 MOI in DMEM containing 2 μg/mL TPCK-Trypsin. CAM and SCM diluted in DMEM to give a final concentration of 20 μM were added to mixture of virus and cells at 0, 0.5, 1, 2, 4, and 12 h post infection. Inhibition activity of CAM and SCM was measured by CPE reduction assay using CCK-8, according to the manufacturer’s instruction. The data were presented as means ± SE of triplicate assays and the experiment was repeated at least twice.

    Journal: Frontiers in Microbiology

    Article Title: The Antihistamine Drugs Carbinoxamine Maleate and Chlorpheniramine Maleate Exhibit Potent Antiviral Activity Against a Broad Spectrum of Influenza Viruses

    doi: 10.3389/fmicb.2018.02643

    Figure Lengend Snippet: (A,B) Time of addition assay for analysis of mechanism of CAM and SCM against influenza A/shanghai/37T (2009H1N1) infection. MDCK cells cultured in DMEM supplemented with 10% FBS (Biowest, France), penicillin (100 U/ml), and streptomycin (10 μg/ml) at 37°C/5% CO 2 were infected with influenza A/shanghai/37T (2009H1N1) at 0.1 MOI in DMEM containing 2 μg/mL TPCK-Trypsin. CAM and SCM diluted in DMEM to give a final concentration of 20 μM were added to mixture of virus and cells at 0, 0.5, 1, 2, 4, and 12 h post infection. Inhibition activity of CAM and SCM was measured by CPE reduction assay using CCK-8, according to the manufacturer’s instruction. The data were presented as means ± SE of triplicate assays and the experiment was repeated at least twice.

    Article Snippet: Cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, United States) containing 10% FBS (Biowest, France), penicillin 100 U/ml, and streptomycin 10 μg/ml.

    Techniques: Chick Chorioallantoic Membrane Assay, Infection, Cell Culture, Concentration Assay, Inhibition, Activity Assay, CCK-8 Assay

    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

    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

    BCR ablation leads to reduced metabolic flexibility. (A) Pictures show Mito Tracker Red-CMXRos staining alone (upper panels) and overlaid with transmitted light (lower panels) of WT and KO cells (magnification 63×, scale bar = 5 μm). (B) Cells were plated on d0 with or without oligomycin and cell numbers were assessed on d1 and d2 using the CCK8-kit. Values were normalized to the measurement obtained on d1. Statistical significance was determines using the ANOVA test. N = 7; * P = 0.0213 and 0.0341, ** P = 0.0012. (C) Cells were left untreated or incubated with oligomycin overnight. Cells were resupsended in medium containing pyruvate, glutamine, and glucose, and OCR was measured using Seahorse flux technology. One of three independent experiments is shown. a, oligomycin, b, FCCP, c, rotenone + antimycin. (D) Cells were cultured in glucose free RPMI+ dialyzed FBS in the presence of 10 mM glucose, 10 mM galactose, or 10 mM GlutaMAX with or without oligomycin. Cell numbers were assessed using the CCK8 kit after overnight incubation. Results are normalized to the values obtained from the untreated sample cultured with glucose. Differences between the indicated pairs of samples were tested for significance using the unpaired t test. N = 3; * P = 0.0242, ** P = 0.0033 and 0.0059, *** P = 0.0005 and 0.0002. KO, BCR-KO Ramos cells; WT, Ramos wild-type cells.

    Journal: Life Science Alliance

    Article Title: Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes

    doi: 10.26508/lsa.202000700

    Figure Lengend Snippet: BCR ablation leads to reduced metabolic flexibility. (A) Pictures show Mito Tracker Red-CMXRos staining alone (upper panels) and overlaid with transmitted light (lower panels) of WT and KO cells (magnification 63×, scale bar = 5 μm). (B) Cells were plated on d0 with or without oligomycin and cell numbers were assessed on d1 and d2 using the CCK8-kit. Values were normalized to the measurement obtained on d1. Statistical significance was determines using the ANOVA test. N = 7; * P = 0.0213 and 0.0341, ** P = 0.0012. (C) Cells were left untreated or incubated with oligomycin overnight. Cells were resupsended in medium containing pyruvate, glutamine, and glucose, and OCR was measured using Seahorse flux technology. One of three independent experiments is shown. a, oligomycin, b, FCCP, c, rotenone + antimycin. (D) Cells were cultured in glucose free RPMI+ dialyzed FBS in the presence of 10 mM glucose, 10 mM galactose, or 10 mM GlutaMAX with or without oligomycin. Cell numbers were assessed using the CCK8 kit after overnight incubation. Results are normalized to the values obtained from the untreated sample cultured with glucose. Differences between the indicated pairs of samples were tested for significance using the unpaired t test. N = 3; * P = 0.0242, ** P = 0.0033 and 0.0059, *** P = 0.0005 and 0.0002. KO, BCR-KO Ramos cells; WT, Ramos wild-type cells.

    Article Snippet: 0.6 × 106 or 1 × 106 cells were incubated in 1 ml RPMI (Thermo Fisher Scientific) + 1% FBS (Biochrom AG) + 15 μl Indo-1 solution at 37°C for 45 min.

    Techniques: Staining, Incubation, Cell Culture

    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

    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