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  • 99
    Thermo Fisher dulbecco s modified eagle s medium
    The role of FASN and related signalling pathways on PCa cell viability. LNCaP and C4-2 cells were cultured in a 96-well plate with <t>Dulbecco's</t> modified <t>Eagle's</t> medium (DMEM) containing 5% fetal bovine serum (FBS), and treated with 50 n M siRNA ( a , c ) and 10 μ M cerulenin ( b , d ) with or without 75 μ M PA for the indicated duration. The MTT assay was performed and cell viability was compared with that of cells treated with control siRNA or FBS ( a , b ). * P
    Dulbecco S Modified Eagle S Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 138312 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dmem
    Osteogenic differentiation of PDLSCs cultured in EHFM, <t>α-MEM</t> and <t>DMEM</t> enriched with dexamethasone, ascorbic acid and β-glycerophosphate. Figure shows the schema of the experimental set up. Detection of the osteogenic differentiation of EHFM-, α-MEM- and DMEM-cultured PDLSCs in basal conditions and in the presence of osteo-inductive factors through ALP activity and Alizarin Red S staining (A). Relative mRNA expression levels of ALP, OPN, OCN, Runx2, ColI, ColII, IBSP and Msx2 in EHFM-, α-MEM- and DMEM-cultured PDLSCs after differentiation culture for 7, 14 and 21 days (B–D). Relative mRNA expression of all osteogenesis markers in EHFM-expanded PDLSCs at 7, 14 and 21 days was expressed as % of DMEM and α-MEM (E). Data are presented as the mean ± SEM. α-MEM: minimum essential medium Eagle, alpha modification; ALP: alkaline phosphatase; Col: collagen; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; IBSP: integrin binding sialoprotein; OCN: osteocalcin; OPN: osteopontin; PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.
    Dmem, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 49883 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dmem
    Populations of <t>PDPN+</t> cells among CAFs affected by environmental conditions. (A) The percentage of PDPN+ cells among CAFs changed in a time-dependent manner by culturing with <t>DMEM-containing</t> growth factors. (B) Populations of PDPN+ cells among CAFs changed by the addition of FBS in concentration- and time-dependent manners. The control (CTRL) condition consisted of DMEM containing 10% FBS and medium changes every day. (C) Populations of PDPN+ cells in cultures of serum-free DMEM increased more rapidly than those in cultures of glucose- and serum-free DMEM.
    Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 148857 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dmem f12
    The effect of solution added TGF-β in <t>DMEM/F12/10%</t> FBS media + DMSO on CSPG production. CSPG staining per cell data were normalized to FBG0/LN100 sample. Error bars indicate ± SEM. Asterisk denotes p
    Dmem F12, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 39224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dmem medium
    PDAC cell supernatants activate macrophages that in turn secrete molecules that promote PDAC cells proliferation. ( a ) Isolated human monocytes ( > 98% CD14 + ) were treated with LPS or rBAG3 for 16 h. Then, supernatants were analysed with human IL-6 ELISA. Data are from duplicate samples and obtained in two separate experiments. Error bars indicate s.d. ( b ) Isolated human monocytes ( > 98% CD14 + ) were cultured in RPMI supplemented with 0.15% FBS (control medium) or in <t>MIA</t> PaCa-2-conditioned medium and treated for 16 h with an anti-BAG3 monoclonal antibody at the indicated concentrations. Unrelated murine IgG1 were used as negative control. After treatment, supernatants were collected and analysed with a human IL-6 ELISA Kit. Data are from triplicate samples and obtained in two separate experiments. Error bars indicate s.d. ( c ) Cells were treated as described above. Cells were then harvested for total RNA extraction and analysed by reverse transcription–PCR (RT–PCR). ( d ) Isolated human monocytes were cultured in RPMI supplemented with 0.15% FBS (control medium) or in MIA PaCa-2-conditioned medium, and treated with the F(ab') 2 fragment of an anti-BAG3 monoclonal antibody. F(ab') 2 fragments of a non-specific murine IgG1 were used as negative control. After treatment, supernatants were collected and analysed with a human IL-6 ELISA Kit. Data are from duplicate samples and repeated two times. Error bars indicate s.d. ( e ) MIA PaCa-2 and CFPAC-1 ( f ) cells were cultured in <t>DMEM</t> supplemented with 0.15% FBS (no donor) or conditioned medium from monocytes treated with LPS or rBAG3 for 16 h at the indicated concentrations. After 72 h incubation, cells were analysed by MTT assay. Data are from duplicate samples. Error bars indicate s.d. ( g ) MIA PaCa-2 cells were incubated with human recombinant ( r ) IL-6 (10 ng ml −1 ) or conditioned medium from donor 4 or donor 5 monocytes stimulated with rBAG3 (6 μg ml −1 ) for 16 h. An anti-IL-6 receptor monoclonal antibody (20 μg ml −1 ) was added to inhibit IL-6 activity. After a 72-h incubation, cells were analysed by MTT assay. Data are from duplicate samples. Error bars indicate s.d. P values were calculated by Student's t -test and represented as follows: * P
    Dmem Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 23297 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dulbecco s modified eagle s medium dmem
    In vitro effects of Euphorbia fischeriana Steud on the growth of B16 cells. B16 cells were cultured for 24 or 48 h in <t>Dulbecco's</t> modified <t>Eagle's</t> medium containing Euphorbia fischeriana Steud at various concentrations. The effects of Euphorbia fischeriana
    Dulbecco S Modified Eagle S Medium Dmem, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10354 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare dmem
    Metabolic inhibition of sulfation of HS chains leads to a loss of sulfated groups of HS, Hsp90α, and Hsp90β from the cell surface ( a ) and to an inhibition of the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with sodium chlorate, after which cells were grown at 37 °C in <t>DMEM-FBS</t> without chlorate. At indicated times, cell surface HS and proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of chlorate-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of chlorate-treated cells. The basal migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p
    Dmem, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 16922 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dmem high glucose
    MCOLN1 is required for MTORC1 activation. ( A ) MCOLN1 ablation compromised MTORC1 reactivation during nutrient refeeding. WT and MCOLN1 − / − human skin fibroblasts were starved for 50 min or followed by nutrient refeeding for 10 or 30 min as indicated. Cell extracts were analyzed by western blotting for the indicated proteins. The graph illustrates the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments), relative to that in WT cells with 30 min refeeding. ( B ) MCOLN1 − / − cells lose MTORC1 activity more easily compared with WT cells when starved. WT and MCOLN1 − / − cells were kept in normal culture medium or starved for 5, 15 or 30 min. Cell extracts were analyzed by western blotting for the indicated proteins. The graph shows the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments), relative to that in fed WT cells. ( C ) Inhibition of MCOLN1 suppressed MTORC1 activity upon starvation and nutrient refeeding. <t>HEK293T</t> cells were kept in normal culture medium, starved for 15 min, or starved for 50 min followed by nutrient refeeding for 5 or 15 min ± ML-SI1 (50 µM) as indicated. MTORC1 activity was assessed by measuring p-RPS6KB (T389) and p-EIF4EBP1 (T37/46) using western blot. The graph shows the mean percentage of the ratio of p-RPS6KB:RPS6KB (left) and p-EIF4EBP1:EIF4EBP1 (right) (mean ± SEM, n = 3 independent experiments), relative to that in nontreated fed cells. ( D, E ) High MTORC1 activity in cells expressing constitutively active RRAGB, which partially mimics nutrient-replete conditions was suppressed by MCOLN1 deletion and BAPTA-AM. HEK293T cells were transfected with RRAGB GTP or together with scramble or MCOLN1 shRNA; 24 h later cells were starved with amino acid-free <t>DMEM</t> for 30 min in the presence or absence of 10 μM BAPTA-AM. ( F ) MCOLN1 inhibition did not alter MTORC1 activity under normal conditions. HEK293T cells were treated with DMSO or ML-SI1 (50 μM) for 1 h in normal medium. The graph illustrates the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments), relative to that in DMSO-treated cells. NS, not significant; *, P
    Dmem High Glucose, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6907 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher high glucose dmem
    CD36 regulates AMPK activation via Fyn-dependent nuclear sequestration of LKB1. A : Suppression of AMPK phosphorylation by CD36 expression. CHO cells lacking CD36 (Vector) or stably expressing CD36 (CHO-CD36) were serum starved (for 16 h in <t>DMEM</t> buffer A), lysed, and probed (in triplicate) for CD36, pAMPK (T172), and GAPDH. Data are representative of two experiments. B : Fyn phosphorylation of LKB1 is enhanced by CD36 expression. Control (Vector) and CD36-expressing cells (CD36) were transiently transfected with Fyn or FynD; and serum-starved and cell lysates (Input) were probed for CD36, LKB1, and Fyn. LKB1 was immunoprecipitated from cell lysates using mouse monoclonal antibody for LKB1 (clone 5c10; Millipore). LKB1 immunoprecipitates (IP:LKB1) were probed with phosphotyrosine PY100 (pLKB1) and LKB1 antibodies. C : CD36 expression induces nuclear sequestration of LKB1 in CHO cells. CHO cells stably expressing CD36 or Vector controls were serum starved, fixed, and processed for IF as described in Research Design and Methods . The cells were stained with mouse monoclonal LKB1 antibody (clone 5c10; Millipore) and with DAPI to visualize the nuclei. Images are representative of multiple fields from three experiments. Scale bar, 10 μm. D : CD36 depletion in <t>C2C12</t> myotubes abolishes nuclear sequestration of LKB1. C2C12 myotubes treated with siCD36 or siCont were serum starved for 16 h in DMEM buffer A. LKB1 and CD36 were detected using mouse monoclonal anti-LKB1 (5c10; Millipore) and rat monoclonal anti-CD36 antibodies (MF3; AbD Serotec). Images are representative of multiple fields from three experiments. Scale bar, 10 μm.
    High Glucose Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5305 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher glucose dmem
    Activator of thyroid and retinoid receptor (ACTR) enhances <t>sorafenib</t> resistance by affecting aerobic glycolysis in vitro. A and B, The relative viability curves of ACTR WT or KO HepG2 cells or ACTR KO HepG2 cells transiently transfected with ACTR, as well as Huh‐7 cells in <t>DMEM</t> with high glucose (25 mmol/L), transfected with ACTR siRNA or ACTR siRNA plus ACTR expression vector or non‐specific control for siRNA (Control siRNA); they were treated with Deoxy‐d‐glucose (2‐DG) (2.5 mmol/L) and increasing concentrations of sorafenib as indicated above. After 72 h, cell viability assays were performed using the CCK‐8. The group without treatment of sorafenib had 100% viable cells and was used as an internal control for comparison. The representative immunoblot with ACTR indicates ACTR expression levels. C and D, The relative viability curves of HepG2 cells (C) or Huh‐7 cells (D) transfected and treated as in (A) or (B) and cultured in DMEM with low glucose (5.5 mmol/L). E and F, Colony formation assays of HepG2 and Huh‐7 cells treated as in (A) and (B) with sorafenib (6 μmol/L) or not. G, Representative flow cytometry analysis of Annexin V (1:1000) and propidium iodide (1:1000) staining was carried out in HepG2 ACTR WT cells, KO cells, WT cells and KO cells treated with 2‐DG (2.5 mmol/L) and sorafenib (6 μmol/L) for 6 h. Data shown are mean ± SD of triplicate measurements that have been repeated three times with similar results. * P
    Glucose Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 4634 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Corning Life Sciences dmem
    A2B receptors are expressed in Schwann cells. (A) Western blot image of A2B expression in RSC-96 cells. GAPDH is used as a housekeeping control. Relative expression of A2B protein in RSC-96 cells co-cultured with <t>DOK</t> or HSC-3 is presented as fold change over <t>DMEM</t> treated RSC-96 cells. (B) Western blot quantification of A2B protein expression in RSC-96 cells. (C) Immunofluorescence labeling of the A2B receptor (green) and nucleus (DAPI, blue) in RSC-96 cells co-cultured with either DOK or HSC-3. Scale: 100 μm. (D) Relative A2B mRNA fold change in RSC-96 cells co-cultured with either DOK or HSC-3 over control RSC-96 cells. No differences are detected across different groups. Kruskal-Wallis test followed by Dunn's multiple comparison analysis.
    Dmem, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 4969 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Mediatech dmem
    Effect of IBTP treatment on mitochondrial respiration of <t>MB231</t> cells. Panel A: Cells plated on XF24 plates were treated with the indicated concentrations of IBTP or BTPP for 4h in 0.5% FBS-containing medium. After treatment, the medium was removed and replaced with XF assay medium <t>(DMEM,</t> containing 5mM glucose, 0.5% FBS, 5mM HEPES without bicarbonate) and equilibrated 1h before OCR measurement. Panel B: Cells plated on 6-well plates were treated with the indicated concentrations of IBTP or BTPP for 4h. After the incubation, the cells were harvested immediately by trypsinization. The harvested cells were replated in XF24 plates and allowed adhere for an additional 20h in complete medium containing 10% FBS (total 24h). The medium was removed and replaced with assay medium and equilibrated 1h before OCR measurement. Panel C : After 4h of IBTP or BTPP treatment, the medium was replaced with complete medium containing 10% FBS, and incubated for 48h. The cells were harvested after 48h, replated in XF24 plates and allowed adhere for an additional 20h in complete medium. The medium was replaced with assay media and incubated 1h before measurement of OCR (total duration 72h).
    Dmem, supplied by Mediatech, used in various techniques. Bioz Stars score: 99/100, based on 4400 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cellgro dmem
    Cell-survival quantification with the XTT assay after treatment with recombinant NGF (open bars) or proNGF123 (filled bars) at the concentrations indicated for 72 h in defined <t>DMEM.</t> RN22 <t>schwannoma</t> cells ( A ), PC12nnr cells ( B ), and C6 glioma cells ( C
    Dmem, supplied by Cellgro, used in various techniques. Bioz Stars score: 92/100, based on 4080 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dulbecco s modified eagle medium dmem
    Schematic diagram of the experiment. Isolation and characterization of A. Umbilical cord blood cluster of differentiation 133 + , B. Rat pancreatic mesenchymal stem cells, C. Culture type, C1; Transwell culture, C2; Direct co-culture and D. Method of differentiation into insulin producing cells. FBS; Fetal bovine serum, <t>DMEM;</t> <t>Dulbecco’s</t> Modified Eagle’s medium, bFGF; Basic fibroblast growth factor, RA; Retinoic acid, NA; Nicotinamide and RT-PCR; Reverse transcription-polymerase chain reaction.
    Dulbecco S Modified Eagle Medium Dmem, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3071 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher knockout dmem
    Schematic illustration of the method for differentiation of ESCs into fibroblasts in three-dimensional type I collagen gel culture. Undifferentiated ESCs are cultured on MEF feeder layer in six-well plate. ESCs are detached with collagenase and re-suspended with differentiation medium. Cells are then placed into a Petri dish and cultured for 4–5 d to allow formation of EBs. EBs are cast into type I collagen gels in a 12-well plate (1.0 ml/well) and cultured for 21 d three-dimensionally in collagen gels with basal medium. Gels are dissolved by collagenase and the cells are suspended in 10% <t>FCS-DMEM.</t> Differentiated fibroblasts are cultured in 100 mm culture dishes. EBs are lost with serial feeding and passaging.
    Knockout Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The role of FASN and related signalling pathways on PCa cell viability. LNCaP and C4-2 cells were cultured in a 96-well plate with Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS), and treated with 50 n M siRNA ( a , c ) and 10 μ M cerulenin ( b , d ) with or without 75 μ M PA for the indicated duration. The MTT assay was performed and cell viability was compared with that of cells treated with control siRNA or FBS ( a , b ). * P

    Journal: Oncogenesis

    Article Title: Diet-induced alteration of fatty acid synthase in prostate cancer progression

    doi: 10.1038/oncsis.2015.42

    Figure Lengend Snippet: The role of FASN and related signalling pathways on PCa cell viability. LNCaP and C4-2 cells were cultured in a 96-well plate with Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS), and treated with 50 n M siRNA ( a , c ) and 10 μ M cerulenin ( b , d ) with or without 75 μ M PA for the indicated duration. The MTT assay was performed and cell viability was compared with that of cells treated with control siRNA or FBS ( a , b ). * P

    Article Snippet: The cells were maintained in RPMI 1640 medium or Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum and 1% penicillin–streptomycin.

    Techniques: Cell Culture, Modification, MTT Assay

    Effect of PI3K (LY294002) and MAPK (U0126) inhibitors and an AMPK activator (AICAR) on FASN expression in LNCaP and C4-2 cells. LNCaP and C4-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) with and without 5 μ M LY294002, 0.5 μ M U0126 ( a – c ), or AICAR ( d ) at the indicated concentration for 24 h. ( a ) An equal amount of protein from the cells and conditioned medium was subjected to anti-human-FASN antibody. Total RNA was extracted from the cells and FASN ( b ) and SREBP-1 ( c ) mRNA levels were measured by quantitative reverse transcription–PCR, and compared with those of untreated cells. ** P

    Journal: Oncogenesis

    Article Title: Diet-induced alteration of fatty acid synthase in prostate cancer progression

    doi: 10.1038/oncsis.2015.42

    Figure Lengend Snippet: Effect of PI3K (LY294002) and MAPK (U0126) inhibitors and an AMPK activator (AICAR) on FASN expression in LNCaP and C4-2 cells. LNCaP and C4-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) with and without 5 μ M LY294002, 0.5 μ M U0126 ( a – c ), or AICAR ( d ) at the indicated concentration for 24 h. ( a ) An equal amount of protein from the cells and conditioned medium was subjected to anti-human-FASN antibody. Total RNA was extracted from the cells and FASN ( b ) and SREBP-1 ( c ) mRNA levels were measured by quantitative reverse transcription–PCR, and compared with those of untreated cells. ** P

    Article Snippet: The cells were maintained in RPMI 1640 medium or Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum and 1% penicillin–streptomycin.

    Techniques: Expressing, Cell Culture, Modification, Concentration Assay, Polymerase Chain Reaction

    Lack of sphingosine-1-phosphate (S1P) induces β-cell dysfunction and apoptosis. Mouse insulinoma 6 (MIN6) cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) media (containing 25 mM glucose and 10% fetal bovine serum). At 50% cell confluence, the cells were incubated in serum-free media with a sphingosine kinase inhibitor (15 µM) and/or S1P (D18:1, 10 µM) for 48 hours. (A, B) Cellular levels of S1P and apoptotic proteins, including cleaved caspase-3 and phosphorylated c-Jun N-terminal kinase (p-JNK), were evaluated by an immunoblotting assay. Quantitative levels of p-JNK and cleaved caspase-3 were normalized to total JNK and caspase-3 expression. (C) Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity is presented as the percent of relative absorbance compared to the vehicle (Veh) group. (D) Transcription of insulin 1 ( INS1 ) was measured by real-time reverse-transcription polymerase chain reaction and normalized to β-actin. (E) Nuclear expression of pancreatic and duodenal homeobox 1 (Pdx1) was analyzed by an immunoblotting assay, and normalized to lamin B1. Each value represents the mean of 3 experiments. (F) The glucose-stimulated (5.5 or 25 mM) insulin secretion of MIN6 cells was evaluated by a mouse insulin enzyme-linked immunosorbent assay kit. The values are representative of four independent experiments. t-JNK, total c-Jun N-terminal kinase; c-casp3, cleaved caspase-3; t-casp3, total caspase-3; SphKi, sphingosine kinase inhibitor. a P

    Journal: Endocrinology and Metabolism

    Article Title: Deficiency of Sphingosine-1-Phosphate Reduces the Expression of Prohibitin and Causes β-Cell Impairment via Mitochondrial Dysregulation

    doi: 10.3803/EnM.2018.33.3.403

    Figure Lengend Snippet: Lack of sphingosine-1-phosphate (S1P) induces β-cell dysfunction and apoptosis. Mouse insulinoma 6 (MIN6) cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) media (containing 25 mM glucose and 10% fetal bovine serum). At 50% cell confluence, the cells were incubated in serum-free media with a sphingosine kinase inhibitor (15 µM) and/or S1P (D18:1, 10 µM) for 48 hours. (A, B) Cellular levels of S1P and apoptotic proteins, including cleaved caspase-3 and phosphorylated c-Jun N-terminal kinase (p-JNK), were evaluated by an immunoblotting assay. Quantitative levels of p-JNK and cleaved caspase-3 were normalized to total JNK and caspase-3 expression. (C) Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity is presented as the percent of relative absorbance compared to the vehicle (Veh) group. (D) Transcription of insulin 1 ( INS1 ) was measured by real-time reverse-transcription polymerase chain reaction and normalized to β-actin. (E) Nuclear expression of pancreatic and duodenal homeobox 1 (Pdx1) was analyzed by an immunoblotting assay, and normalized to lamin B1. Each value represents the mean of 3 experiments. (F) The glucose-stimulated (5.5 or 25 mM) insulin secretion of MIN6 cells was evaluated by a mouse insulin enzyme-linked immunosorbent assay kit. The values are representative of four independent experiments. t-JNK, total c-Jun N-terminal kinase; c-casp3, cleaved caspase-3; t-casp3, total caspase-3; SphKi, sphingosine kinase inhibitor. a P

    Article Snippet: Cell culture, treatment, and transfection MIN6 cell lines were maintained in Dulbecco's Modified Eagle's medium (DMEM) containing 25 mmol/L glucose, mixed with heat-inactivated 10% fetal bovine serum, 100 IU/mL penicillin, 50 µg/mL streptomycin, and 50 µM 2-mercaptoethanol (Life Technologies Corporation, Paisley, UK), in a 37℃ humidified incubator with 5% CO2 .

    Techniques: Modification, Incubation, Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Serum-dependent activation of NF-κB by PEX. 293 cells were transiently transfected with plasmids encoding CD14 (0.1 μg) and TLR2 (0.1 μg), together with an NF-κB-dependent luciferase reporter plasmid. After 24 h, cells were washed with phosphate-buffered saline twice and stimulated for 6 h with PEX (A), a synthetic murein lipopeptide (Pam 3 CSSNA; B), or peptidoglycan (PG; B) in Dulbecco's modified Eagle medium in the absence (open bars) or presence (hatched bars) of LBP (100 ng/ml) or FCS (10% [vol/vol]) (solid bars), and luciferase activity was then measured. Values are means ± standard errors from at least four independent experiments. Responses were compared with responses in the absence of LBP or serum by a two-tailed Student t test (∗, P

    Journal: Infection and Immunity

    Article Title: Lipopolysaccharide-Mimetic Activities of a Toll-Like Receptor 2-Stimulatory Substance(s) in Enterobacterial Lipopolysaccharide Preparations

    doi: 10.1128/IAI.71.6.3221-3226.2003

    Figure Lengend Snippet: Serum-dependent activation of NF-κB by PEX. 293 cells were transiently transfected with plasmids encoding CD14 (0.1 μg) and TLR2 (0.1 μg), together with an NF-κB-dependent luciferase reporter plasmid. After 24 h, cells were washed with phosphate-buffered saline twice and stimulated for 6 h with PEX (A), a synthetic murein lipopeptide (Pam 3 CSSNA; B), or peptidoglycan (PG; B) in Dulbecco's modified Eagle medium in the absence (open bars) or presence (hatched bars) of LBP (100 ng/ml) or FCS (10% [vol/vol]) (solid bars), and luciferase activity was then measured. Values are means ± standard errors from at least four independent experiments. Responses were compared with responses in the absence of LBP or serum by a two-tailed Student t test (∗, P

    Article Snippet: The human embryonic 293 cell line (obtained from the Human Science Research Resources Bank, Tokyo, Japan) was grown in Dulbecco's modified Eagle medium (Gibco BRL, Rockville, Md.) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (FCS; Gibco BRL), penicillin (100 U/ml), and streptomycin (100 μg/ml).

    Techniques: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Modification, Activity Assay, Two Tailed Test

    Data for the µCT-characteristics for TV ( a ) SV ( b ), SA ( c ), and TMD_SV ( d ) of the analyzed scaffolds (groups A and B) over time in Dulbecco’s Modified Eagle Medium (DMEM) (T0: initial scan at the beginning of the immersion period, followed by scans after seven (T1), 14 (T2), and 56 (T3) days in immersion), shown as boxplots (mean values with SEM and range). Significant changes over time are marked by (*).

    Journal: Materials

    Article Title: Micro-Computed-Tomography-Guided Analysis of In Vitro Structural Modifications in Two Types of 45S5 Bioactive Glass Based Scaffolds

    doi: 10.3390/ma10121341

    Figure Lengend Snippet: Data for the µCT-characteristics for TV ( a ) SV ( b ), SA ( c ), and TMD_SV ( d ) of the analyzed scaffolds (groups A and B) over time in Dulbecco’s Modified Eagle Medium (DMEM) (T0: initial scan at the beginning of the immersion period, followed by scans after seven (T1), 14 (T2), and 56 (T3) days in immersion), shown as boxplots (mean values with SEM and range). Significant changes over time are marked by (*).

    Article Snippet: Immersion Method and pH Measurement The BG-based scaffolds were stored in cryo-vials (Greiner Bio-One, Frickenhausen, Germany) containing 2.25 mL Dulbecco’s Modified Eagle Medium (DMEM), 4.5 g/L Glucose, 0.11 g/L Sodium Pyruvate, no L-Glutamine (all Thermo Fisher Scientific, Dreieich, Germany) under standard static cell-culture conditions (37 °C, 74% N2 , 21% O2 , and 5% CO2 ).

    Techniques: Modification

    Viability of HCT116 cells after treatment with selected inhibitor candidate compounds, against the mTOR rapamycin binding site, as measured by both the MTT assay (symbols in red color) and counting Hoechst stained-nuclei (symbols in blue color). Each compound was always compared with rapamycin in parallel experiments. Each panel included a legend with the compounds analyzed and methodology followed (MTT or Hoechst stained-nuclei). The values were normalized with respect to media cultured only Dulbecco’s Modified Eagle Medium (DMEM).

    Journal: Marine Drugs

    Article Title: New Mammalian Target of Rapamycin (mTOR) Modulators Derived from Natural Product Databases and Marine Extracts by Using Molecular Docking Techniques

    doi: 10.3390/md16100385

    Figure Lengend Snippet: Viability of HCT116 cells after treatment with selected inhibitor candidate compounds, against the mTOR rapamycin binding site, as measured by both the MTT assay (symbols in red color) and counting Hoechst stained-nuclei (symbols in blue color). Each compound was always compared with rapamycin in parallel experiments. Each panel included a legend with the compounds analyzed and methodology followed (MTT or Hoechst stained-nuclei). The values were normalized with respect to media cultured only Dulbecco’s Modified Eagle Medium (DMEM).

    Article Snippet: High-glucose Dulbecco’s Modified Eagle’s Medium, penicillin-streptomycin, fetal bovine serum and 0.05x trypsin/ethylene diamine tetra acetic acid was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA).

    Techniques: Binding Assay, MTT Assay, Staining, Cell Culture, Modification

    Osteogenic differentiation of PDLSCs cultured in EHFM, α-MEM and DMEM enriched with dexamethasone, ascorbic acid and β-glycerophosphate. Figure shows the schema of the experimental set up. Detection of the osteogenic differentiation of EHFM-, α-MEM- and DMEM-cultured PDLSCs in basal conditions and in the presence of osteo-inductive factors through ALP activity and Alizarin Red S staining (A). Relative mRNA expression levels of ALP, OPN, OCN, Runx2, ColI, ColII, IBSP and Msx2 in EHFM-, α-MEM- and DMEM-cultured PDLSCs after differentiation culture for 7, 14 and 21 days (B–D). Relative mRNA expression of all osteogenesis markers in EHFM-expanded PDLSCs at 7, 14 and 21 days was expressed as % of DMEM and α-MEM (E). Data are presented as the mean ± SEM. α-MEM: minimum essential medium Eagle, alpha modification; ALP: alkaline phosphatase; Col: collagen; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; IBSP: integrin binding sialoprotein; OCN: osteocalcin; OPN: osteopontin; PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.

    Journal: Cell Transplantation

    Article Title: In Vitro Long-Term Expansion and High Osteogenic Potential of Periodontal Ligament Stem Cells: More Than a Mirage

    doi: 10.1177/0963689718807680

    Figure Lengend Snippet: Osteogenic differentiation of PDLSCs cultured in EHFM, α-MEM and DMEM enriched with dexamethasone, ascorbic acid and β-glycerophosphate. Figure shows the schema of the experimental set up. Detection of the osteogenic differentiation of EHFM-, α-MEM- and DMEM-cultured PDLSCs in basal conditions and in the presence of osteo-inductive factors through ALP activity and Alizarin Red S staining (A). Relative mRNA expression levels of ALP, OPN, OCN, Runx2, ColI, ColII, IBSP and Msx2 in EHFM-, α-MEM- and DMEM-cultured PDLSCs after differentiation culture for 7, 14 and 21 days (B–D). Relative mRNA expression of all osteogenesis markers in EHFM-expanded PDLSCs at 7, 14 and 21 days was expressed as % of DMEM and α-MEM (E). Data are presented as the mean ± SEM. α-MEM: minimum essential medium Eagle, alpha modification; ALP: alkaline phosphatase; Col: collagen; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; IBSP: integrin binding sialoprotein; OCN: osteocalcin; OPN: osteopontin; PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.

    Article Snippet: In this study, we used and compared three different culture media: α-MEM (no. 22561021; Life Technologies, Milan, Italy) and DMEM (no. D5796; Sigma Aldrich, Milan, Italy), which were supplemented with 10% fetal bovine serum (FBS; no. 10270106; Life Technologies), 100 U/ml penicillin and 100 μmol/ml streptomycin (no. P4333; Sigma Aldrich); and Ham’s F12 medium (no. 21765037; Gibco, Life Technologies) supplemented with 10% FBS, 0.5 U/ml heparin, 50 ng/ml epidermal growth factor (EGF), 25 ng/ml fibroblast growth factor (FGF), 1% bovine serum albumin (BSA), 100 U/ml penicillin, and 100 μmol/ml streptomycin (EHFM) , .

    Techniques: Cell Culture, ALP Assay, Activity Assay, Staining, Expressing, Modification, Binding Assay

    Characterization of PDLSCs. PDLSCs maintained in EHFM, α-MEM and DMEM were characterized for the expression of hematopoietic and mesenchymal markers. Flow-cytometric analysis of CD14, CD34, CD45, CD73, CD90 and CD105 expression in PDLSCs at passage 3 and passage 8 (A). The percentages of positive cells are indicated. The expression levels of CD73 (B), CD90 (C) and CD105 (D) in PDLSCs maintained in EHFM, α-MEM and DMEM are shown until passage 9 and expressed as the mean fluorescence intensity. Data are presented as the mean ± SEM. For statistical analysis, a one-way analysis of variance followed by a Student’s t test was applied. α-MEM: minimum essential medium Eagle, alpha modification; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.

    Journal: Cell Transplantation

    Article Title: In Vitro Long-Term Expansion and High Osteogenic Potential of Periodontal Ligament Stem Cells: More Than a Mirage

    doi: 10.1177/0963689718807680

    Figure Lengend Snippet: Characterization of PDLSCs. PDLSCs maintained in EHFM, α-MEM and DMEM were characterized for the expression of hematopoietic and mesenchymal markers. Flow-cytometric analysis of CD14, CD34, CD45, CD73, CD90 and CD105 expression in PDLSCs at passage 3 and passage 8 (A). The percentages of positive cells are indicated. The expression levels of CD73 (B), CD90 (C) and CD105 (D) in PDLSCs maintained in EHFM, α-MEM and DMEM are shown until passage 9 and expressed as the mean fluorescence intensity. Data are presented as the mean ± SEM. For statistical analysis, a one-way analysis of variance followed by a Student’s t test was applied. α-MEM: minimum essential medium Eagle, alpha modification; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.

    Article Snippet: In this study, we used and compared three different culture media: α-MEM (no. 22561021; Life Technologies, Milan, Italy) and DMEM (no. D5796; Sigma Aldrich, Milan, Italy), which were supplemented with 10% fetal bovine serum (FBS; no. 10270106; Life Technologies), 100 U/ml penicillin and 100 μmol/ml streptomycin (no. P4333; Sigma Aldrich); and Ham’s F12 medium (no. 21765037; Gibco, Life Technologies) supplemented with 10% FBS, 0.5 U/ml heparin, 50 ng/ml epidermal growth factor (EGF), 25 ng/ml fibroblast growth factor (FGF), 1% bovine serum albumin (BSA), 100 U/ml penicillin, and 100 μmol/ml streptomycin (EHFM) , .

    Techniques: Expressing, Flow Cytometry, Fluorescence, Modification

    Osteogenic differentiation of EHFM-, α-MEM- and DMEM-expanded PDLSCs in standardized culture medium for the osteogenesis (Life Technologies). Figure shows the schema of the experimental set up. Detection of the osteogenic differentiation of EHFM-, α-MEM- and DMEM-expanded PDLSCs in the StemPro™ Osteogenesis Differentiation Kit through ALP activity (A) and Alizarin Red S staining (A, F). Relative mRNA expression levels of ALP, OPN, OCN, Runx2, ColI, ColII, IBSP and Msx2 in EHFM-, α-MEM- and DMEM-cultured PDLSCs after differentiation culture for 7, 14 and 21 days (B–D). Relative mRNA expression of all osteogenesis markers in the EHFM-expanded PDLSCs at 7, 14 and 21 days is expressed as % of DMEM and α-MEM (E). Data are presented as the mean ± SEM. α-MEM: minimum essential medium Eagle, alpha modification; ALP: alkaline phosphatase; Col: collagen; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; IBSP: integrin binding sialoprotein; OCN: osteocalcin; OPN: osteopontin; PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.

    Journal: Cell Transplantation

    Article Title: In Vitro Long-Term Expansion and High Osteogenic Potential of Periodontal Ligament Stem Cells: More Than a Mirage

    doi: 10.1177/0963689718807680

    Figure Lengend Snippet: Osteogenic differentiation of EHFM-, α-MEM- and DMEM-expanded PDLSCs in standardized culture medium for the osteogenesis (Life Technologies). Figure shows the schema of the experimental set up. Detection of the osteogenic differentiation of EHFM-, α-MEM- and DMEM-expanded PDLSCs in the StemPro™ Osteogenesis Differentiation Kit through ALP activity (A) and Alizarin Red S staining (A, F). Relative mRNA expression levels of ALP, OPN, OCN, Runx2, ColI, ColII, IBSP and Msx2 in EHFM-, α-MEM- and DMEM-cultured PDLSCs after differentiation culture for 7, 14 and 21 days (B–D). Relative mRNA expression of all osteogenesis markers in the EHFM-expanded PDLSCs at 7, 14 and 21 days is expressed as % of DMEM and α-MEM (E). Data are presented as the mean ± SEM. α-MEM: minimum essential medium Eagle, alpha modification; ALP: alkaline phosphatase; Col: collagen; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; IBSP: integrin binding sialoprotein; OCN: osteocalcin; OPN: osteopontin; PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.

    Article Snippet: In this study, we used and compared three different culture media: α-MEM (no. 22561021; Life Technologies, Milan, Italy) and DMEM (no. D5796; Sigma Aldrich, Milan, Italy), which were supplemented with 10% fetal bovine serum (FBS; no. 10270106; Life Technologies), 100 U/ml penicillin and 100 μmol/ml streptomycin (no. P4333; Sigma Aldrich); and Ham’s F12 medium (no. 21765037; Gibco, Life Technologies) supplemented with 10% FBS, 0.5 U/ml heparin, 50 ng/ml epidermal growth factor (EGF), 25 ng/ml fibroblast growth factor (FGF), 1% bovine serum albumin (BSA), 100 U/ml penicillin, and 100 μmol/ml streptomycin (EHFM) , .

    Techniques: ALP Assay, Activity Assay, Staining, Expressing, Cell Culture, Modification, Binding Assay

    Cell proliferation assay. The proliferation of PDLSCs was determined by the means of population doubling (A) and population doubling time (B) at all passages, mean score and final score. The growth curve of PDLSCs was determined until passage 9 (C). MTT analysis showed the proliferation of PDLSCs at DIV3 and DIV7 in EHFM, α-MEM and DMEM (D). Data are presented as the mean ± SEM. α-MEM: minimum essential medium Eagle, alpha modification; DIV: day in vitro; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.

    Journal: Cell Transplantation

    Article Title: In Vitro Long-Term Expansion and High Osteogenic Potential of Periodontal Ligament Stem Cells: More Than a Mirage

    doi: 10.1177/0963689718807680

    Figure Lengend Snippet: Cell proliferation assay. The proliferation of PDLSCs was determined by the means of population doubling (A) and population doubling time (B) at all passages, mean score and final score. The growth curve of PDLSCs was determined until passage 9 (C). MTT analysis showed the proliferation of PDLSCs at DIV3 and DIV7 in EHFM, α-MEM and DMEM (D). Data are presented as the mean ± SEM. α-MEM: minimum essential medium Eagle, alpha modification; DIV: day in vitro; DMEM: Dulbecco’s modified Eagle’s medium; EHFM: enriched Ham’s F12 medium; MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); PDLSC: periodontal ligament stem cell; SEM: standard error of the mean.

    Article Snippet: In this study, we used and compared three different culture media: α-MEM (no. 22561021; Life Technologies, Milan, Italy) and DMEM (no. D5796; Sigma Aldrich, Milan, Italy), which were supplemented with 10% fetal bovine serum (FBS; no. 10270106; Life Technologies), 100 U/ml penicillin and 100 μmol/ml streptomycin (no. P4333; Sigma Aldrich); and Ham’s F12 medium (no. 21765037; Gibco, Life Technologies) supplemented with 10% FBS, 0.5 U/ml heparin, 50 ng/ml epidermal growth factor (EGF), 25 ng/ml fibroblast growth factor (FGF), 1% bovine serum albumin (BSA), 100 U/ml penicillin, and 100 μmol/ml streptomycin (EHFM) , .

    Techniques: Proliferation Assay, MTT Assay, Modification, In Vitro

    Populations of PDPN+ cells among CAFs affected by environmental conditions. (A) The percentage of PDPN+ cells among CAFs changed in a time-dependent manner by culturing with DMEM-containing growth factors. (B) Populations of PDPN+ cells among CAFs changed by the addition of FBS in concentration- and time-dependent manners. The control (CTRL) condition consisted of DMEM containing 10% FBS and medium changes every day. (C) Populations of PDPN+ cells in cultures of serum-free DMEM increased more rapidly than those in cultures of glucose- and serum-free DMEM.

    Journal: Molecular Cancer

    Article Title: Podoplanin expression in cancer-associated fibroblasts enhances tumor progression of invasive ductal carcinoma of the pancreas

    doi: 10.1186/1476-4598-12-168

    Figure Lengend Snippet: Populations of PDPN+ cells among CAFs affected by environmental conditions. (A) The percentage of PDPN+ cells among CAFs changed in a time-dependent manner by culturing with DMEM-containing growth factors. (B) Populations of PDPN+ cells among CAFs changed by the addition of FBS in concentration- and time-dependent manners. The control (CTRL) condition consisted of DMEM containing 10% FBS and medium changes every day. (C) Populations of PDPN+ cells in cultures of serum-free DMEM increased more rapidly than those in cultures of glucose- and serum-free DMEM.

    Article Snippet: In the PDPN induction assay, DMEM (Sigma Chemical Co., St. Louis, MO) or DMEM containing no glucose (Invitrogen, Carlsbad, CA) was used for cell culture.

    Techniques: Concentration Assay

    Activation of ERK and JNK in the NF1 −/− MPNST cell line ST8814 by shDUSP1 and shDUSP6 knockdown. A , ST8814 MPNST cells infected with shRNA targeting DUSP1 (sh1–D1) or B , shRNA targeting DUSP6 (sh2–D6) were starved overnight in media without serum. Cells were then stimulated with DMEM and 10% FBS for designated minutes (min). Immunoblots were analyzed for p-ERK, p-JNK, p-c-jun and p-p38 with β-actin as a loading control. C , MAPK signaling effects of DUSP1 shRNA-2 (sh2–D1) alone or in combination with DUSP6 shRNA2 (sh2–D6) in ST8814. D , Immunoblot analysis of TP53, p-ATM, cyclin D1 and PARP in ST8814 cells infected with DUSP1 shRNA-2 (sh2–D1) alone and in combination with DUSP6 shRNA2 (sh2–D6). Immunoblots were quantified by densitometry and expressed as percentages relative to NT2 controls (see text). E , Cell death analyzed with TUNEL fluorescence microscopy (red signal) with nuclei stained with DAPI (blue signal). Arrows indicate dying cells. Scale bar is 25 um.

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Targeted inhibition of the dual specificity phosphatases DUSP1 and DUSP6 suppress MPNST growth via JNK

    doi: 10.1158/1078-0432.CCR-18-3224

    Figure Lengend Snippet: Activation of ERK and JNK in the NF1 −/− MPNST cell line ST8814 by shDUSP1 and shDUSP6 knockdown. A , ST8814 MPNST cells infected with shRNA targeting DUSP1 (sh1–D1) or B , shRNA targeting DUSP6 (sh2–D6) were starved overnight in media without serum. Cells were then stimulated with DMEM and 10% FBS for designated minutes (min). Immunoblots were analyzed for p-ERK, p-JNK, p-c-jun and p-p38 with β-actin as a loading control. C , MAPK signaling effects of DUSP1 shRNA-2 (sh2–D1) alone or in combination with DUSP6 shRNA2 (sh2–D6) in ST8814. D , Immunoblot analysis of TP53, p-ATM, cyclin D1 and PARP in ST8814 cells infected with DUSP1 shRNA-2 (sh2–D1) alone and in combination with DUSP6 shRNA2 (sh2–D6). Immunoblots were quantified by densitometry and expressed as percentages relative to NT2 controls (see text). E , Cell death analyzed with TUNEL fluorescence microscopy (red signal) with nuclei stained with DAPI (blue signal). Arrows indicate dying cells. Scale bar is 25 um.

    Article Snippet: In vitro signaling experiments and TUNEL assaysCells were serum starved for 16 hours in DMEM media (Life Technologies).

    Techniques: Activation Assay, Infection, shRNA, Western Blot, TUNEL Assay, Fluorescence, Microscopy, Staining

    The effect of solution added TGF-β in DMEM/F12/10% FBS media + DMSO on CSPG production. CSPG staining per cell data were normalized to FBG0/LN100 sample. Error bars indicate ± SEM. Asterisk denotes p

    Journal: Acta biomaterialia

    Article Title: Astrocytes Specifically Remove Surface-Adsorbed Fibrinogen and Locally Express Chondroitin Sulfate Proteoglycans

    doi: 10.1016/j.actbio.2013.02.047

    Figure Lengend Snippet: The effect of solution added TGF-β in DMEM/F12/10% FBS media + DMSO on CSPG production. CSPG staining per cell data were normalized to FBG0/LN100 sample. Error bars indicate ± SEM. Asterisk denotes p

    Article Snippet: Cells were maintained in DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma).

    Techniques: Staining

    Sphere formation and differentiation from the sciatic nerve in suspension culture. (A) The sciatic nerve was cultured in DMEM-F12 medium containing bFGF. The ND-GFP-expressing cells proliferated and formed spheres by day 32. Bar: 100 µm. (B) The spheres expressing ND-GFP co-expressed p75 NTR and CD34 but did not express β-III tubulin S100 and GFAP. Bar: 100 µm. (C) The ND-GFP-expressing spheres were switched to RPMI 1640 medium containing 10% FBS from DMEM-F12 containing B-27, N2 and bFGF and began to differentiate. At 7 days after switching into medium containing FBS, β-III tubulin-positive neuronal cells which expressed ND-GFP were observed. Bar: 10 µm. (D) At 14 days, the ND-GFP-expressing cells differentiated to GFAP-positive glial cells. Bar: 10 µm (E) At 7 days after culture in FBS medium, the ND-GFP-expressing cells differentiated to K15-positive keratinocytes, some of which still expressed nestin. Bar: 10 µm. (F) At 30 days after culture in FBS medium, the ND-GFP-expressing cells differentiated to α-SMA-positive smooth muscle cells. Bar: 50 µm.

    Journal: PLoS ONE

    Article Title: Nestin-Expressing Stem Cells Promote Nerve Growth in Long-Term 3-Dimensional Gelfoam(R)-Supported Histoculture

    doi: 10.1371/journal.pone.0067153

    Figure Lengend Snippet: Sphere formation and differentiation from the sciatic nerve in suspension culture. (A) The sciatic nerve was cultured in DMEM-F12 medium containing bFGF. The ND-GFP-expressing cells proliferated and formed spheres by day 32. Bar: 100 µm. (B) The spheres expressing ND-GFP co-expressed p75 NTR and CD34 but did not express β-III tubulin S100 and GFAP. Bar: 100 µm. (C) The ND-GFP-expressing spheres were switched to RPMI 1640 medium containing 10% FBS from DMEM-F12 containing B-27, N2 and bFGF and began to differentiate. At 7 days after switching into medium containing FBS, β-III tubulin-positive neuronal cells which expressed ND-GFP were observed. Bar: 10 µm. (D) At 14 days, the ND-GFP-expressing cells differentiated to GFAP-positive glial cells. Bar: 10 µm (E) At 7 days after culture in FBS medium, the ND-GFP-expressing cells differentiated to K15-positive keratinocytes, some of which still expressed nestin. Bar: 10 µm. (F) At 30 days after culture in FBS medium, the ND-GFP-expressing cells differentiated to α-SMA-positive smooth muscle cells. Bar: 50 µm.

    Article Snippet: Culture medium used was DMEM-F12 medium (GIBCO/BRL) containing B-27 (2.5%) (GIBCO/BRL), N2 (1%) (GIBCO/BRL) and 1% penicillin and streptomycin (GIBCO/BRL).

    Techniques: Cell Culture, Expressing

    IL-12 p40 induces the activation of NF- κ B in BV-2 glial cells A , cells incubated in serum-free DMEM/F-12 were treated with different concentrations of p40 2 .” B, lanes 1–3 represent nuclear extract of control cells, nuclear extract of p40 2 -treated cells, and nuclear extract of p40 2 -treated cells incubated with a 100-fold excess of unlabeled oligonucleotide. The concentration of p40 2 used in this experiment was 5 ng/ml. The upper arrow indicates the induced NF- κ B band, and the lower arrow indicates the unbound probe. C , cells plated at 50–60% confluence in 6-well plates were cotransfected with 1 μ g of pNF- κ B-Luc (an NF- κ B-dependent reporter construct) and 50 ng of pRL-TK (a plasmid encoding Renilla .” After 24 h of transfection, cells were stimulated with different concentrations of p40 2 for 6 h under serum-free conditions. Firefly and Renilla .”

    Journal: The Journal of biological chemistry

    Article Title: Induction of Nitric-oxide Synthase and Activation of NF-κB by Interleukin-12 p40 in Microglial Cells *

    doi: 10.1074/jbc.M008262200

    Figure Lengend Snippet: IL-12 p40 induces the activation of NF- κ B in BV-2 glial cells A , cells incubated in serum-free DMEM/F-12 were treated with different concentrations of p40 2 .” B, lanes 1–3 represent nuclear extract of control cells, nuclear extract of p40 2 -treated cells, and nuclear extract of p40 2 -treated cells incubated with a 100-fold excess of unlabeled oligonucleotide. The concentration of p40 2 used in this experiment was 5 ng/ml. The upper arrow indicates the induced NF- κ B band, and the lower arrow indicates the unbound probe. C , cells plated at 50–60% confluence in 6-well plates were cotransfected with 1 μ g of pNF- κ B-Luc (an NF- κ B-dependent reporter construct) and 50 ng of pRL-TK (a plasmid encoding Renilla .” After 24 h of transfection, cells were stimulated with different concentrations of p40 2 for 6 h under serum-free conditions. Firefly and Renilla .”

    Article Snippet: Fetal bovine serum, Hank's balanced salt solution and DMEM/F-12 were from Life Technologies, Inc. L- N G -Monomethylarginine (L-NMMA) and D- N G -monomethylarginine (D-NMMA), NF- κ B SN50, and NF- κ B SN50 M were purchased from Biomol.

    Techniques: Activation Assay, Incubation, Concentration Assay, Construct, Plasmid Preparation, Transfection

    Rod-shaped crystals develop in CHO cells overexpressing recombinant human IgG. A and B , suspension-cultured CHO cells were withdrawn from a 50-liter bioreactor bag on day 5 and immobilized on 3-triethoxysilylpropylamine-coated glass coverslips. Cells were paraformaldehyde-fixed and visualized using a DIC microscope. Scale bars , 50 μm. C , suspension-cultured CHO cells were seeded onto polylysine-coated coverslips and statically cultured for 24 h using DMEM/F-12 medium supplemented with 10% FBS in a humidified incubator at 37 °C, 5% CO 2 . Cells were paraformaldehyde-fixed and visualized using a DIC microscope. Scale bar , 20 or 50 μm as indicated.

    Journal: The Journal of Biological Chemistry

    Article Title: In Vivo

    doi: 10.1074/jbc.M110.204362

    Figure Lengend Snippet: Rod-shaped crystals develop in CHO cells overexpressing recombinant human IgG. A and B , suspension-cultured CHO cells were withdrawn from a 50-liter bioreactor bag on day 5 and immobilized on 3-triethoxysilylpropylamine-coated glass coverslips. Cells were paraformaldehyde-fixed and visualized using a DIC microscope. Scale bars , 50 μm. C , suspension-cultured CHO cells were seeded onto polylysine-coated coverslips and statically cultured for 24 h using DMEM/F-12 medium supplemented with 10% FBS in a humidified incubator at 37 °C, 5% CO 2 . Cells were paraformaldehyde-fixed and visualized using a DIC microscope. Scale bar , 20 or 50 μm as indicated.

    Article Snippet: Suspension-cultured CHO cells were withdrawn from the CultiBag via a sampling portal on day 5 or 6 and seeded onto polylysine-coated glass coverslips using DMEM/F-12 medium (Invitrogen) supplemented with 10% FBS.

    Techniques: Recombinant, Cell Culture, Microscopy

    Overexpression of AIB1 localizes AIB1-Δ4 to the nucleus. a , CHO cells were transfected with FLAG AIB1-Δ4 alone or with an equal amount of HA-AIB1 and plated on glass coverslips in DMEM F-12 + 10% FBS. Cells were fixed and permeabilized

    Journal: The Journal of Biological Chemistry

    Article Title: Role of the Nuclear Receptor Coactivator AIB1-?4 Splice Variant in the Control of Gene Transcription *

    doi: 10.1074/jbc.M110.216200

    Figure Lengend Snippet: Overexpression of AIB1 localizes AIB1-Δ4 to the nucleus. a , CHO cells were transfected with FLAG AIB1-Δ4 alone or with an equal amount of HA-AIB1 and plated on glass coverslips in DMEM F-12 + 10% FBS. Cells were fixed and permeabilized

    Article Snippet: CHO cells were grown in DMEM F-12 (Invitrogen) with 10% FBS.

    Techniques: Over Expression, Transfection

    PDAC cell supernatants activate macrophages that in turn secrete molecules that promote PDAC cells proliferation. ( a ) Isolated human monocytes ( > 98% CD14 + ) were treated with LPS or rBAG3 for 16 h. Then, supernatants were analysed with human IL-6 ELISA. Data are from duplicate samples and obtained in two separate experiments. Error bars indicate s.d. ( b ) Isolated human monocytes ( > 98% CD14 + ) were cultured in RPMI supplemented with 0.15% FBS (control medium) or in MIA PaCa-2-conditioned medium and treated for 16 h with an anti-BAG3 monoclonal antibody at the indicated concentrations. Unrelated murine IgG1 were used as negative control. After treatment, supernatants were collected and analysed with a human IL-6 ELISA Kit. Data are from triplicate samples and obtained in two separate experiments. Error bars indicate s.d. ( c ) Cells were treated as described above. Cells were then harvested for total RNA extraction and analysed by reverse transcription–PCR (RT–PCR). ( d ) Isolated human monocytes were cultured in RPMI supplemented with 0.15% FBS (control medium) or in MIA PaCa-2-conditioned medium, and treated with the F(ab') 2 fragment of an anti-BAG3 monoclonal antibody. F(ab') 2 fragments of a non-specific murine IgG1 were used as negative control. After treatment, supernatants were collected and analysed with a human IL-6 ELISA Kit. Data are from duplicate samples and repeated two times. Error bars indicate s.d. ( e ) MIA PaCa-2 and CFPAC-1 ( f ) cells were cultured in DMEM supplemented with 0.15% FBS (no donor) or conditioned medium from monocytes treated with LPS or rBAG3 for 16 h at the indicated concentrations. After 72 h incubation, cells were analysed by MTT assay. Data are from duplicate samples. Error bars indicate s.d. ( g ) MIA PaCa-2 cells were incubated with human recombinant ( r ) IL-6 (10 ng ml −1 ) or conditioned medium from donor 4 or donor 5 monocytes stimulated with rBAG3 (6 μg ml −1 ) for 16 h. An anti-IL-6 receptor monoclonal antibody (20 μg ml −1 ) was added to inhibit IL-6 activity. After a 72-h incubation, cells were analysed by MTT assay. Data are from duplicate samples. Error bars indicate s.d. P values were calculated by Student's t -test and represented as follows: * P

    Journal: Nature Communications

    Article Title: BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages

    doi: 10.1038/ncomms9695

    Figure Lengend Snippet: PDAC cell supernatants activate macrophages that in turn secrete molecules that promote PDAC cells proliferation. ( a ) Isolated human monocytes ( > 98% CD14 + ) were treated with LPS or rBAG3 for 16 h. Then, supernatants were analysed with human IL-6 ELISA. Data are from duplicate samples and obtained in two separate experiments. Error bars indicate s.d. ( b ) Isolated human monocytes ( > 98% CD14 + ) were cultured in RPMI supplemented with 0.15% FBS (control medium) or in MIA PaCa-2-conditioned medium and treated for 16 h with an anti-BAG3 monoclonal antibody at the indicated concentrations. Unrelated murine IgG1 were used as negative control. After treatment, supernatants were collected and analysed with a human IL-6 ELISA Kit. Data are from triplicate samples and obtained in two separate experiments. Error bars indicate s.d. ( c ) Cells were treated as described above. Cells were then harvested for total RNA extraction and analysed by reverse transcription–PCR (RT–PCR). ( d ) Isolated human monocytes were cultured in RPMI supplemented with 0.15% FBS (control medium) or in MIA PaCa-2-conditioned medium, and treated with the F(ab') 2 fragment of an anti-BAG3 monoclonal antibody. F(ab') 2 fragments of a non-specific murine IgG1 were used as negative control. After treatment, supernatants were collected and analysed with a human IL-6 ELISA Kit. Data are from duplicate samples and repeated two times. Error bars indicate s.d. ( e ) MIA PaCa-2 and CFPAC-1 ( f ) cells were cultured in DMEM supplemented with 0.15% FBS (no donor) or conditioned medium from monocytes treated with LPS or rBAG3 for 16 h at the indicated concentrations. After 72 h incubation, cells were analysed by MTT assay. Data are from duplicate samples. Error bars indicate s.d. ( g ) MIA PaCa-2 cells were incubated with human recombinant ( r ) IL-6 (10 ng ml −1 ) or conditioned medium from donor 4 or donor 5 monocytes stimulated with rBAG3 (6 μg ml −1 ) for 16 h. An anti-IL-6 receptor monoclonal antibody (20 μg ml −1 ) was added to inhibit IL-6 activity. After a 72-h incubation, cells were analysed by MTT assay. Data are from duplicate samples. Error bars indicate s.d. P values were calculated by Student's t -test and represented as follows: * P

    Article Snippet: PANC-1 and MIA PaCa-2 were cultured in DMEM medium containing 10% FBS and 1% P/S; 2.5% of horse serum (Gibco) was added in MIA PaCa-2 culturing medium.

    Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture, Negative Control, RNA Extraction, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Incubation, MTT Assay, Recombinant, Activity Assay

    In vitro effects of Euphorbia fischeriana Steud on the growth of B16 cells. B16 cells were cultured for 24 or 48 h in Dulbecco's modified Eagle's medium containing Euphorbia fischeriana Steud at various concentrations. The effects of Euphorbia fischeriana

    Journal: Experimental and Therapeutic Medicine

    Article Title: Euphorbia fischeriana Steud inhibits malignant melanoma via modulation of the phosphoinositide-3-kinase/Akt signaling pathway

    doi: 10.3892/etm.2016.3061

    Figure Lengend Snippet: In vitro effects of Euphorbia fischeriana Steud on the growth of B16 cells. B16 cells were cultured for 24 or 48 h in Dulbecco's modified Eagle's medium containing Euphorbia fischeriana Steud at various concentrations. The effects of Euphorbia fischeriana

    Article Snippet: Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypsin-EDTA and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: In Vitro, Cell Culture, Modification

    Metabolic inhibition of sulfation of HS chains leads to a loss of sulfated groups of HS, Hsp90α, and Hsp90β from the cell surface ( a ) and to an inhibition of the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with sodium chlorate, after which cells were grown at 37 °C in DMEM-FBS without chlorate. At indicated times, cell surface HS and proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of chlorate-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of chlorate-treated cells. The basal migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p

    Journal: Cell Stress & Chaperones

    Article Title: Cell surface heparan sulfate proteoglycans are involved in the extracellular Hsp90-stimulated migration and invasion of cancer cells

    doi: 10.1007/s12192-018-0955-5

    Figure Lengend Snippet: Metabolic inhibition of sulfation of HS chains leads to a loss of sulfated groups of HS, Hsp90α, and Hsp90β from the cell surface ( a ) and to an inhibition of the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with sodium chlorate, after which cells were grown at 37 °C in DMEM-FBS without chlorate. At indicated times, cell surface HS and proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of chlorate-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of chlorate-treated cells. The basal migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p

    Article Snippet: FBS and DMEM were purchased from GE Healthcare.

    Techniques: Inhibition, Migration, Staining, Flow Cytometry, Cytometry, Wound Healing Assay

    Digestion of HS moieties of HSPGs with a heparinase reduces the level of HS and Hsp90s at the cell surface ( a ) and inhibits the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with a heparinase, after which cells were grown at 37 °C in DMEM-FBS. At indicated times, cell surface proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of heparinase-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of heparinase-treated cells. The migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p

    Journal: Cell Stress & Chaperones

    Article Title: Cell surface heparan sulfate proteoglycans are involved in the extracellular Hsp90-stimulated migration and invasion of cancer cells

    doi: 10.1007/s12192-018-0955-5

    Figure Lengend Snippet: Digestion of HS moieties of HSPGs with a heparinase reduces the level of HS and Hsp90s at the cell surface ( a ) and inhibits the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with a heparinase, after which cells were grown at 37 °C in DMEM-FBS. At indicated times, cell surface proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of heparinase-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of heparinase-treated cells. The migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p

    Article Snippet: FBS and DMEM were purchased from GE Healthcare.

    Techniques: Migration, Staining, Flow Cytometry, Cytometry, Wound Healing Assay

    MCOLN1 is required for MTORC1 activation. ( A ) MCOLN1 ablation compromised MTORC1 reactivation during nutrient refeeding. WT and MCOLN1 − / − human skin fibroblasts were starved for 50 min or followed by nutrient refeeding for 10 or 30 min as indicated. Cell extracts were analyzed by western blotting for the indicated proteins. The graph illustrates the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments), relative to that in WT cells with 30 min refeeding. ( B ) MCOLN1 − / − cells lose MTORC1 activity more easily compared with WT cells when starved. WT and MCOLN1 − / − cells were kept in normal culture medium or starved for 5, 15 or 30 min. Cell extracts were analyzed by western blotting for the indicated proteins. The graph shows the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments), relative to that in fed WT cells. ( C ) Inhibition of MCOLN1 suppressed MTORC1 activity upon starvation and nutrient refeeding. HEK293T cells were kept in normal culture medium, starved for 15 min, or starved for 50 min followed by nutrient refeeding for 5 or 15 min ± ML-SI1 (50 µM) as indicated. MTORC1 activity was assessed by measuring p-RPS6KB (T389) and p-EIF4EBP1 (T37/46) using western blot. The graph shows the mean percentage of the ratio of p-RPS6KB:RPS6KB (left) and p-EIF4EBP1:EIF4EBP1 (right) (mean ± SEM, n = 3 independent experiments), relative to that in nontreated fed cells. ( D, E ) High MTORC1 activity in cells expressing constitutively active RRAGB, which partially mimics nutrient-replete conditions was suppressed by MCOLN1 deletion and BAPTA-AM. HEK293T cells were transfected with RRAGB GTP or together with scramble or MCOLN1 shRNA; 24 h later cells were starved with amino acid-free DMEM for 30 min in the presence or absence of 10 μM BAPTA-AM. ( F ) MCOLN1 inhibition did not alter MTORC1 activity under normal conditions. HEK293T cells were treated with DMSO or ML-SI1 (50 μM) for 1 h in normal medium. The graph illustrates the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments), relative to that in DMSO-treated cells. NS, not significant; *, P

    Journal: Autophagy

    Article Title: A negative feedback regulation of MTORC1 activity by the lysosomal Ca2+ channel MCOLN1 (mucolipin 1) using a CALM (calmodulin)-dependent mechanism

    doi: 10.1080/15548627.2017.1389822

    Figure Lengend Snippet: MCOLN1 is required for MTORC1 activation. ( A ) MCOLN1 ablation compromised MTORC1 reactivation during nutrient refeeding. WT and MCOLN1 − / − human skin fibroblasts were starved for 50 min or followed by nutrient refeeding for 10 or 30 min as indicated. Cell extracts were analyzed by western blotting for the indicated proteins. The graph illustrates the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments), relative to that in WT cells with 30 min refeeding. ( B ) MCOLN1 − / − cells lose MTORC1 activity more easily compared with WT cells when starved. WT and MCOLN1 − / − cells were kept in normal culture medium or starved for 5, 15 or 30 min. Cell extracts were analyzed by western blotting for the indicated proteins. The graph shows the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments), relative to that in fed WT cells. ( C ) Inhibition of MCOLN1 suppressed MTORC1 activity upon starvation and nutrient refeeding. HEK293T cells were kept in normal culture medium, starved for 15 min, or starved for 50 min followed by nutrient refeeding for 5 or 15 min ± ML-SI1 (50 µM) as indicated. MTORC1 activity was assessed by measuring p-RPS6KB (T389) and p-EIF4EBP1 (T37/46) using western blot. The graph shows the mean percentage of the ratio of p-RPS6KB:RPS6KB (left) and p-EIF4EBP1:EIF4EBP1 (right) (mean ± SEM, n = 3 independent experiments), relative to that in nontreated fed cells. ( D, E ) High MTORC1 activity in cells expressing constitutively active RRAGB, which partially mimics nutrient-replete conditions was suppressed by MCOLN1 deletion and BAPTA-AM. HEK293T cells were transfected with RRAGB GTP or together with scramble or MCOLN1 shRNA; 24 h later cells were starved with amino acid-free DMEM for 30 min in the presence or absence of 10 μM BAPTA-AM. ( F ) MCOLN1 inhibition did not alter MTORC1 activity under normal conditions. HEK293T cells were treated with DMSO or ML-SI1 (50 μM) for 1 h in normal medium. The graph illustrates the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments), relative to that in DMSO-treated cells. NS, not significant; *, P

    Article Snippet: HEK293T cells (ATCC, CRL-3216™) and HeLa cells (ATCC, CRL-3216™) were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Thermo Fisher Scientific, 11995065) supplemented with 10% FBS (Thermo Fisher Scientific, 12483020).

    Techniques: Activation Assay, Western Blot, Activity Assay, Inhibition, Expressing, Transfection, shRNA

    MTORC1-dependent regulation of MCOLN1-mediated lysosomal Ca 2+ release. ( A ) Starvation increased MCOLN1 activity as indicated by elevated GECO-MCOLN1 responses to ML-SA1 in HEK293T cells expressing GECO-MCOLN1. Cells were kept in normal culture medium, starved for 50 min or followed by nutrient refeeding for 15 min prior to the measurement. ( B ) Summary of ML-SA1-induced GECO-MCOLN1 responses as in ( A ). ( C, D ) Starvation or refeeding did not affect GECO-MCOLN1 response to GPN (200 µM) ( C ) or ionomycin (Iono, 2 µM) ( D ). ( E ) RRAGB GTP decreased GECO-MCOLN1 responses in starved cells, whereas RRAGB GDP increased GECO-MCOLN1 responses in refeeding cells. HEK293T cells expressing GECO-MCOLN1 and RRAGB WT together with RRAGB GTP or RRAGB GDP were subjected to starvation, or nutrient refeeding (DMEM + 10% FBS, 15 min). ( F, G ) GECO-MCOLN1 response to GPN (200 µM) ( F ) and Ionomycin (2 µM) ( G ) was comparable to conditions in ( E ). ( H-J ) Inhibition of MTOR with AZD8055 (1 µM) induced GECO responses in HEK293T cells expressing GECO-MCOLN1 but not GECO-MCOLN1-DDKK, a nonconducting mutant of MCOLN1. GECO signals were measured in the absence of external Ca 2+ . GECO responses to ionomycin (2 µM, with 2 mM Ca 2+ in the bath) was used to compare the expression levels of GECO-MCOLN1 and GECO-MCOLN1-DDKK. ( K ) S571,576E phosphomimetic mutation of GECO-MCOLN1 (GECO-MCOLN1-SSEE) decreased ML-SA1-induced GECO responses upon starvation compared to GECO-MCOLN1. HEK293T cells expressing GECO-MCOLN1 and GECO-MCOLN1-SSEE, respectively, were subjected to starvation (50 min) prior to the measurement. ( L, M ) GECO-MCOLN1 response to GPN (200 µM) ( I ) or ionomycin (2 µM) ( M ) was comparable in conditions as in ( H ). ( N ) WT MCOLN1 but not MCOLN1-SSEE and MCOLN1-DDKK increased MTORC1 activity. HEK293T cells expressing LAMP1-GFP, MCOLN1-EGFP, MCOLN1 S51E -EGFP, MCOLN1-SSEE-EGFP and MCOLN1-DDKK-GFP, respectively, were starved for 30 min. MCOLN1-DDKK-GFP (MCOLN1 D471,472K MCOLN1 S51E Cell extracts were analyzed by western blotting using anti-p-RPS6KB (T389) and anti-RPS6KB antibodies, and anti-GAPDH antibody was used as a loading control. Histograms represent the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments) in the indicated conditions, relative to that of cells expressing LAMP1. ( O-Q ) Summary of ML-SA1-induced GECO responses in HEK293T cells expressing GECO-MCOLN1 and GECO-MCOLN1-SSAA under normal fed conditions. MCOLN1-SSAA displayed a higher activity in normal fed conditions compared to MCOLN1. However, MCOLN1-SSAA did not have an effect on lysosomal Ca 2+ content. These findings suggest that the lysosome must have a mechanism to maintain its Ca 2+ homeostasis. In this case, although MCOLN1-SSAA increases Ca 2+ release, a compensational mechanism exists to increase Ca 2+ uptake that depends on the endoplasmic reticulum Ca 2+ NS, not significant; *, P

    Journal: Autophagy

    Article Title: A negative feedback regulation of MTORC1 activity by the lysosomal Ca2+ channel MCOLN1 (mucolipin 1) using a CALM (calmodulin)-dependent mechanism

    doi: 10.1080/15548627.2017.1389822

    Figure Lengend Snippet: MTORC1-dependent regulation of MCOLN1-mediated lysosomal Ca 2+ release. ( A ) Starvation increased MCOLN1 activity as indicated by elevated GECO-MCOLN1 responses to ML-SA1 in HEK293T cells expressing GECO-MCOLN1. Cells were kept in normal culture medium, starved for 50 min or followed by nutrient refeeding for 15 min prior to the measurement. ( B ) Summary of ML-SA1-induced GECO-MCOLN1 responses as in ( A ). ( C, D ) Starvation or refeeding did not affect GECO-MCOLN1 response to GPN (200 µM) ( C ) or ionomycin (Iono, 2 µM) ( D ). ( E ) RRAGB GTP decreased GECO-MCOLN1 responses in starved cells, whereas RRAGB GDP increased GECO-MCOLN1 responses in refeeding cells. HEK293T cells expressing GECO-MCOLN1 and RRAGB WT together with RRAGB GTP or RRAGB GDP were subjected to starvation, or nutrient refeeding (DMEM + 10% FBS, 15 min). ( F, G ) GECO-MCOLN1 response to GPN (200 µM) ( F ) and Ionomycin (2 µM) ( G ) was comparable to conditions in ( E ). ( H-J ) Inhibition of MTOR with AZD8055 (1 µM) induced GECO responses in HEK293T cells expressing GECO-MCOLN1 but not GECO-MCOLN1-DDKK, a nonconducting mutant of MCOLN1. GECO signals were measured in the absence of external Ca 2+ . GECO responses to ionomycin (2 µM, with 2 mM Ca 2+ in the bath) was used to compare the expression levels of GECO-MCOLN1 and GECO-MCOLN1-DDKK. ( K ) S571,576E phosphomimetic mutation of GECO-MCOLN1 (GECO-MCOLN1-SSEE) decreased ML-SA1-induced GECO responses upon starvation compared to GECO-MCOLN1. HEK293T cells expressing GECO-MCOLN1 and GECO-MCOLN1-SSEE, respectively, were subjected to starvation (50 min) prior to the measurement. ( L, M ) GECO-MCOLN1 response to GPN (200 µM) ( I ) or ionomycin (2 µM) ( M ) was comparable in conditions as in ( H ). ( N ) WT MCOLN1 but not MCOLN1-SSEE and MCOLN1-DDKK increased MTORC1 activity. HEK293T cells expressing LAMP1-GFP, MCOLN1-EGFP, MCOLN1 S51E -EGFP, MCOLN1-SSEE-EGFP and MCOLN1-DDKK-GFP, respectively, were starved for 30 min. MCOLN1-DDKK-GFP (MCOLN1 D471,472K MCOLN1 S51E Cell extracts were analyzed by western blotting using anti-p-RPS6KB (T389) and anti-RPS6KB antibodies, and anti-GAPDH antibody was used as a loading control. Histograms represent the mean percentage of the ratio of p-RPS6KB:RPS6KB (mean ± SEM, n = 3 independent experiments) in the indicated conditions, relative to that of cells expressing LAMP1. ( O-Q ) Summary of ML-SA1-induced GECO responses in HEK293T cells expressing GECO-MCOLN1 and GECO-MCOLN1-SSAA under normal fed conditions. MCOLN1-SSAA displayed a higher activity in normal fed conditions compared to MCOLN1. However, MCOLN1-SSAA did not have an effect on lysosomal Ca 2+ content. These findings suggest that the lysosome must have a mechanism to maintain its Ca 2+ homeostasis. In this case, although MCOLN1-SSAA increases Ca 2+ release, a compensational mechanism exists to increase Ca 2+ uptake that depends on the endoplasmic reticulum Ca 2+ NS, not significant; *, P

    Article Snippet: HEK293T cells (ATCC, CRL-3216™) and HeLa cells (ATCC, CRL-3216™) were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Thermo Fisher Scientific, 11995065) supplemented with 10% FBS (Thermo Fisher Scientific, 12483020).

    Techniques: Activity Assay, Expressing, Inhibition, Mutagenesis, Western Blot

    CD36 regulates AMPK activation via Fyn-dependent nuclear sequestration of LKB1. A : Suppression of AMPK phosphorylation by CD36 expression. CHO cells lacking CD36 (Vector) or stably expressing CD36 (CHO-CD36) were serum starved (for 16 h in DMEM buffer A), lysed, and probed (in triplicate) for CD36, pAMPK (T172), and GAPDH. Data are representative of two experiments. B : Fyn phosphorylation of LKB1 is enhanced by CD36 expression. Control (Vector) and CD36-expressing cells (CD36) were transiently transfected with Fyn or FynD; and serum-starved and cell lysates (Input) were probed for CD36, LKB1, and Fyn. LKB1 was immunoprecipitated from cell lysates using mouse monoclonal antibody for LKB1 (clone 5c10; Millipore). LKB1 immunoprecipitates (IP:LKB1) were probed with phosphotyrosine PY100 (pLKB1) and LKB1 antibodies. C : CD36 expression induces nuclear sequestration of LKB1 in CHO cells. CHO cells stably expressing CD36 or Vector controls were serum starved, fixed, and processed for IF as described in Research Design and Methods . The cells were stained with mouse monoclonal LKB1 antibody (clone 5c10; Millipore) and with DAPI to visualize the nuclei. Images are representative of multiple fields from three experiments. Scale bar, 10 μm. D : CD36 depletion in C2C12 myotubes abolishes nuclear sequestration of LKB1. C2C12 myotubes treated with siCD36 or siCont were serum starved for 16 h in DMEM buffer A. LKB1 and CD36 were detected using mouse monoclonal anti-LKB1 (5c10; Millipore) and rat monoclonal anti-CD36 antibodies (MF3; AbD Serotec). Images are representative of multiple fields from three experiments. Scale bar, 10 μm.

    Journal: Diabetes

    Article Title: Regulation of AMPK Activation by CD36 Links Fatty Acid Uptake to β-Oxidation

    doi: 10.2337/db14-0582

    Figure Lengend Snippet: CD36 regulates AMPK activation via Fyn-dependent nuclear sequestration of LKB1. A : Suppression of AMPK phosphorylation by CD36 expression. CHO cells lacking CD36 (Vector) or stably expressing CD36 (CHO-CD36) were serum starved (for 16 h in DMEM buffer A), lysed, and probed (in triplicate) for CD36, pAMPK (T172), and GAPDH. Data are representative of two experiments. B : Fyn phosphorylation of LKB1 is enhanced by CD36 expression. Control (Vector) and CD36-expressing cells (CD36) were transiently transfected with Fyn or FynD; and serum-starved and cell lysates (Input) were probed for CD36, LKB1, and Fyn. LKB1 was immunoprecipitated from cell lysates using mouse monoclonal antibody for LKB1 (clone 5c10; Millipore). LKB1 immunoprecipitates (IP:LKB1) were probed with phosphotyrosine PY100 (pLKB1) and LKB1 antibodies. C : CD36 expression induces nuclear sequestration of LKB1 in CHO cells. CHO cells stably expressing CD36 or Vector controls were serum starved, fixed, and processed for IF as described in Research Design and Methods . The cells were stained with mouse monoclonal LKB1 antibody (clone 5c10; Millipore) and with DAPI to visualize the nuclei. Images are representative of multiple fields from three experiments. Scale bar, 10 μm. D : CD36 depletion in C2C12 myotubes abolishes nuclear sequestration of LKB1. C2C12 myotubes treated with siCD36 or siCont were serum starved for 16 h in DMEM buffer A. LKB1 and CD36 were detected using mouse monoclonal anti-LKB1 (5c10; Millipore) and rat monoclonal anti-CD36 antibodies (MF3; AbD Serotec). Images are representative of multiple fields from three experiments. Scale bar, 10 μm.

    Article Snippet: Cells C2C12 myoblasts grown in high-glucose DMEM with 10% FBS (Life Technologies) supplemented with l -glutamine (2 mmol/L) were differentiated in DMEM containing 2% horse serum.

    Techniques: Activation Assay, Expressing, Plasmid Preparation, Stable Transfection, Transfection, Immunoprecipitation, Staining

    Activator of thyroid and retinoid receptor (ACTR) enhances sorafenib resistance by affecting aerobic glycolysis in vitro. A and B, The relative viability curves of ACTR WT or KO HepG2 cells or ACTR KO HepG2 cells transiently transfected with ACTR, as well as Huh‐7 cells in DMEM with high glucose (25 mmol/L), transfected with ACTR siRNA or ACTR siRNA plus ACTR expression vector or non‐specific control for siRNA (Control siRNA); they were treated with Deoxy‐d‐glucose (2‐DG) (2.5 mmol/L) and increasing concentrations of sorafenib as indicated above. After 72 h, cell viability assays were performed using the CCK‐8. The group without treatment of sorafenib had 100% viable cells and was used as an internal control for comparison. The representative immunoblot with ACTR indicates ACTR expression levels. C and D, The relative viability curves of HepG2 cells (C) or Huh‐7 cells (D) transfected and treated as in (A) or (B) and cultured in DMEM with low glucose (5.5 mmol/L). E and F, Colony formation assays of HepG2 and Huh‐7 cells treated as in (A) and (B) with sorafenib (6 μmol/L) or not. G, Representative flow cytometry analysis of Annexin V (1:1000) and propidium iodide (1:1000) staining was carried out in HepG2 ACTR WT cells, KO cells, WT cells and KO cells treated with 2‐DG (2.5 mmol/L) and sorafenib (6 μmol/L) for 6 h. Data shown are mean ± SD of triplicate measurements that have been repeated three times with similar results. * P

    Journal: Cancer Science

    Article Title: Activator of thyroid and retinoid receptor increases sorafenib resistance in hepatocellular carcinoma by facilitating the Warburg effect, et al. Activator of thyroid and retinoid receptor increases sorafenib resistance in hepatocellular carcinoma by facilitating the Warburg effect

    doi: 10.1111/cas.14412

    Figure Lengend Snippet: Activator of thyroid and retinoid receptor (ACTR) enhances sorafenib resistance by affecting aerobic glycolysis in vitro. A and B, The relative viability curves of ACTR WT or KO HepG2 cells or ACTR KO HepG2 cells transiently transfected with ACTR, as well as Huh‐7 cells in DMEM with high glucose (25 mmol/L), transfected with ACTR siRNA or ACTR siRNA plus ACTR expression vector or non‐specific control for siRNA (Control siRNA); they were treated with Deoxy‐d‐glucose (2‐DG) (2.5 mmol/L) and increasing concentrations of sorafenib as indicated above. After 72 h, cell viability assays were performed using the CCK‐8. The group without treatment of sorafenib had 100% viable cells and was used as an internal control for comparison. The representative immunoblot with ACTR indicates ACTR expression levels. C and D, The relative viability curves of HepG2 cells (C) or Huh‐7 cells (D) transfected and treated as in (A) or (B) and cultured in DMEM with low glucose (5.5 mmol/L). E and F, Colony formation assays of HepG2 and Huh‐7 cells treated as in (A) and (B) with sorafenib (6 μmol/L) or not. G, Representative flow cytometry analysis of Annexin V (1:1000) and propidium iodide (1:1000) staining was carried out in HepG2 ACTR WT cells, KO cells, WT cells and KO cells treated with 2‐DG (2.5 mmol/L) and sorafenib (6 μmol/L) for 6 h. Data shown are mean ± SD of triplicate measurements that have been repeated three times with similar results. * P

    Article Snippet: At the same time, the sensitivity of low glucose DMEM (5.5 mM, Gibco) to sorafenib was detected.

    Techniques: In Vitro, Transfection, Expressing, Plasmid Preparation, CCK-8 Assay, Cell Culture, Flow Cytometry, Staining

    A2B receptors are expressed in Schwann cells. (A) Western blot image of A2B expression in RSC-96 cells. GAPDH is used as a housekeeping control. Relative expression of A2B protein in RSC-96 cells co-cultured with DOK or HSC-3 is presented as fold change over DMEM treated RSC-96 cells. (B) Western blot quantification of A2B protein expression in RSC-96 cells. (C) Immunofluorescence labeling of the A2B receptor (green) and nucleus (DAPI, blue) in RSC-96 cells co-cultured with either DOK or HSC-3. Scale: 100 μm. (D) Relative A2B mRNA fold change in RSC-96 cells co-cultured with either DOK or HSC-3 over control RSC-96 cells. No differences are detected across different groups. Kruskal-Wallis test followed by Dunn's multiple comparison analysis.

    Journal: Heliyon

    Article Title: Reciprocal interactions between cancer and Schwann cells contribute to oral cancer progression and pain

    doi: 10.1016/j.heliyon.2019.e01223

    Figure Lengend Snippet: A2B receptors are expressed in Schwann cells. (A) Western blot image of A2B expression in RSC-96 cells. GAPDH is used as a housekeeping control. Relative expression of A2B protein in RSC-96 cells co-cultured with DOK or HSC-3 is presented as fold change over DMEM treated RSC-96 cells. (B) Western blot quantification of A2B protein expression in RSC-96 cells. (C) Immunofluorescence labeling of the A2B receptor (green) and nucleus (DAPI, blue) in RSC-96 cells co-cultured with either DOK or HSC-3. Scale: 100 μm. (D) Relative A2B mRNA fold change in RSC-96 cells co-cultured with either DOK or HSC-3 over control RSC-96 cells. No differences are detected across different groups. Kruskal-Wallis test followed by Dunn's multiple comparison analysis.

    Article Snippet: To study the effect of cancer cells on Schwann cell intracellular Ca2+ levels, RSC-96 cells were seeded onto glass coverslips and co-cultured with either inserts (3-μm pore size, Corning) containing DMEM alone, inserts with DOK culture, or inserts with HSC-3 culture ( A).

    Techniques: Western Blot, Expressing, Cell Culture, Immunofluorescence, Labeling

    Oral SCC induces Schwann cell hypertrophy and increased Ca 2+ influx. (A) Co-culture model. To study Schwann cell morphology and basal intracellular Ca 2+ levels following exposure to cancer cells, RSC-96 cells were cultured in the lower chamber, while either DOK or HSC-3 cells were cultured in the cell inserts. The inserts have 3 μm-sized pores that allow free exchange of media but do not allow cells to migrate through. (B) Representative images of RSC-96 cells cultured with inserts containing DMEM, DOK or HSC-3. Scale: 100 μm. (C) The mean size of RSC-96 cells was greater when co-cultured with HSC-3 cells, compared to RSC-96 cells co-culture with DOK or with DMEM alone. (D) Intracellular Ca 2+ concentration was higher in Schwann cells co-cultured with HSC-3 cells compared with co-culture with DOK or with DMEM alone. (E) Representative Ca 2+ responses of RSC-96 cells to DMEM, HSC-3 supernatant, and 100 μM ATP. Each color represents a different cell. One-way ANOVA with Tukey's post hoc analysis.

    Journal: Heliyon

    Article Title: Reciprocal interactions between cancer and Schwann cells contribute to oral cancer progression and pain

    doi: 10.1016/j.heliyon.2019.e01223

    Figure Lengend Snippet: Oral SCC induces Schwann cell hypertrophy and increased Ca 2+ influx. (A) Co-culture model. To study Schwann cell morphology and basal intracellular Ca 2+ levels following exposure to cancer cells, RSC-96 cells were cultured in the lower chamber, while either DOK or HSC-3 cells were cultured in the cell inserts. The inserts have 3 μm-sized pores that allow free exchange of media but do not allow cells to migrate through. (B) Representative images of RSC-96 cells cultured with inserts containing DMEM, DOK or HSC-3. Scale: 100 μm. (C) The mean size of RSC-96 cells was greater when co-cultured with HSC-3 cells, compared to RSC-96 cells co-culture with DOK or with DMEM alone. (D) Intracellular Ca 2+ concentration was higher in Schwann cells co-cultured with HSC-3 cells compared with co-culture with DOK or with DMEM alone. (E) Representative Ca 2+ responses of RSC-96 cells to DMEM, HSC-3 supernatant, and 100 μM ATP. Each color represents a different cell. One-way ANOVA with Tukey's post hoc analysis.

    Article Snippet: To study the effect of cancer cells on Schwann cell intracellular Ca2+ levels, RSC-96 cells were seeded onto glass coverslips and co-cultured with either inserts (3-μm pore size, Corning) containing DMEM alone, inserts with DOK culture, or inserts with HSC-3 culture ( A).

    Techniques: Co-Culture Assay, Cell Culture, Concentration Assay

    Oral SCC promotes Schwann cell proliferation, migration, and invasion. (A) To study the effect of HSC-3 or DOK cells on Schwann cell proliferation, RSC-96 cells were cultured in the lower chamber, while either DOK or HSC-3 cells were cultured in the cell inserts. (B) Growth rate of RSC-96 cells measured by the RTCA, increased with HSC-3 cell number. (C) Optical density (OD) measured using the MTS assay increased when RSC-96 cells were co-cultured with DOK or HSC-3 cells compared to DMEM alone. Kruskal-Wallis with Dunn's test. (D) To study the effect of HSC-3 or DOK cells on RSC-96 migration, RSC-96 cells were cultured in a migration chamber. Either HSC-3 or DOK cells were seeded in the bottom chamber. (E) Migration of RSC-96 cells towards HSC-3 cells increased in a cell number dependent manner. (F) Increased numbers of RSC-96 cells migrated toward HSC-3 cells compared to DOK or DMEM. (G) To study effect of HSC-3 or DOK cells on RSC-96 invasion, RSC-96 cells were cultured in invasion chambers. Either HSC-3 or DOK cells were seeded in the bottom chamber. (H) Increased numbers of invaded RSC-96 cells towards HSC-3 compared to DOK or DMEM. One-way ANOVA with Tukey's post hoc analysis.

    Journal: Heliyon

    Article Title: Reciprocal interactions between cancer and Schwann cells contribute to oral cancer progression and pain

    doi: 10.1016/j.heliyon.2019.e01223

    Figure Lengend Snippet: Oral SCC promotes Schwann cell proliferation, migration, and invasion. (A) To study the effect of HSC-3 or DOK cells on Schwann cell proliferation, RSC-96 cells were cultured in the lower chamber, while either DOK or HSC-3 cells were cultured in the cell inserts. (B) Growth rate of RSC-96 cells measured by the RTCA, increased with HSC-3 cell number. (C) Optical density (OD) measured using the MTS assay increased when RSC-96 cells were co-cultured with DOK or HSC-3 cells compared to DMEM alone. Kruskal-Wallis with Dunn's test. (D) To study the effect of HSC-3 or DOK cells on RSC-96 migration, RSC-96 cells were cultured in a migration chamber. Either HSC-3 or DOK cells were seeded in the bottom chamber. (E) Migration of RSC-96 cells towards HSC-3 cells increased in a cell number dependent manner. (F) Increased numbers of RSC-96 cells migrated toward HSC-3 cells compared to DOK or DMEM. (G) To study effect of HSC-3 or DOK cells on RSC-96 invasion, RSC-96 cells were cultured in invasion chambers. Either HSC-3 or DOK cells were seeded in the bottom chamber. (H) Increased numbers of invaded RSC-96 cells towards HSC-3 compared to DOK or DMEM. One-way ANOVA with Tukey's post hoc analysis.

    Article Snippet: To study the effect of cancer cells on Schwann cell intracellular Ca2+ levels, RSC-96 cells were seeded onto glass coverslips and co-cultured with either inserts (3-μm pore size, Corning) containing DMEM alone, inserts with DOK culture, or inserts with HSC-3 culture ( A).

    Techniques: Migration, Cell Culture, MTS Assay

    Schwann cells promote oral SCC proliferation, migration, and invasion. (A) To study the effect of RSC-96 cells on proliferation, HSC-3 or DOK cells were cultured in the lower chamber, and RSC-96 cells were cultured in the cell inserts. Cell culture media DMEM in the lower chamber was used as control. (B) Growth rate of HSC-3 cells, measured with the RTCA, increased with RSC-96 cell number. (C) HSC-3 cells proliferated more than DOK when co-cultured with RSC-96 cells. Data are presented as a percentage increase in OD from DMEM treated controls. (D) To study the effect of RSC-96 on cancer cell migration, HSC-3 or DOK cells were cultured in migration chambers with RSC-96 cells or DMEM (control) in the bottom chamber. (E) HSC-3 cells migrated towards RSC-96 cells in a cell number dependent manner. (F) HSC-3 cells migrated more than DOK towards RSC-96 cells. Data are presented as a percentage increase in number of migrated cells towards RSC-96 relative to DMEM controls. (G) To study effect of RSC-96 cells on cancer cell invasion, HSC-3 and DOK cells were cultured in invasion chambers with RSC-96 cells seeded at the bottom chamber. Bottom chambers containing DMEM alone were used as controls. (H) Increased invasion of HSC-3 cells compared to DOK cells in the presence of RSC-96 cells. Data are presented as percentage increase in number of invaded cells towards RSC-96 relative to DMEM treated controls. B, E, one-way ANOVA with Tukey's post hoc analysis; C, Mann-Whitney U-test, F, H, student's t-test.

    Journal: Heliyon

    Article Title: Reciprocal interactions between cancer and Schwann cells contribute to oral cancer progression and pain

    doi: 10.1016/j.heliyon.2019.e01223

    Figure Lengend Snippet: Schwann cells promote oral SCC proliferation, migration, and invasion. (A) To study the effect of RSC-96 cells on proliferation, HSC-3 or DOK cells were cultured in the lower chamber, and RSC-96 cells were cultured in the cell inserts. Cell culture media DMEM in the lower chamber was used as control. (B) Growth rate of HSC-3 cells, measured with the RTCA, increased with RSC-96 cell number. (C) HSC-3 cells proliferated more than DOK when co-cultured with RSC-96 cells. Data are presented as a percentage increase in OD from DMEM treated controls. (D) To study the effect of RSC-96 on cancer cell migration, HSC-3 or DOK cells were cultured in migration chambers with RSC-96 cells or DMEM (control) in the bottom chamber. (E) HSC-3 cells migrated towards RSC-96 cells in a cell number dependent manner. (F) HSC-3 cells migrated more than DOK towards RSC-96 cells. Data are presented as a percentage increase in number of migrated cells towards RSC-96 relative to DMEM controls. (G) To study effect of RSC-96 cells on cancer cell invasion, HSC-3 and DOK cells were cultured in invasion chambers with RSC-96 cells seeded at the bottom chamber. Bottom chambers containing DMEM alone were used as controls. (H) Increased invasion of HSC-3 cells compared to DOK cells in the presence of RSC-96 cells. Data are presented as percentage increase in number of invaded cells towards RSC-96 relative to DMEM treated controls. B, E, one-way ANOVA with Tukey's post hoc analysis; C, Mann-Whitney U-test, F, H, student's t-test.

    Article Snippet: To study the effect of cancer cells on Schwann cell intracellular Ca2+ levels, RSC-96 cells were seeded onto glass coverslips and co-cultured with either inserts (3-μm pore size, Corning) containing DMEM alone, inserts with DOK culture, or inserts with HSC-3 culture ( A).

    Techniques: Migration, Cell Culture, MANN-WHITNEY

    Effect of IBTP treatment on mitochondrial respiration of MB231 cells. Panel A: Cells plated on XF24 plates were treated with the indicated concentrations of IBTP or BTPP for 4h in 0.5% FBS-containing medium. After treatment, the medium was removed and replaced with XF assay medium (DMEM, containing 5mM glucose, 0.5% FBS, 5mM HEPES without bicarbonate) and equilibrated 1h before OCR measurement. Panel B: Cells plated on 6-well plates were treated with the indicated concentrations of IBTP or BTPP for 4h. After the incubation, the cells were harvested immediately by trypsinization. The harvested cells were replated in XF24 plates and allowed adhere for an additional 20h in complete medium containing 10% FBS (total 24h). The medium was removed and replaced with assay medium and equilibrated 1h before OCR measurement. Panel C : After 4h of IBTP or BTPP treatment, the medium was replaced with complete medium containing 10% FBS, and incubated for 48h. The cells were harvested after 48h, replated in XF24 plates and allowed adhere for an additional 20h in complete medium. The medium was replaced with assay media and incubated 1h before measurement of OCR (total duration 72h).

    Journal: PLoS ONE

    Article Title: A Novel Class of Mitochondria-Targeted Soft Electrophiles Modifies Mitochondrial Proteins and Inhibits Mitochondrial Metabolism in Breast Cancer Cells through Redox Mechanisms

    doi: 10.1371/journal.pone.0120460

    Figure Lengend Snippet: Effect of IBTP treatment on mitochondrial respiration of MB231 cells. Panel A: Cells plated on XF24 plates were treated with the indicated concentrations of IBTP or BTPP for 4h in 0.5% FBS-containing medium. After treatment, the medium was removed and replaced with XF assay medium (DMEM, containing 5mM glucose, 0.5% FBS, 5mM HEPES without bicarbonate) and equilibrated 1h before OCR measurement. Panel B: Cells plated on 6-well plates were treated with the indicated concentrations of IBTP or BTPP for 4h. After the incubation, the cells were harvested immediately by trypsinization. The harvested cells were replated in XF24 plates and allowed adhere for an additional 20h in complete medium containing 10% FBS (total 24h). The medium was removed and replaced with assay medium and equilibrated 1h before OCR measurement. Panel C : After 4h of IBTP or BTPP treatment, the medium was replaced with complete medium containing 10% FBS, and incubated for 48h. The cells were harvested after 48h, replated in XF24 plates and allowed adhere for an additional 20h in complete medium. The medium was replaced with assay media and incubated 1h before measurement of OCR (total duration 72h).

    Article Snippet: Breast cancer cell lines MDA-MB-231 (MB231) and MDA-MB-468 (MB468) human breast adenocarcinoma cells were cultured in DMEM (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA).

    Techniques: XF Assay, Incubation

    Cell-survival quantification with the XTT assay after treatment with recombinant NGF (open bars) or proNGF123 (filled bars) at the concentrations indicated for 72 h in defined DMEM. RN22 schwannoma cells ( A ), PC12nnr cells ( B ), and C6 glioma cells ( C

    Journal:

    Article Title: Construction of a mutated pro-nerve growth factor resistant to degradation and suitable for biophysical and cellular utilization

    doi: 10.1073/pnas.0604139103

    Figure Lengend Snippet: Cell-survival quantification with the XTT assay after treatment with recombinant NGF (open bars) or proNGF123 (filled bars) at the concentrations indicated for 72 h in defined DMEM. RN22 schwannoma cells ( A ), PC12nnr cells ( B ), and C6 glioma cells ( C

    Article Snippet: PC12 (from Lloyd A. Greene, Columbia University, New York, NY), PC12nnr (from Phil Barker, McGill University, Montreal, CA), C6 glioma (from ATCC, Manassas, VA), and RN22 schwannoma (from Bruce Carter, Vanderbilt University, Nashville, TN) cells were grown in DMEM (Cellgro; Voigt Global Distribution, Kansas City, MO) supplemented with 10% horse serum, 5% FBS, 4.5 mg/ml glucose, 4.0 mM l -glutamine, 100 units/ml penicillin, 100 pg/ml streptomycin, and 0.25 pg/ml amphotericin-B25 at 37°C in a humid atmosphere containing 5% CO2 .

    Techniques: XTT Assay, Recombinant

    Schematic diagram of the experiment. Isolation and characterization of A. Umbilical cord blood cluster of differentiation 133 + , B. Rat pancreatic mesenchymal stem cells, C. Culture type, C1; Transwell culture, C2; Direct co-culture and D. Method of differentiation into insulin producing cells. FBS; Fetal bovine serum, DMEM; Dulbecco’s Modified Eagle’s medium, bFGF; Basic fibroblast growth factor, RA; Retinoic acid, NA; Nicotinamide and RT-PCR; Reverse transcription-polymerase chain reaction.

    Journal: Cell Journal (Yakhteh)

    Article Title: In Vitro Differentiation of Human Umbilical Cord Blood CD133+Cells into Insulin Producing Cells in Co-Culture with Rat Pancreatic Mesenchymal Stem Cells

    doi:

    Figure Lengend Snippet: Schematic diagram of the experiment. Isolation and characterization of A. Umbilical cord blood cluster of differentiation 133 + , B. Rat pancreatic mesenchymal stem cells, C. Culture type, C1; Transwell culture, C2; Direct co-culture and D. Method of differentiation into insulin producing cells. FBS; Fetal bovine serum, DMEM; Dulbecco’s Modified Eagle’s medium, bFGF; Basic fibroblast growth factor, RA; Retinoic acid, NA; Nicotinamide and RT-PCR; Reverse transcription-polymerase chain reaction.

    Article Snippet: Rat PMCs were cultured for 21 days in Dulbecco’s Modified Eagle Medium (DMEM) that contained 10% FBS, 50 mg/ml ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM b-glycerol phosphate (all purchased from Sigma, Germany).

    Techniques: Isolation, Co-Culture Assay, Modification, Reverse Transcription Polymerase Chain Reaction

    Schematic illustration of the method for differentiation of ESCs into fibroblasts in three-dimensional type I collagen gel culture. Undifferentiated ESCs are cultured on MEF feeder layer in six-well plate. ESCs are detached with collagenase and re-suspended with differentiation medium. Cells are then placed into a Petri dish and cultured for 4–5 d to allow formation of EBs. EBs are cast into type I collagen gels in a 12-well plate (1.0 ml/well) and cultured for 21 d three-dimensionally in collagen gels with basal medium. Gels are dissolved by collagenase and the cells are suspended in 10% FCS-DMEM. Differentiated fibroblasts are cultured in 100 mm culture dishes. EBs are lost with serial feeding and passaging.

    Journal: In Vitro Cellular & Developmental Biology. Animal

    Article Title: Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type I collagen gel cultures

    doi: 10.1007/s11626-010-9367-2

    Figure Lengend Snippet: Schematic illustration of the method for differentiation of ESCs into fibroblasts in three-dimensional type I collagen gel culture. Undifferentiated ESCs are cultured on MEF feeder layer in six-well plate. ESCs are detached with collagenase and re-suspended with differentiation medium. Cells are then placed into a Petri dish and cultured for 4–5 d to allow formation of EBs. EBs are cast into type I collagen gels in a 12-well plate (1.0 ml/well) and cultured for 21 d three-dimensionally in collagen gels with basal medium. Gels are dissolved by collagenase and the cells are suspended in 10% FCS-DMEM. Differentiated fibroblasts are cultured in 100 mm culture dishes. EBs are lost with serial feeding and passaging.

    Article Snippet: Commercially available reagents were obtained as follows: transforming growth factor (TGF)-β1 was from R & D Systems (Minneapolis, MN); prostaglandin E2 (PGE2 ), monoclonal anti-α-smooth muscle actin (SMA), anti-pan cytokeratin monoclonal, anti-vimentin monoclonal antibodies, anti-mouse IgG FITC (fluorescein isothiocyanate stain-green immunofluorescence) conjugate, propidium iodide and 2-mercaptoethanol were from Sigma (St. Louis, MO); ESGRO® (leukemia inhibitory factor; LIF), anti-stage specific embryonic antigen (SSEA)-1 and 4 monoclonal antibodies were from Chemicon International (Temecula, CA); Dulbecco’s modified eagle’s medium (DMEM), fetal calf serum (FCS), DMEM/F12 [1:1 mixture], KnockOut™ serum replacement, KnockOut™ DMEM, non-essential amino acids, l -glutamine, basic fibroblast growth factor (bFGF), collagenase type IV, and 0.05% Trypsin-EDTA were from Invitrogen (Carlsbad, CA).

    Techniques: Cell Culture, Passaging

    Effects of TGF-β1 and PGE2 on proliferation of fibroblasts derived from human ESCs. Fibroblasts were cultured in monolayers in 10% FCS-DMEM with or without PGE 2 (10 −7 mol/l) or TGF-β1 (10 −10 mol/l). Cells were detached with trypsin/EDTA and cell numbers were determined using a Coulter electronic cell counter. Vertical axis cell number (×10 5 cells/ml); horizontal axis , time (d). Each point shows mean ± SEM of three separate experiments, each of which included triplicated wells. Circles control, triangles PGE 2 , squares TGF-β1; * p

    Journal: In Vitro Cellular & Developmental Biology. Animal

    Article Title: Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type I collagen gel cultures

    doi: 10.1007/s11626-010-9367-2

    Figure Lengend Snippet: Effects of TGF-β1 and PGE2 on proliferation of fibroblasts derived from human ESCs. Fibroblasts were cultured in monolayers in 10% FCS-DMEM with or without PGE 2 (10 −7 mol/l) or TGF-β1 (10 −10 mol/l). Cells were detached with trypsin/EDTA and cell numbers were determined using a Coulter electronic cell counter. Vertical axis cell number (×10 5 cells/ml); horizontal axis , time (d). Each point shows mean ± SEM of three separate experiments, each of which included triplicated wells. Circles control, triangles PGE 2 , squares TGF-β1; * p

    Article Snippet: Commercially available reagents were obtained as follows: transforming growth factor (TGF)-β1 was from R & D Systems (Minneapolis, MN); prostaglandin E2 (PGE2 ), monoclonal anti-α-smooth muscle actin (SMA), anti-pan cytokeratin monoclonal, anti-vimentin monoclonal antibodies, anti-mouse IgG FITC (fluorescein isothiocyanate stain-green immunofluorescence) conjugate, propidium iodide and 2-mercaptoethanol were from Sigma (St. Louis, MO); ESGRO® (leukemia inhibitory factor; LIF), anti-stage specific embryonic antigen (SSEA)-1 and 4 monoclonal antibodies were from Chemicon International (Temecula, CA); Dulbecco’s modified eagle’s medium (DMEM), fetal calf serum (FCS), DMEM/F12 [1:1 mixture], KnockOut™ serum replacement, KnockOut™ DMEM, non-essential amino acids, l -glutamine, basic fibroblast growth factor (bFGF), collagenase type IV, and 0.05% Trypsin-EDTA were from Invitrogen (Carlsbad, CA).

    Techniques: Derivative Assay, Cell Culture