Structured Review

FUJIFILM dulbecco s modified eagle s medium
Effect of conventional anticancer agents against PANC-1 cells after 24 hours in NDM and DMEM. Data are expressed as the mean ± standard deviation, n=3. Abbreviations: NDM, nutrient-deprived medium; DMEM, <t>Dulbecco’s</t> Modified <t>Eagle’s</t> Medium; 5-FU, 5-fluorouracil; 2-DG, 2-deoxyglucose.
Dulbecco S Modified Eagle S Medium, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "(+)-Grandifloracin, an antiausterity agent, induces autophagic PANC-1 pancreatic cancer cell death"

Article Title: (+)-Grandifloracin, an antiausterity agent, induces autophagic PANC-1 pancreatic cancer cell death

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S52168

Effect of conventional anticancer agents against PANC-1 cells after 24 hours in NDM and DMEM. Data are expressed as the mean ± standard deviation, n=3. Abbreviations: NDM, nutrient-deprived medium; DMEM, Dulbecco’s Modified Eagle’s Medium; 5-FU, 5-fluorouracil; 2-DG, 2-deoxyglucose.
Figure Legend Snippet: Effect of conventional anticancer agents against PANC-1 cells after 24 hours in NDM and DMEM. Data are expressed as the mean ± standard deviation, n=3. Abbreviations: NDM, nutrient-deprived medium; DMEM, Dulbecco’s Modified Eagle’s Medium; 5-FU, 5-fluorouracil; 2-DG, 2-deoxyglucose.

Techniques Used: Standard Deviation, Modification

Assessment of cytotoxicity of conventional anticancer agents against PANC-1 cells in Dulbecco’s Modified Eagle’s Medium. Data are expressed as the mean ± standard deviation, n=3. * P
Figure Legend Snippet: Assessment of cytotoxicity of conventional anticancer agents against PANC-1 cells in Dulbecco’s Modified Eagle’s Medium. Data are expressed as the mean ± standard deviation, n=3. * P

Techniques Used: Modification, Standard Deviation

Effect of GF against Akt, mTOR, LC3A/B I, and LC3A/B II. Abbreviations: GF, (+)-grandifloracin; NDM, nutrient-deprived medium; DMEM, Dulbecco’s Modified Eagle’s Medium.
Figure Legend Snippet: Effect of GF against Akt, mTOR, LC3A/B I, and LC3A/B II. Abbreviations: GF, (+)-grandifloracin; NDM, nutrient-deprived medium; DMEM, Dulbecco’s Modified Eagle’s Medium.

Techniques Used: Modification

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Cell Culture:

Article Title: Efficient differentiation and polarization of primary cultured neurons on poly(lactic acid) scaffolds with microgrooved structures
Article Snippet: All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Animals for Scientific Purposes at Tokai University, which was established based on Act on Welfare and Management of Animals in Japan. .. Cell cultures PC12 cells were cultured in Dulbecco’s Modified Eagle’s medium with High Glucose (Wako) supplemented with 7.5% (w/v) heat-inactivated fetal bovine serum (FBS; PAA Laboratories), 7.5% (w/v) heat-inactivated horse serum (Gibco), 100 U/mL penicillin G, 100 µg/mL streptomycin, and 100 µg/mL sodium pyruvate. .. Mouse primary cortical neurons were cultured as previously reported , .

Article Title: Photodynamic Therapy Using a Novel Phosphorus Tetraphenylporphyrin Induces an Anticancer Effect via Bax/Bcl-xL-related Mitochondrial Apoptosis in Biliary Cancer Cells
Article Snippet: .. Cell culture Human biliary cancer cells (NOZ), mouse embryonic fibroblasts (MEF) (Japanese Collection of Research Bioresources, Tokyo, Japan), human breast cancer cells (MCF7), human hepatocyte carcinoma cells (HepG2) and human embryonic kidney 293 cells (HEK293) (Riken Cell Bank, Ibaraki, Japan) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with L-glutamine and phenol red (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) in a humidified atmosphere with 5% CO2 at 37°C. .. The medium was supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and penicillin-streptomycin-amphotericin B (Fujifilm Wako).

Article Title: Increase in Toxicity of Anticancer Drugs by PMTPV, a Claudin-1-Binding Peptide, Mediated via Down-Regulation of Claudin-1 in Human Lung Adenocarcinoma A549 Cells
Article Snippet: Cell Culture and TransfectionA549 cells derived from human lung adenocarcinoma were purchased from the RIKEN BRC through the National Bio-Resource Project of the MEXT (Ibaraki, Japan). .. The cells were cultured in Dulbecco’s modified Eagle’s medium (Fujifilm Wako Pure Chemical) supplemented with 5% fetal calf serum (HyClone, Logan, UT, USA), 0.07 mg/mL penicillin-G potassium, and 0.14 mg/mL streptomycin sulfate in a 5% CO2 atmosphere at 37 °C as described previously [ ]. ..

Article Title: N-Aryl Pyrido Cyanine derivatives: nuclear and organelle DNA markers for two-photon and super-resolution imaging
Article Snippet: The fluorescent intensity of equal amount mixture of oligonucleotide and PC dye solution was measured by EnSpire (PerkinElmer). .. Animal and plant cell cultures for fluorescence imaging Cell culture lines (HeLa, U-2OS, C6, NIH3T3) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Wako) containing 10% fetal bovine serum at 37 °C in a 5% CO2 /95% air incubator. .. These lines (2 × 104 cells /mL) were transferred on each well of a glass-bottom 8-well slide and cultured 1day before imaging.

Modification:

Article Title: Efficient differentiation and polarization of primary cultured neurons on poly(lactic acid) scaffolds with microgrooved structures
Article Snippet: All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Animals for Scientific Purposes at Tokai University, which was established based on Act on Welfare and Management of Animals in Japan. .. Cell cultures PC12 cells were cultured in Dulbecco’s Modified Eagle’s medium with High Glucose (Wako) supplemented with 7.5% (w/v) heat-inactivated fetal bovine serum (FBS; PAA Laboratories), 7.5% (w/v) heat-inactivated horse serum (Gibco), 100 U/mL penicillin G, 100 µg/mL streptomycin, and 100 µg/mL sodium pyruvate. .. Mouse primary cortical neurons were cultured as previously reported , .

Article Title: Tissue-scale tensional homeostasis in skin regulates structure and physiological function
Article Snippet: .. Normal human dermal fibroblasts (NHDFs) were purchased from KURABO and were maintained in Dulbecco’s modified Eagle’s medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, JAPAN) that was supplemented with 10% foetal bovine serum (Biowest, Rue du Vieux Bour, Nuaillé, France), 100 units mL−1 penicillin, 100 µg mL−1 streptomycin (Thermo Fisher Scientific, MA, USA) and 10 ng ml−1 bFGF (PeproTech, NJ, USA). ..

Article Title: Photodynamic Therapy Using a Novel Phosphorus Tetraphenylporphyrin Induces an Anticancer Effect via Bax/Bcl-xL-related Mitochondrial Apoptosis in Biliary Cancer Cells
Article Snippet: .. Cell culture Human biliary cancer cells (NOZ), mouse embryonic fibroblasts (MEF) (Japanese Collection of Research Bioresources, Tokyo, Japan), human breast cancer cells (MCF7), human hepatocyte carcinoma cells (HepG2) and human embryonic kidney 293 cells (HEK293) (Riken Cell Bank, Ibaraki, Japan) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with L-glutamine and phenol red (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) in a humidified atmosphere with 5% CO2 at 37°C. .. The medium was supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and penicillin-streptomycin-amphotericin B (Fujifilm Wako).

Article Title: Identification of novel methylation markers in HPV-associated oropharyngeal cancer: genome-wide discovery, tissue verification and validation testing in ctDNA
Article Snippet: Twelve hours after plating, cultures were incubated either for 48 h with 5-Aza-CdR (5 μM; Sigma, St. Louis, MO). .. The medium was then removed, and cultures were maintained in standard Dulbecco’s modified Eagle’s medium, which was replaced every other day. .. Clinical tumour samplesSurgical HNSCC tumour and matched adjacent non-tumour tissues were obtained from 252 patients who underwent surgical resection at the Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine (Hamamatsu, Shizuoka, Japan).

Article Title: Increase in Toxicity of Anticancer Drugs by PMTPV, a Claudin-1-Binding Peptide, Mediated via Down-Regulation of Claudin-1 in Human Lung Adenocarcinoma A549 Cells
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Article Title: Negative chronotropic and inotropic effects of lubiprostone on iPS cell-derived cardiomyocytes via activation of CFTR
Article Snippet: .. Dulbecco’s Modified Eagle Medium ..

Article Title: Pemafibrate, a selective PPARα modulator, and fenofibrate suppress microglial activation through distinct PPARα and SIRT1-dependent pathways.
Article Snippet: .. Pemafibrate, a selective peroxisome proliferator-activated receptor (PPAR) α modulator, is a new drug that specifically modulates PPARα conformation and co-activator recruitment, thereby lowers plasma triglycerides with less off-target effects. .. Pemafibrate, a selective peroxisome proliferator-activated receptor (PPAR) α modulator, is a new drug that specifically modulates PPARα conformation and co-activator recruitment, thereby lowers plasma triglycerides with less off-target effects.

Article Title: N-Aryl Pyrido Cyanine derivatives: nuclear and organelle DNA markers for two-photon and super-resolution imaging
Article Snippet: The fluorescent intensity of equal amount mixture of oligonucleotide and PC dye solution was measured by EnSpire (PerkinElmer). .. Animal and plant cell cultures for fluorescence imaging Cell culture lines (HeLa, U-2OS, C6, NIH3T3) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Wako) containing 10% fetal bovine serum at 37 °C in a 5% CO2 /95% air incubator. .. These lines (2 × 104 cells /mL) were transferred on each well of a glass-bottom 8-well slide and cultured 1day before imaging.

Fluorescence:

Article Title: N-Aryl Pyrido Cyanine derivatives: nuclear and organelle DNA markers for two-photon and super-resolution imaging
Article Snippet: The fluorescent intensity of equal amount mixture of oligonucleotide and PC dye solution was measured by EnSpire (PerkinElmer). .. Animal and plant cell cultures for fluorescence imaging Cell culture lines (HeLa, U-2OS, C6, NIH3T3) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Wako) containing 10% fetal bovine serum at 37 °C in a 5% CO2 /95% air incubator. .. These lines (2 × 104 cells /mL) were transferred on each well of a glass-bottom 8-well slide and cultured 1day before imaging.

Imaging:

Article Title: N-Aryl Pyrido Cyanine derivatives: nuclear and organelle DNA markers for two-photon and super-resolution imaging
Article Snippet: The fluorescent intensity of equal amount mixture of oligonucleotide and PC dye solution was measured by EnSpire (PerkinElmer). .. Animal and plant cell cultures for fluorescence imaging Cell culture lines (HeLa, U-2OS, C6, NIH3T3) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Wako) containing 10% fetal bovine serum at 37 °C in a 5% CO2 /95% air incubator. .. These lines (2 × 104 cells /mL) were transferred on each well of a glass-bottom 8-well slide and cultured 1day before imaging.

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    FUJIFILM dulbecco modified eagle s medium dmem
    Culture media (CM) derived from POSTN-stimulated RVFbs inhibited Ca 2+ inflow through l -type Ca 2+ channel (LTCC) in H9c2 cardiomyoblasts. RVFbs were stimulated for 24 h with recombinant rat POSTN (1000 ng/mL) or solvent (CONT). Then, the RVFbs were incubated in new serum-free <t>Dulbecco</t> Modified <t>Eagle’s</t> Medium containing l -arginine (1 mM) for 24 h in the presence or absence of pretreatment with l -NAME (600 μM, 10 min). CM derived from solvent-stimulated RVFbs (CONT-CM) and from POSTN-stimulated RVFbs in the presence (+ l -NAME) or absence (POSTN-CM) of l -NAME were collected. H9c2 cardiomyoblasts grown to confluent on a coverslip were treated with the CM. Ca 2+ inflow through LTCC was measured by a fluorescent calcium measuring system using Fura-2 AM, a fluorescent calcium indicator. F340/F380 ratio (F), was calculated and normalized by the basal fluorescence (F 0 ) at 30 seconds before KCl (30 mM)-stimulation (F/F 0 ). ( a ) Average time course of F/F 0 in H9c2 cardiomyoblasts treated for 1 h with milliQ (Cont), CONT-CM, POSTN-CM or sodium nitroprusside (SNP, 100 μM). The fluorescence was recorded every 0.1 s. ( b ) The maximum F/F 0 change (ΔF/F 0 ) by KCl-induced Ca 2+ inflow was shown as mean ± S.E.M. (Cont, SNP: n = 6; CONT-CM, POSTN-CM: n = 4). n.s.: not significant, *, ** p
    Dulbecco Modified Eagle S Medium Dmem, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM phenol red free dmem
    Nedd4-1 regulates HER3 and ER degradation in the presence of estradiol. (A) sh-control <t>MCF-7</t> cells and sh-Nedd4-1 knockdown MCF-7 cells were incubated with serum-starved <t>PRF-DMEM</t> for 1.5 h. The cells were then treated with 50 µg/ml CHX for 30 min, followed by treatment with EtOH or 1 nM estradiol in the presence of CHX. All protein levels were assessed by immunoblotting at indicated time points. Quantification of the HER3 (B, C) and ER (D, E) protein levels were done using ImageJ software. The protein levels were normalized to β actin levels. All values are shown as means ± SD of three independent experiments. *P
    Phenol Red Free Dmem, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    FUJIFILM serum free dmem
    Progesterone promotes ERK phosphorylation via non-GPCR signaling in <t>NGF-induced</t> neuronal PC12 cells. ( a ) Mobilization of [Ca 2+ ] i induced by progesterone was monitored in PC12 cells, and data are presented as relative Ca 2+ intensity. After 2 h in culture, cells were treated with NGF (50 ng/mL) and further cultured in <t>DMEM</t> containing 1% FBS for 24 h. (n = 3). ( b ) cAMP levels in response to progesterone treatment in PC12 cells. After 24 h in culture, NGF-induced PC12 cells pre-cultured with IBMX for 30 min were cultured in the presence of progesterone for 10 min. The cAMP levels in the cells were determined by using a cAMP EIA kit. (n = 3). ( c ) Effects of progesterone on AMPK phosphorylation in PC12 cells. After 24 h of culture, NGF-induced neuronal PC12 cells were further cultured for 3 h in serum-free DMEM. The cells were cultured in the presence of progesterone for 10 min. AMPK and its phosphorylated form were detected by western blotting with specific antibodies. (n = 5) ( d ) Agonistic effects of progesterone on ERK1/2 phosphorylation in PC12 cells. After 24 h of culture, NGF-induced neuronal PC12 cells were further cultured for 3 h in serum-free DMEM. The cells were cultured in the presence of progesterone for 10 min. ERK1/2 and its phosphorylated form were detected by western blotting with specific antibodies. (n = 3). Statistical analysis was performed by using one-way analysis of variance followed by Tukey-Kramer’s post hoc test, compared with control. FSK: Forskolin. Results are presented as means ± S.E.M. of independent wells.
    Serum Free Dmem, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/serum free dmem/product/FUJIFILM
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    FUJIFILM dmem medium
    Intestinal epithelial cell-specific <t>FXR</t> activation of two bacterial metabolites. FXR-expressed or FXR-null SW480 cells were treated with bacterial culture supernatants (10 % v/v) for 24 h incubation before measurement of by administration of luciferase assay reagent. ( a ) The activation of FXR in each cell was measured by luciferase reporter construct FXRE-Luc ( n = 2). The induction of FXR target gene by bacterial culture supernatants was determined with quantitative real-time reverse transcription-PCR analysis ( n = 1): ( b ) Ibabp gene, ( c ) Ostα gene. ( d , e ) The culture supernatant derived from B. dorei transactivated FXR target gene ( Ibabp ) in Caco-2 cells ( n = 1). Before the treatment with bacterial supernatants, cells were cultured for 7 days or 21 days. The induction of FXR target gene ( Ibabp ) by bacterial culture supernatant were determined in undifferentiated ( d ) or fully differentiated Caco-2 cells ( e ). ( f ) The FXR stimulatory potential of bacterial supernatants FXR in HepG2 cells. Shp gene expression levels in HepG2 cells treated with bacterial culture supernatant were determined ( n = 1). mRNA levels were normalized to Gapdh mRNA levels via the relative standard curve method. Relative mRNA expression: Compared to <t>DMEM</t> medium group. Experiments were performed in triplicate. Values are the mean ± SD. Differences were calculated using Student’s t -test (* p
    Dmem Medium, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Culture media (CM) derived from POSTN-stimulated RVFbs inhibited Ca 2+ inflow through l -type Ca 2+ channel (LTCC) in H9c2 cardiomyoblasts. RVFbs were stimulated for 24 h with recombinant rat POSTN (1000 ng/mL) or solvent (CONT). Then, the RVFbs were incubated in new serum-free Dulbecco Modified Eagle’s Medium containing l -arginine (1 mM) for 24 h in the presence or absence of pretreatment with l -NAME (600 μM, 10 min). CM derived from solvent-stimulated RVFbs (CONT-CM) and from POSTN-stimulated RVFbs in the presence (+ l -NAME) or absence (POSTN-CM) of l -NAME were collected. H9c2 cardiomyoblasts grown to confluent on a coverslip were treated with the CM. Ca 2+ inflow through LTCC was measured by a fluorescent calcium measuring system using Fura-2 AM, a fluorescent calcium indicator. F340/F380 ratio (F), was calculated and normalized by the basal fluorescence (F 0 ) at 30 seconds before KCl (30 mM)-stimulation (F/F 0 ). ( a ) Average time course of F/F 0 in H9c2 cardiomyoblasts treated for 1 h with milliQ (Cont), CONT-CM, POSTN-CM or sodium nitroprusside (SNP, 100 μM). The fluorescence was recorded every 0.1 s. ( b ) The maximum F/F 0 change (ΔF/F 0 ) by KCl-induced Ca 2+ inflow was shown as mean ± S.E.M. (Cont, SNP: n = 6; CONT-CM, POSTN-CM: n = 4). n.s.: not significant, *, ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Periostin Mediates Right Ventricular Failure through Induction of Inducible Nitric Oxide Synthase Expression in Right Ventricular Fibroblasts from Monocrotaline-Induced Pulmonary Arterial Hypertensive Rats

    doi: 10.3390/ijms20010062

    Figure Lengend Snippet: Culture media (CM) derived from POSTN-stimulated RVFbs inhibited Ca 2+ inflow through l -type Ca 2+ channel (LTCC) in H9c2 cardiomyoblasts. RVFbs were stimulated for 24 h with recombinant rat POSTN (1000 ng/mL) or solvent (CONT). Then, the RVFbs were incubated in new serum-free Dulbecco Modified Eagle’s Medium containing l -arginine (1 mM) for 24 h in the presence or absence of pretreatment with l -NAME (600 μM, 10 min). CM derived from solvent-stimulated RVFbs (CONT-CM) and from POSTN-stimulated RVFbs in the presence (+ l -NAME) or absence (POSTN-CM) of l -NAME were collected. H9c2 cardiomyoblasts grown to confluent on a coverslip were treated with the CM. Ca 2+ inflow through LTCC was measured by a fluorescent calcium measuring system using Fura-2 AM, a fluorescent calcium indicator. F340/F380 ratio (F), was calculated and normalized by the basal fluorescence (F 0 ) at 30 seconds before KCl (30 mM)-stimulation (F/F 0 ). ( a ) Average time course of F/F 0 in H9c2 cardiomyoblasts treated for 1 h with milliQ (Cont), CONT-CM, POSTN-CM or sodium nitroprusside (SNP, 100 μM). The fluorescence was recorded every 0.1 s. ( b ) The maximum F/F 0 change (ΔF/F 0 ) by KCl-induced Ca 2+ inflow was shown as mean ± S.E.M. (Cont, SNP: n = 6; CONT-CM, POSTN-CM: n = 4). n.s.: not significant, *, ** p

    Article Snippet: The isolated RVs were minced and suspended in Dulbecco Modified Eagle’s Medium (DMEM) (Wako Pure Chemical Industries, Ltd.).

    Techniques: Derivative Assay, Recombinant, Incubation, Modification, Fluorescence

    Nedd4-1 regulates HER3 and ER degradation in the presence of estradiol. (A) sh-control MCF-7 cells and sh-Nedd4-1 knockdown MCF-7 cells were incubated with serum-starved PRF-DMEM for 1.5 h. The cells were then treated with 50 µg/ml CHX for 30 min, followed by treatment with EtOH or 1 nM estradiol in the presence of CHX. All protein levels were assessed by immunoblotting at indicated time points. Quantification of the HER3 (B, C) and ER (D, E) protein levels were done using ImageJ software. The protein levels were normalized to β actin levels. All values are shown as means ± SD of three independent experiments. *P

    Journal: Biochemistry and Biophysics Reports

    Article Title: Estradiol promotes rapid degradation of HER3 in ER-positive breast cancer cell line MCF-7

    doi: 10.1016/j.bbrep.2018.10.008

    Figure Lengend Snippet: Nedd4-1 regulates HER3 and ER degradation in the presence of estradiol. (A) sh-control MCF-7 cells and sh-Nedd4-1 knockdown MCF-7 cells were incubated with serum-starved PRF-DMEM for 1.5 h. The cells were then treated with 50 µg/ml CHX for 30 min, followed by treatment with EtOH or 1 nM estradiol in the presence of CHX. All protein levels were assessed by immunoblotting at indicated time points. Quantification of the HER3 (B, C) and ER (D, E) protein levels were done using ImageJ software. The protein levels were normalized to β actin levels. All values are shown as means ± SD of three independent experiments. *P

    Article Snippet: For experiments evaluating the effect of 17β-estradiol (estradiol, Sigma-Aldrich), the MCF-7 cells were cultured for two days in phenol red-free DMEM (PRF-DMEM, Wako) containing 10% heat-inactivated FBS stripped of steroids by absorption to dextran-coated charcoal (DCC-FBS, Biological Industries).

    Techniques: Incubation, Software

    Estradiol induces rapid degradation of HER3 via proteasome pathway. (A) MCF-7 cells were incubated with serum-starved PRF-DMEM for 1.5 h. The cells were then treated with 50 µg/ml cycloheximide (CHX) for 30 min, followed by treatment with indicated concentrations of estradiol. The cells were lysed at indicated time points and subjected to immunoblotting for anti-HER3, anti-ER and anti-β actin antibodies. (B) The quantification of the HER3 protein levels was done using ImageJ software. The protein levels were normalized to β actin levels. The results are shown as means ± SD of three independent experiments. *P

    Journal: Biochemistry and Biophysics Reports

    Article Title: Estradiol promotes rapid degradation of HER3 in ER-positive breast cancer cell line MCF-7

    doi: 10.1016/j.bbrep.2018.10.008

    Figure Lengend Snippet: Estradiol induces rapid degradation of HER3 via proteasome pathway. (A) MCF-7 cells were incubated with serum-starved PRF-DMEM for 1.5 h. The cells were then treated with 50 µg/ml cycloheximide (CHX) for 30 min, followed by treatment with indicated concentrations of estradiol. The cells were lysed at indicated time points and subjected to immunoblotting for anti-HER3, anti-ER and anti-β actin antibodies. (B) The quantification of the HER3 protein levels was done using ImageJ software. The protein levels were normalized to β actin levels. The results are shown as means ± SD of three independent experiments. *P

    Article Snippet: For experiments evaluating the effect of 17β-estradiol (estradiol, Sigma-Aldrich), the MCF-7 cells were cultured for two days in phenol red-free DMEM (PRF-DMEM, Wako) containing 10% heat-inactivated FBS stripped of steroids by absorption to dextran-coated charcoal (DCC-FBS, Biological Industries).

    Techniques: Incubation, Software

    Depletion of ER promotes the rapid degradation of HER3. si-control MCF-7 cells and si-ER knockdown MCF-7 cells were incubated with serum-starved PRF-DMEM for 1.5 h. The cells were then treated with 50 µg/ml CHX for 30 min, followed by treatment with EtOH or 1 nM estradiol in the presence of CHX. All protein levels were assessed using immunoblotting at indicated time points. (B, C) Quantification of the HER3 protein levels was done using ImageJ software. All data from the three experiments were normalized to β actin. Mean values ± SD were plotted. *P

    Journal: Biochemistry and Biophysics Reports

    Article Title: Estradiol promotes rapid degradation of HER3 in ER-positive breast cancer cell line MCF-7

    doi: 10.1016/j.bbrep.2018.10.008

    Figure Lengend Snippet: Depletion of ER promotes the rapid degradation of HER3. si-control MCF-7 cells and si-ER knockdown MCF-7 cells were incubated with serum-starved PRF-DMEM for 1.5 h. The cells were then treated with 50 µg/ml CHX for 30 min, followed by treatment with EtOH or 1 nM estradiol in the presence of CHX. All protein levels were assessed using immunoblotting at indicated time points. (B, C) Quantification of the HER3 protein levels was done using ImageJ software. All data from the three experiments were normalized to β actin. Mean values ± SD were plotted. *P

    Article Snippet: For experiments evaluating the effect of 17β-estradiol (estradiol, Sigma-Aldrich), the MCF-7 cells were cultured for two days in phenol red-free DMEM (PRF-DMEM, Wako) containing 10% heat-inactivated FBS stripped of steroids by absorption to dextran-coated charcoal (DCC-FBS, Biological Industries).

    Techniques: Incubation, Software

    Progesterone promotes ERK phosphorylation via non-GPCR signaling in NGF-induced neuronal PC12 cells. ( a ) Mobilization of [Ca 2+ ] i induced by progesterone was monitored in PC12 cells, and data are presented as relative Ca 2+ intensity. After 2 h in culture, cells were treated with NGF (50 ng/mL) and further cultured in DMEM containing 1% FBS for 24 h. (n = 3). ( b ) cAMP levels in response to progesterone treatment in PC12 cells. After 24 h in culture, NGF-induced PC12 cells pre-cultured with IBMX for 30 min were cultured in the presence of progesterone for 10 min. The cAMP levels in the cells were determined by using a cAMP EIA kit. (n = 3). ( c ) Effects of progesterone on AMPK phosphorylation in PC12 cells. After 24 h of culture, NGF-induced neuronal PC12 cells were further cultured for 3 h in serum-free DMEM. The cells were cultured in the presence of progesterone for 10 min. AMPK and its phosphorylated form were detected by western blotting with specific antibodies. (n = 5) ( d ) Agonistic effects of progesterone on ERK1/2 phosphorylation in PC12 cells. After 24 h of culture, NGF-induced neuronal PC12 cells were further cultured for 3 h in serum-free DMEM. The cells were cultured in the presence of progesterone for 10 min. ERK1/2 and its phosphorylated form were detected by western blotting with specific antibodies. (n = 3). Statistical analysis was performed by using one-way analysis of variance followed by Tukey-Kramer’s post hoc test, compared with control. FSK: Forskolin. Results are presented as means ± S.E.M. of independent wells.

    Journal: Scientific Reports

    Article Title: Membrane progesterone receptor beta (mPRβ/Paqr8) promotes progesterone-dependent neurite outgrowth in PC12 neuronal cells via non-G protein-coupled receptor (GPCR) signaling

    doi: 10.1038/s41598-017-05423-9

    Figure Lengend Snippet: Progesterone promotes ERK phosphorylation via non-GPCR signaling in NGF-induced neuronal PC12 cells. ( a ) Mobilization of [Ca 2+ ] i induced by progesterone was monitored in PC12 cells, and data are presented as relative Ca 2+ intensity. After 2 h in culture, cells were treated with NGF (50 ng/mL) and further cultured in DMEM containing 1% FBS for 24 h. (n = 3). ( b ) cAMP levels in response to progesterone treatment in PC12 cells. After 24 h in culture, NGF-induced PC12 cells pre-cultured with IBMX for 30 min were cultured in the presence of progesterone for 10 min. The cAMP levels in the cells were determined by using a cAMP EIA kit. (n = 3). ( c ) Effects of progesterone on AMPK phosphorylation in PC12 cells. After 24 h of culture, NGF-induced neuronal PC12 cells were further cultured for 3 h in serum-free DMEM. The cells were cultured in the presence of progesterone for 10 min. AMPK and its phosphorylated form were detected by western blotting with specific antibodies. (n = 5) ( d ) Agonistic effects of progesterone on ERK1/2 phosphorylation in PC12 cells. After 24 h of culture, NGF-induced neuronal PC12 cells were further cultured for 3 h in serum-free DMEM. The cells were cultured in the presence of progesterone for 10 min. ERK1/2 and its phosphorylated form were detected by western blotting with specific antibodies. (n = 3). Statistical analysis was performed by using one-way analysis of variance followed by Tukey-Kramer’s post hoc test, compared with control. FSK: Forskolin. Results are presented as means ± S.E.M. of independent wells.

    Article Snippet: The cells were cultured in DMEM containing NGF (50 ng/mL) and 1% FBS for 24 h, and then in serum-free DMEM for 3 h. The cells were further cultured for 10 min in the presence of progesterone (10 μM; Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

    Inhibition of progesterone-mPRβ-MAPK signaling in PC12 cells suppresses neurite outgrowth. ( a ) Inhibitory effects of MEK inhibitor (U0126) on progesterone-induced neurite outgrowth in NGF-induced neuronal PC12 cells. After 24 h in culture, cells were further cultured in DMEM containing NGF (50 ng/mL), 1% FBS, with or without U0126 (10 μM) and progesterone (10 μM) for 3 days. Scale bar = 200 μm. (n = 3–5). Statistical analysis was performed by using one-way analysis of variance followed by Tukey-Kramer’s post hoc test. ( b ) Inhibitory effects of mPRβ siRNA on the phosphorylation of ERK1/2 in NGF-induced neuronal PC12 cells. After being treated with Control siRNA or mPRβ siRNA, cells were cultured for 3 days in DMEM containing 1% FBS, NGF (50 ng/mL) and with or without progesterone (10 μM). ERK1/2 and phosphorylated ERK1/2 in cells were detected by western blotting with specific antibodies. (n = 3). Statistical analysis was performed by using Student’s t-test. ( c ) Effects of progesterone (10 μM) on the phosphorylation of ERK1/2 in the presence or absence of RU486 (10 μM) and AG205 (10 μM) in PC12 cells. After 24 h in culture, cells were cultured in DMEM containing NGF (50 ng/mL) and 1% FBS. Cells were further cultured for 3 h in serum-free DMEM. After precultured with RU486 (10 μM) or AG205 (10 μM) for 30 min, cells were cultured in the presence or absence of progesterone (10 μM) for 10 min. (n = 5) Statistical analysis was performed by using one-way analysis of variance followed by Tukey-Kramer’s post hoc test. ( d ) Cells were left untreated or treated for 5 min with the progesterone (10 μM) or NGF (100 ng/ml). Lysate proteins were immune-precipitated with anti-mPRβ antibodies. The anti-mPRβ antibodies was used to detect mPRβ and anti-TrkA antibodies was used to detect TrkA. ( e ) Effects of progesterone on Akt phosphorylation in PC12 cells. After 24 h of culture, NGF-induced neuronal PC12 cells were further cultured for 3 h in serum-free DMEM. The cells were cultured in the presence of progesterone for 10 min. AKT and its phosphorylated form were detected by western blotting with specific antibodies. (n = 6) Statistical analysis was performed by using one-way analysis of variance followed by Tukey-Kramer’s post hoc test. ( f ) Effects of progesterone on Rac1 activation in PC12 cells. After 24 h of culture, NGF-induced neuronal PC12 cells were further cultured for 3 h in serum-free DMEM. The cells were cultured in the presence of progesterone or NGF (50 ng/ml) for 10 min. Rac activation was analyzed by pull-down assay. Active (Rac-GTP) or total Rac (Rac1) was detected by Western blot. ( g ) Effects of progesterone (10 μM) on the phosphorylation of ERK1/2 in the presence or absence of LY294002 (10 μM) in PC12 cells. After 24 h in culture, cells were cultured in DMEM containing NGF (50 ng/mL) and 1% FBS. Cells were further cultured for 3 h in serum-free DMEM. After precultured with LY294002 for 30 min, cells were cultured in the presence or absence of progesterone (10 μM) for 10 min. (n = 5). Statistical analysis was performed by using Student’s t-test. Results are presented as means ± S.E.M. * p

    Journal: Scientific Reports

    Article Title: Membrane progesterone receptor beta (mPRβ/Paqr8) promotes progesterone-dependent neurite outgrowth in PC12 neuronal cells via non-G protein-coupled receptor (GPCR) signaling

    doi: 10.1038/s41598-017-05423-9

    Figure Lengend Snippet: Inhibition of progesterone-mPRβ-MAPK signaling in PC12 cells suppresses neurite outgrowth. ( a ) Inhibitory effects of MEK inhibitor (U0126) on progesterone-induced neurite outgrowth in NGF-induced neuronal PC12 cells. After 24 h in culture, cells were further cultured in DMEM containing NGF (50 ng/mL), 1% FBS, with or without U0126 (10 μM) and progesterone (10 μM) for 3 days. Scale bar = 200 μm. (n = 3–5). Statistical analysis was performed by using one-way analysis of variance followed by Tukey-Kramer’s post hoc test. ( b ) Inhibitory effects of mPRβ siRNA on the phosphorylation of ERK1/2 in NGF-induced neuronal PC12 cells. After being treated with Control siRNA or mPRβ siRNA, cells were cultured for 3 days in DMEM containing 1% FBS, NGF (50 ng/mL) and with or without progesterone (10 μM). ERK1/2 and phosphorylated ERK1/2 in cells were detected by western blotting with specific antibodies. (n = 3). Statistical analysis was performed by using Student’s t-test. ( c ) Effects of progesterone (10 μM) on the phosphorylation of ERK1/2 in the presence or absence of RU486 (10 μM) and AG205 (10 μM) in PC12 cells. After 24 h in culture, cells were cultured in DMEM containing NGF (50 ng/mL) and 1% FBS. Cells were further cultured for 3 h in serum-free DMEM. After precultured with RU486 (10 μM) or AG205 (10 μM) for 30 min, cells were cultured in the presence or absence of progesterone (10 μM) for 10 min. (n = 5) Statistical analysis was performed by using one-way analysis of variance followed by Tukey-Kramer’s post hoc test. ( d ) Cells were left untreated or treated for 5 min with the progesterone (10 μM) or NGF (100 ng/ml). Lysate proteins were immune-precipitated with anti-mPRβ antibodies. The anti-mPRβ antibodies was used to detect mPRβ and anti-TrkA antibodies was used to detect TrkA. ( e ) Effects of progesterone on Akt phosphorylation in PC12 cells. After 24 h of culture, NGF-induced neuronal PC12 cells were further cultured for 3 h in serum-free DMEM. The cells were cultured in the presence of progesterone for 10 min. AKT and its phosphorylated form were detected by western blotting with specific antibodies. (n = 6) Statistical analysis was performed by using one-way analysis of variance followed by Tukey-Kramer’s post hoc test. ( f ) Effects of progesterone on Rac1 activation in PC12 cells. After 24 h of culture, NGF-induced neuronal PC12 cells were further cultured for 3 h in serum-free DMEM. The cells were cultured in the presence of progesterone or NGF (50 ng/ml) for 10 min. Rac activation was analyzed by pull-down assay. Active (Rac-GTP) or total Rac (Rac1) was detected by Western blot. ( g ) Effects of progesterone (10 μM) on the phosphorylation of ERK1/2 in the presence or absence of LY294002 (10 μM) in PC12 cells. After 24 h in culture, cells were cultured in DMEM containing NGF (50 ng/mL) and 1% FBS. Cells were further cultured for 3 h in serum-free DMEM. After precultured with LY294002 for 30 min, cells were cultured in the presence or absence of progesterone (10 μM) for 10 min. (n = 5). Statistical analysis was performed by using Student’s t-test. Results are presented as means ± S.E.M. * p

    Article Snippet: The cells were cultured in DMEM containing NGF (50 ng/mL) and 1% FBS for 24 h, and then in serum-free DMEM for 3 h. The cells were further cultured for 10 min in the presence of progesterone (10 μM; Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Inhibition, Cell Culture, Western Blot, Activation Assay, Pull Down Assay

    Effects of progesterone on neurite outgrowth via mPRβ in NGF-induced neuronal PC12 cells. ( a ) Effects of progesterone on neurite outgrowth. After 24 h in culture, PC12 cells were treated with NGF (50 ng/mL) or co-stimulated with NGF and progesterone (10 μM) for 3 days. (n = 3). Scale bar = 100 μm. ( b ) After being treated with Control siRNA or mPRβ siRNA, PC12 cells were cultured for 3 days in DMEM containing 1% FBS, NGF (50 ng/mL) and progesterone (10 μM) (n = 3). ( c ) Effects of progesterone on neurite outgrowth. After 24 h in culture, SH-SY5Y cells were treated with NGF (50 ng/mL) or co-stimulated with NGF and progesterone (10 μM) for 12 h. (n = 4–8). Scale bar = 100 μm. The graph reports the average length of neurites. Results are presented as means ± S.E.M. * p

    Journal: Scientific Reports

    Article Title: Membrane progesterone receptor beta (mPRβ/Paqr8) promotes progesterone-dependent neurite outgrowth in PC12 neuronal cells via non-G protein-coupled receptor (GPCR) signaling

    doi: 10.1038/s41598-017-05423-9

    Figure Lengend Snippet: Effects of progesterone on neurite outgrowth via mPRβ in NGF-induced neuronal PC12 cells. ( a ) Effects of progesterone on neurite outgrowth. After 24 h in culture, PC12 cells were treated with NGF (50 ng/mL) or co-stimulated with NGF and progesterone (10 μM) for 3 days. (n = 3). Scale bar = 100 μm. ( b ) After being treated with Control siRNA or mPRβ siRNA, PC12 cells were cultured for 3 days in DMEM containing 1% FBS, NGF (50 ng/mL) and progesterone (10 μM) (n = 3). ( c ) Effects of progesterone on neurite outgrowth. After 24 h in culture, SH-SY5Y cells were treated with NGF (50 ng/mL) or co-stimulated with NGF and progesterone (10 μM) for 12 h. (n = 4–8). Scale bar = 100 μm. The graph reports the average length of neurites. Results are presented as means ± S.E.M. * p

    Article Snippet: The cells were cultured in DMEM containing NGF (50 ng/mL) and 1% FBS for 24 h, and then in serum-free DMEM for 3 h. The cells were further cultured for 10 min in the presence of progesterone (10 μM; Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Cell Culture

    Intestinal epithelial cell-specific FXR activation of two bacterial metabolites. FXR-expressed or FXR-null SW480 cells were treated with bacterial culture supernatants (10 % v/v) for 24 h incubation before measurement of by administration of luciferase assay reagent. ( a ) The activation of FXR in each cell was measured by luciferase reporter construct FXRE-Luc ( n = 2). The induction of FXR target gene by bacterial culture supernatants was determined with quantitative real-time reverse transcription-PCR analysis ( n = 1): ( b ) Ibabp gene, ( c ) Ostα gene. ( d , e ) The culture supernatant derived from B. dorei transactivated FXR target gene ( Ibabp ) in Caco-2 cells ( n = 1). Before the treatment with bacterial supernatants, cells were cultured for 7 days or 21 days. The induction of FXR target gene ( Ibabp ) by bacterial culture supernatant were determined in undifferentiated ( d ) or fully differentiated Caco-2 cells ( e ). ( f ) The FXR stimulatory potential of bacterial supernatants FXR in HepG2 cells. Shp gene expression levels in HepG2 cells treated with bacterial culture supernatant were determined ( n = 1). mRNA levels were normalized to Gapdh mRNA levels via the relative standard curve method. Relative mRNA expression: Compared to DMEM medium group. Experiments were performed in triplicate. Values are the mean ± SD. Differences were calculated using Student’s t -test (* p

    Journal: Nutrition & Metabolism

    Article Title: Bacterial metabolites directly modulate farnesoid X receptor activity

    doi: 10.1186/s12986-015-0045-y

    Figure Lengend Snippet: Intestinal epithelial cell-specific FXR activation of two bacterial metabolites. FXR-expressed or FXR-null SW480 cells were treated with bacterial culture supernatants (10 % v/v) for 24 h incubation before measurement of by administration of luciferase assay reagent. ( a ) The activation of FXR in each cell was measured by luciferase reporter construct FXRE-Luc ( n = 2). The induction of FXR target gene by bacterial culture supernatants was determined with quantitative real-time reverse transcription-PCR analysis ( n = 1): ( b ) Ibabp gene, ( c ) Ostα gene. ( d , e ) The culture supernatant derived from B. dorei transactivated FXR target gene ( Ibabp ) in Caco-2 cells ( n = 1). Before the treatment with bacterial supernatants, cells were cultured for 7 days or 21 days. The induction of FXR target gene ( Ibabp ) by bacterial culture supernatant were determined in undifferentiated ( d ) or fully differentiated Caco-2 cells ( e ). ( f ) The FXR stimulatory potential of bacterial supernatants FXR in HepG2 cells. Shp gene expression levels in HepG2 cells treated with bacterial culture supernatant were determined ( n = 1). mRNA levels were normalized to Gapdh mRNA levels via the relative standard curve method. Relative mRNA expression: Compared to DMEM medium group. Experiments were performed in triplicate. Values are the mean ± SD. Differences were calculated using Student’s t -test (* p

    Article Snippet: For the selection of stable FXR expressing cells, cells were cultured in DMEM medium containing 800 μg/ml G418 (Wako).

    Techniques: Activation Assay, Incubation, Luciferase, Construct, Polymerase Chain Reaction, Derivative Assay, Cell Culture, Expressing