dulbecco s modified eagle s medium  (FUJIFILM)

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

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

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

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

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

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

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

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