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Welgene inc fetal bovine serum fbs
GW501516-activated PPARδ suppresses Ang II-induced ROS generation and [ 3 H]-leucine incorporation in a PI3K/Akt-dependent manner. (A) VSMCs were treated with Ang II for the indicated durations. (B and C) VSMCs pretreated with LY294002 for 30 min (B) or transfected with PPARδ-targeting siRNA for 48 h (C) were incubated in the presence or absence of GW501516 for 24 h and then exposed to Ang II for 24 h. Levels of phosphorylated and total Akt were determined by western blotting. Representative blots from three independent experiments and densitometric measurements are shown. (D and E) VSMCs pretreated with LY294002 for 30 min were incubated in the presence or absence of GW501516 for 24 h and then exposed to Ang II for 24 h. Intracellular ROS were detected by confocal laser fluorescence microscopy using CM-H 2 DCF-DA (D), and the fluorescence intensity was quantified (E). Bars, 200 μm. (F) VSMCs pretreated with LY294002 for 30 min were incubated in the presence or absence of GW501516 for 24 h in <t>DMEM</t> containing 0.1% <t>FBS.</t> Thereafter, cells were exposed to Ang II for 24 h and pulsed with 1 μCi/mL [ 3 H]-leucine for the final 6 h. Data represent means ± SE (n = 3 or 4). ** p
Fetal Bovine Serum Fbs, supplied by Welgene inc, used in various techniques. Bioz Stars score: 92/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Ligand-activated PPARδ inhibits angiotensin II-stimulated hypertrophy of vascular smooth muscle cells by targeting ROS"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0210482

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

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

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

Techniques Used: Transfection, Incubation, Fluorescence, Microscopy

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

Techniques Used: Transfection

2) Product Images from "Ligand-activated PPARδ inhibits angiotensin II-stimulated hypertrophy of vascular smooth muscle cells by targeting ROS"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0210482

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

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

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

Techniques Used: Transfection, Incubation, Fluorescence, Microscopy

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

Techniques Used: Transfection

3) Product Images from "Serum Starvation-Induced Voltage-Gated Potassium Channel Kv7.5 Expression and Its Regulation by Sp1 in Canine Osteosarcoma Cells"

Article Title: Serum Starvation-Induced Voltage-Gated Potassium Channel Kv7.5 Expression and Its Regulation by Sp1 in Canine Osteosarcoma Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms15010977

Upregulation of Kv7.5 expression levels by serum deprivation. ( A ) The cells were seeded onto plates and incubated overnight before serum withdrawal. On the following day, one plate of cells was harvested as a control for the experiments (0 h), and the other subconfluent proliferating cells were washed two times with warm PBS and transferred into serum-free DMEM. The cells were further incubated for 6, 10, 20, 30, 44, 54, and 68 h (prefixed with “−” to imply the withdrawal of serum) and harvested. Three plates of cells were transferred into complete growth medium after 30 h of serum deprivation to induce the cell progression into the G 1 –S transition and incubated for 14, 24, and 38 h (prefixed with “+” to imply the re-addition of serum); The relative expression levels of Kv7.5 in the presence or absence of FBS were analyzed by qPCR ( B ) and western blot analysis ( C ). The values are the mean ± SEM of five ( B ) and four ( C ) independent experiments. The asterisks denote values significantly different from the control (0 h). * p
Figure Legend Snippet: Upregulation of Kv7.5 expression levels by serum deprivation. ( A ) The cells were seeded onto plates and incubated overnight before serum withdrawal. On the following day, one plate of cells was harvested as a control for the experiments (0 h), and the other subconfluent proliferating cells were washed two times with warm PBS and transferred into serum-free DMEM. The cells were further incubated for 6, 10, 20, 30, 44, 54, and 68 h (prefixed with “−” to imply the withdrawal of serum) and harvested. Three plates of cells were transferred into complete growth medium after 30 h of serum deprivation to induce the cell progression into the G 1 –S transition and incubated for 14, 24, and 38 h (prefixed with “+” to imply the re-addition of serum); The relative expression levels of Kv7.5 in the presence or absence of FBS were analyzed by qPCR ( B ) and western blot analysis ( C ). The values are the mean ± SEM of five ( B ) and four ( C ) independent experiments. The asterisks denote values significantly different from the control (0 h). * p

Techniques Used: Expressing, Incubation, Real-time Polymerase Chain Reaction, Western Blot

4) Product Images from "Resveratrol inhibits the protein expression of transcription factors related adipocyte differentiation and the activity of matrix metalloproteinase in mouse fibroblast 3T3-L1 preadipocytes"

Article Title: Resveratrol inhibits the protein expression of transcription factors related adipocyte differentiation and the activity of matrix metalloproteinase in mouse fibroblast 3T3-L1 preadipocytes

Journal: Nutrition Research and Practice

doi: 10.4162/nrp.2012.6.6.499

Effect of resveratrol on triglyceride and GPDH activity in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 2.0 × 10 4 cells/mLin a 6 well plate with DMEM supplemented with 10% BCS for 2 days, the monolayers were applied to differentiation induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 days. After differentiation induction, the monolayer was incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. Triglyceride accumulation was estimated by the commercial triglyceride assay kit (A). GPDH activity was estimated by a commercial GPDH activity assay kit (B). Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences ( P
Figure Legend Snippet: Effect of resveratrol on triglyceride and GPDH activity in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 2.0 × 10 4 cells/mLin a 6 well plate with DMEM supplemented with 10% BCS for 2 days, the monolayers were applied to differentiation induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 days. After differentiation induction, the monolayer was incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. Triglyceride accumulation was estimated by the commercial triglyceride assay kit (A). GPDH activity was estimated by a commercial GPDH activity assay kit (B). Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences ( P

Techniques Used: Activity Assay, Incubation

Effect of resveratrol on quantification of lipid content in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 1 × 10 4 cells/mLin a 96 well plate with DMEM supplemented with 10% BCS for 2 days, the monolayers were differentiation induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 days. After differentiation induction, the monolayer was incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. Lipid accumulation was estimated by the Oil red O staining. Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences ( P
Figure Legend Snippet: Effect of resveratrol on quantification of lipid content in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 1 × 10 4 cells/mLin a 96 well plate with DMEM supplemented with 10% BCS for 2 days, the monolayers were differentiation induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 days. After differentiation induction, the monolayer was incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. Lipid accumulation was estimated by the Oil red O staining. Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences ( P

Techniques Used: Incubation, Staining

Effect of resveratrol on cell proliferation in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 1.5 × 10 4 cells/mLin 24 well plates with DMEM supplemented with 10% BCS for 2 days, the monolayers were differentiated induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 days. After differentiation induction, the monolayer was incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. Viable cell numbers were estimated by MTT assay. Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences ( P
Figure Legend Snippet: Effect of resveratrol on cell proliferation in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 1.5 × 10 4 cells/mLin 24 well plates with DMEM supplemented with 10% BCS for 2 days, the monolayers were differentiated induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 days. After differentiation induction, the monolayer was incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. Viable cell numbers were estimated by MTT assay. Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences ( P

Techniques Used: Incubation, MTT Assay

Effect of resveratrol on the protein expression of transcription factors in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 2.5 × 10 4 cells/dish with DMEM supplemented with 10% BCS for 2 days, the monolayers were applied to differentiation induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 day. After differentiation induction, the monolayers were incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. Chemiluminescent detection and quantitative analysis of western blots were performed for three independent experiments. The protein expression of C/EBPβ (A), PPARγ (B), C/EBPα (C), and FABP (D) are shown above. Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences ( P
Figure Legend Snippet: Effect of resveratrol on the protein expression of transcription factors in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 2.5 × 10 4 cells/dish with DMEM supplemented with 10% BCS for 2 days, the monolayers were applied to differentiation induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 day. After differentiation induction, the monolayers were incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. Chemiluminescent detection and quantitative analysis of western blots were performed for three independent experiments. The protein expression of C/EBPβ (A), PPARγ (B), C/EBPα (C), and FABP (D) are shown above. Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences ( P

Techniques Used: Expressing, Incubation, Western Blot

Effect of resveratrol on MMP 2 activity in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 2.0 × 10 4 cells/dish with DMEM supplemented with 10% BCS for 2 days, the monolayers were differentiation induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 days. After differentiation induction, the monolayer was incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. The media were collected and concentrated for zymography. MMP-2 bands (A) and MMP-9 bands (B), which were representative of three independent experiments, are shown. Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences ( P
Figure Legend Snippet: Effect of resveratrol on MMP 2 activity in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 2.0 × 10 4 cells/dish with DMEM supplemented with 10% BCS for 2 days, the monolayers were differentiation induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 days. After differentiation induction, the monolayer was incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. The media were collected and concentrated for zymography. MMP-2 bands (A) and MMP-9 bands (B), which were representative of three independent experiments, are shown. Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences ( P

Techniques Used: Activity Assay, Incubation, Zymography

5) Product Images from "Role of l-carnitine and oleate in myogenic differentiation: implications for myofiber regeneration"

Article Title: Role of l-carnitine and oleate in myogenic differentiation: implications for myofiber regeneration

Journal: Journal of Exercise Nutrition & Biochemistry

doi: 10.20463/jenb.2018.0015

The effects of oleate and L-carnitine on myoblast proliferation and viability. (A and B) Proliferating myoblasts (5 × 104/ml) were seeded onto non-coated 6-well culture dishes and incubated for 48 h in DMEM (10% FBS) supplemented with 300 μM oleate and/or 5 mM L-carnitine. The cells were trypsinized and stained with trypan blue. Non-stained viable cells were counted using a hemocytometer and expressed as %NT. The bar graph represents mean ± SEM. The data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. *p
Figure Legend Snippet: The effects of oleate and L-carnitine on myoblast proliferation and viability. (A and B) Proliferating myoblasts (5 × 104/ml) were seeded onto non-coated 6-well culture dishes and incubated for 48 h in DMEM (10% FBS) supplemented with 300 μM oleate and/or 5 mM L-carnitine. The cells were trypsinized and stained with trypan blue. Non-stained viable cells were counted using a hemocytometer and expressed as %NT. The bar graph represents mean ± SEM. The data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. *p

Techniques Used: Incubation, Staining

6) Product Images from "Analysis of endogenous lipids during intestinal wound healing"

Article Title: Analysis of endogenous lipids during intestinal wound healing

Journal: PLoS ONE

doi: 10.1371/journal.pone.0183028

Standard curves for quantification of fish oil fatty acids were determined and tandem mass spectra of fish oil fatty acid standards were assigned. (A) To determine the efficiency of tC18-SPE of fish oil fatty acids spiked into fetal bovine serum, TLC was performed. (B) Standard curves for quantification of fish oil fatty acids relative to internal standard LB4-d4 by mass spectrometry was shown. (C) Results of tandem mass spectra of fish oil fatty acid standards were shown. Tandem mass spectra were assigned to the predicted fragment ions generated from collision-induced dissociation of the precursor ions. The major fragment ions of ARA: m/z 303.25 [M-H], m/z 285.27 [M-H-H 2 O], m/z 259.23 [M-COOH], and m/z 205.11. EPA: m/z 301.25 [M-H], m/z 283.29 [M-H-H 2 O], m/z 257.27 [M-COOH], and m/z 203.16. DTA: m/z 331.26 [M-H], m/z 313.27 [M-H-H 2 O], m/z 287.29 [M-COOH], and m/z 233.15. DHA: m/z 327.21 [M-H], m/z 309.27 [M-H-H 2 O], m/z 283.15 [M-COOH], m/z 229.13, and m/z 191.13. 12HETE: m/z 319.25 [M-H], m/z 301.14 [M-H-H 2 O], m/z 275.24 [M-COOH], m/z 257.14 [M-H 2 O-COOH], and m/z 179.04. LB4-d4: m/z 339.21 [M-H], m/z 321.18 [M-H-H 2 O], m/z 295.26 [M-COOH], m/z 277.20 [M-H 2 O-COOH], and m/z 197.03.
Figure Legend Snippet: Standard curves for quantification of fish oil fatty acids were determined and tandem mass spectra of fish oil fatty acid standards were assigned. (A) To determine the efficiency of tC18-SPE of fish oil fatty acids spiked into fetal bovine serum, TLC was performed. (B) Standard curves for quantification of fish oil fatty acids relative to internal standard LB4-d4 by mass spectrometry was shown. (C) Results of tandem mass spectra of fish oil fatty acid standards were shown. Tandem mass spectra were assigned to the predicted fragment ions generated from collision-induced dissociation of the precursor ions. The major fragment ions of ARA: m/z 303.25 [M-H], m/z 285.27 [M-H-H 2 O], m/z 259.23 [M-COOH], and m/z 205.11. EPA: m/z 301.25 [M-H], m/z 283.29 [M-H-H 2 O], m/z 257.27 [M-COOH], and m/z 203.16. DTA: m/z 331.26 [M-H], m/z 313.27 [M-H-H 2 O], m/z 287.29 [M-COOH], and m/z 233.15. DHA: m/z 327.21 [M-H], m/z 309.27 [M-H-H 2 O], m/z 283.15 [M-COOH], m/z 229.13, and m/z 191.13. 12HETE: m/z 319.25 [M-H], m/z 301.14 [M-H-H 2 O], m/z 275.24 [M-COOH], m/z 257.14 [M-H 2 O-COOH], and m/z 179.04. LB4-d4: m/z 339.21 [M-H], m/z 321.18 [M-H-H 2 O], m/z 295.26 [M-COOH], m/z 277.20 [M-H 2 O-COOH], and m/z 197.03.

Techniques Used: Fluorescence In Situ Hybridization, Thin Layer Chromatography, Mass Spectrometry, Generated, Acetylene Reduction Assay

7) Product Images from "Role of l-carnitine and oleate in myogenic differentiation: implications for myofiber regeneration"

Article Title: Role of l-carnitine and oleate in myogenic differentiation: implications for myofiber regeneration

Journal: Journal of Exercise Nutrition & Biochemistry

doi: 10.20463/jenb.2018.0015

The effects of oleate and L-carnitine on myoblast proliferation and viability. (A and B) Proliferating myoblasts (5 × 104/ml) were seeded onto non-coated 6-well culture dishes and incubated for 48 h in DMEM (10% FBS) supplemented with 300 μM oleate and/or 5 mM L-carnitine. The cells were trypsinized and stained with trypan blue. Non-stained viable cells were counted using a hemocytometer and expressed as %NT. The bar graph represents mean ± SEM. The data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. *p
Figure Legend Snippet: The effects of oleate and L-carnitine on myoblast proliferation and viability. (A and B) Proliferating myoblasts (5 × 104/ml) were seeded onto non-coated 6-well culture dishes and incubated for 48 h in DMEM (10% FBS) supplemented with 300 μM oleate and/or 5 mM L-carnitine. The cells were trypsinized and stained with trypan blue. Non-stained viable cells were counted using a hemocytometer and expressed as %NT. The bar graph represents mean ± SEM. The data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. *p

Techniques Used: Incubation, Staining

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Acetylene Reduction Assay:

Article Title: Analysis of endogenous lipids during intestinal wound healing
Article Snippet: .. Thin-layer chromatography (TLC) To optimize a solid-phase extraction (SPE) method for fish oil fatty acids in serum, each 0.1 μg of fish oil fatty acids, including ARA, EPA, cis-7,10,13,16-docosatetraenoic acid (DTA), DHA, 12(S )-hydroxy-eicosatetraenoic acid (12HETE), 20-hydroxy-eicosatetraenoic acid (20HETE) and 5S ,12R -dihydroxy-eicosatetraenoic acid-d4 (LB4-d4) (internal standard), were spiked into 0.5 mL fetal bovine serum (FBS) (WelGene, Daegu, Korea). .. The fish oil fatty acid standards were obtained from Sigma-Aldrich.

Transfection:

Article Title: Ligand-activated PPARδ inhibits angiotensin II-stimulated hypertrophy of vascular smooth muscle cells by targeting ROS
Article Snippet: .. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and WelFect-si transfection reagent were obtained from WelGENE Inc. (Gyeongsan-si, Gyeongsangbuk-do, Republic of Korea). .. Chloromethyl-2′,7′-dichlorofluorescein diacetate (CM-H2 DCF-DA) was purchased from Molecular Probes (Eugene, OR, USA).

Cell Culture:

Article Title: Serum Starvation-Induced Voltage-Gated Potassium Channel Kv7.5 Expression and Its Regulation by Sp1 in Canine Osteosarcoma Cells
Article Snippet: .. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Welgene, Daegu, Korea) supplemented with 10% (v /v ) fetal bovine serum (FBS) (Welgene, Daegu, Korea), antibiotics, and an antimycotic solution (antibiotics): 10 U/mL penicillin, 10 μg/mL streptomycin, and 25 ng/mL amphotericin B (Sigma Aldrich, St. Louis, MO, USA). ..

Article Title: Role of l-carnitine and oleate in myogenic differentiation: implications for myofiber regeneration
Article Snippet: .. Cell culture C2C12 myoblasts (ATCC) were seeded onto non-coated 6-well plates and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Korea) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Welgene, Korea) in a humidified atmosphere of 95% air and 5% CO2 at 37°C. ..

Thin Layer Chromatography:

Article Title: Analysis of endogenous lipids during intestinal wound healing
Article Snippet: .. Thin-layer chromatography (TLC) To optimize a solid-phase extraction (SPE) method for fish oil fatty acids in serum, each 0.1 μg of fish oil fatty acids, including ARA, EPA, cis-7,10,13,16-docosatetraenoic acid (DTA), DHA, 12(S )-hydroxy-eicosatetraenoic acid (12HETE), 20-hydroxy-eicosatetraenoic acid (20HETE) and 5S ,12R -dihydroxy-eicosatetraenoic acid-d4 (LB4-d4) (internal standard), were spiked into 0.5 mL fetal bovine serum (FBS) (WelGene, Daegu, Korea). .. The fish oil fatty acid standards were obtained from Sigma-Aldrich.

Modification:

Article Title: Serum Starvation-Induced Voltage-Gated Potassium Channel Kv7.5 Expression and Its Regulation by Sp1 in Canine Osteosarcoma Cells
Article Snippet: .. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Welgene, Daegu, Korea) supplemented with 10% (v /v ) fetal bovine serum (FBS) (Welgene, Daegu, Korea), antibiotics, and an antimycotic solution (antibiotics): 10 U/mL penicillin, 10 μg/mL streptomycin, and 25 ng/mL amphotericin B (Sigma Aldrich, St. Louis, MO, USA). ..

Article Title: Microgravity inhibits decidualization via decreasing Akt activity and FOXO3a expression in human endometrial stromal cells
Article Snippet: .. Human eSCs were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 1 g/L glucose with 10% fetal bovine serum (FBS) at 37 °C and 5% CO2 and detached from the plate using 0.05% trypsin-EDTA (Welgene, Gyeongsangbuk-do, Korea). .. To induce in vitro decidualization, cells were plated, grown to 100% confluence, treated with DMEM with 10% FBS containing 0.5 mM 8-Br-cAMP, and replenished with fresh medium every other day.

Article Title: Ligand-activated PPARδ inhibits angiotensin II-stimulated hypertrophy of vascular smooth muscle cells by targeting ROS
Article Snippet: .. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and WelFect-si transfection reagent were obtained from WelGENE Inc. (Gyeongsan-si, Gyeongsangbuk-do, Republic of Korea). .. Chloromethyl-2′,7′-dichlorofluorescein diacetate (CM-H2 DCF-DA) was purchased from Molecular Probes (Eugene, OR, USA).

Article Title: Role of l-carnitine and oleate in myogenic differentiation: implications for myofiber regeneration
Article Snippet: .. Cell culture C2C12 myoblasts (ATCC) were seeded onto non-coated 6-well plates and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Korea) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Welgene, Korea) in a humidified atmosphere of 95% air and 5% CO2 at 37°C. ..

Article Title: Role of l-carnitine and oleate in myogenic differentiation: implications for myofiber regeneration
Article Snippet: .. C2C12 myoblasts (ATCC) were seeded onto non-coated 6-well plates and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Korea) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Welgene, Korea) in a humidified atmosphere of 95% air and 5% CO2 at 37°C. ..

Fluorescence In Situ Hybridization:

Article Title: Analysis of endogenous lipids during intestinal wound healing
Article Snippet: .. Thin-layer chromatography (TLC) To optimize a solid-phase extraction (SPE) method for fish oil fatty acids in serum, each 0.1 μg of fish oil fatty acids, including ARA, EPA, cis-7,10,13,16-docosatetraenoic acid (DTA), DHA, 12(S )-hydroxy-eicosatetraenoic acid (12HETE), 20-hydroxy-eicosatetraenoic acid (20HETE) and 5S ,12R -dihydroxy-eicosatetraenoic acid-d4 (LB4-d4) (internal standard), were spiked into 0.5 mL fetal bovine serum (FBS) (WelGene, Daegu, Korea). .. The fish oil fatty acid standards were obtained from Sigma-Aldrich.

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    Welgene inc fetal bovine serum fbs
    GW501516-activated PPARδ suppresses Ang II-induced ROS generation and [ 3 H]-leucine incorporation in a PI3K/Akt-dependent manner. (A) VSMCs were treated with Ang II for the indicated durations. (B and C) VSMCs pretreated with LY294002 for 30 min (B) or transfected with PPARδ-targeting siRNA for 48 h (C) were incubated in the presence or absence of GW501516 for 24 h and then exposed to Ang II for 24 h. Levels of phosphorylated and total Akt were determined by western blotting. Representative blots from three independent experiments and densitometric measurements are shown. (D and E) VSMCs pretreated with LY294002 for 30 min were incubated in the presence or absence of GW501516 for 24 h and then exposed to Ang II for 24 h. Intracellular ROS were detected by confocal laser fluorescence microscopy using CM-H 2 DCF-DA (D), and the fluorescence intensity was quantified (E). Bars, 200 μm. (F) VSMCs pretreated with LY294002 for 30 min were incubated in the presence or absence of GW501516 for 24 h in <t>DMEM</t> containing 0.1% <t>FBS.</t> Thereafter, cells were exposed to Ang II for 24 h and pulsed with 1 μCi/mL [ 3 H]-leucine for the final 6 h. Data represent means ± SE (n = 3 or 4). ** p
    Fetal Bovine Serum Fbs, supplied by Welgene inc, used in various techniques. Bioz Stars score: 92/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GW501516-activated PPARδ suppresses Ang II-induced ROS generation and [ 3 H]-leucine incorporation in a PI3K/Akt-dependent manner. (A) VSMCs were treated with Ang II for the indicated durations. (B and C) VSMCs pretreated with LY294002 for 30 min (B) or transfected with PPARδ-targeting siRNA for 48 h (C) were incubated in the presence or absence of GW501516 for 24 h and then exposed to Ang II for 24 h. Levels of phosphorylated and total Akt were determined by western blotting. Representative blots from three independent experiments and densitometric measurements are shown. (D and E) VSMCs pretreated with LY294002 for 30 min were incubated in the presence or absence of GW501516 for 24 h and then exposed to Ang II for 24 h. Intracellular ROS were detected by confocal laser fluorescence microscopy using CM-H 2 DCF-DA (D), and the fluorescence intensity was quantified (E). Bars, 200 μm. (F) VSMCs pretreated with LY294002 for 30 min were incubated in the presence or absence of GW501516 for 24 h in DMEM containing 0.1% FBS. Thereafter, cells were exposed to Ang II for 24 h and pulsed with 1 μCi/mL [ 3 H]-leucine for the final 6 h. Data represent means ± SE (n = 3 or 4). ** p

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0210482

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0210482

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

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

    Techniques: Transfection, Incubation, Fluorescence, Microscopy

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0210482

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

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

    Techniques: Transfection

    CCM1 and DDX5 regulate Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif signaling. (A) RNAi-mediated suppression of DDX5 or YAP1 alone or co-suppression of DDX5 and YAP1 was performed, and TEAD reporter activity was analyzed in C4-2 (left) and C4-2B (right) cells grown in medium supplemented with charcoal-stripped FBS and stimulated with DHT for 18 h. (B) PC3 cells co-transfected with overexpression plasmids for Flag-YAP, myc-TEAD1, and siDDX5, were harvested, and nuclear lysates were generated. Shown are immunoprecipitation input samples from the nuclear lysates used in Figure 5H . Paxillin, a cytosolic marker, is not shown in these nuclear lysates. (C) WT or Y593F DDX5 was overexpressed in LNCaP (left), C4-2B (middle), and CWR22r (right) cells, and TEAD reporter activity was measured. (D) Subcellular localization of YAP was analyzed in DDX5-suppressed PC3 cells. (E) YAP expression was analyzed in the total lysates of CCM1-suppressed PC3 and LNCaP cells. (F) Subcellular localization of YAP was analyzed in CCM1-suppressed PC3 cells. Cyt: cytosolic fraction, Nuc: nuclear fraction.

    Journal: bioRxiv

    Article Title: Cerebral cavernous malformation 1 determines YAP/TAZ signaling dependent metastatic hallmarks of prostate cancer cells

    doi: 10.1101/2020.04.23.057778

    Figure Lengend Snippet: CCM1 and DDX5 regulate Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif signaling. (A) RNAi-mediated suppression of DDX5 or YAP1 alone or co-suppression of DDX5 and YAP1 was performed, and TEAD reporter activity was analyzed in C4-2 (left) and C4-2B (right) cells grown in medium supplemented with charcoal-stripped FBS and stimulated with DHT for 18 h. (B) PC3 cells co-transfected with overexpression plasmids for Flag-YAP, myc-TEAD1, and siDDX5, were harvested, and nuclear lysates were generated. Shown are immunoprecipitation input samples from the nuclear lysates used in Figure 5H . Paxillin, a cytosolic marker, is not shown in these nuclear lysates. (C) WT or Y593F DDX5 was overexpressed in LNCaP (left), C4-2B (middle), and CWR22r (right) cells, and TEAD reporter activity was measured. (D) Subcellular localization of YAP was analyzed in DDX5-suppressed PC3 cells. (E) YAP expression was analyzed in the total lysates of CCM1-suppressed PC3 and LNCaP cells. (F) Subcellular localization of YAP was analyzed in CCM1-suppressed PC3 cells. Cyt: cytosolic fraction, Nuc: nuclear fraction.

    Article Snippet: Cell culture and reagents Human DU145, PC3, LNCaP, C4-2, and CWR22r cells were grown in RPMI medium (Welgene) supplemented with 10% FBS (Welgene) and 1% penicillin/streptomycin (Invitrogen) at a temperature of 37°C in an atmosphere of 5% CO2 .

    Techniques: Binding Assay, Activity Assay, Transfection, Over Expression, Generated, Immunoprecipitation, Marker, Expressing

    CCM1-mediated regulation of Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) signaling. (A) An androgen receptor (AR) overexpression plasmid was stably transfected in WT PC3 cells, and individual PC3 clones were sorted into AR high and AR low groups according to the expression level of ectopic AR. (B) PC3 AR high (red box) or low (white box) cells were grown in medium supplemented with 10% charcoal-stripped FBS and stimulated with 1 µM DHT for the indicated time points, and ARR reporter activities were measured using luciferase assays. (C) CCM1 was RNAi-silenced in DU145 cells grown in medium containing 10% FBS, and TEAD reporter activity was analyzed. (D) CCM1 was RNAi-silenced in AR low (left two panels) or AR high (right two panels) PC3 cells grown in 10% charcoal-stripped FBS supplemented medium with or without DHT stimulation as indicated, and TEAD reporter activity was analyzed. Reporter values are presented relative to that in each scramble control. (E) YAP1 was suppressed via RNAi in LNCaP cells, and TEAD reporter activity was analyzed. Suppression of YAP1 was confirmed via immunoblotting (Inset). (F) The representative degree of CCM1 suppression in LNCaP, C4-2, and CWR22r cells.

    Journal: bioRxiv

    Article Title: Cerebral cavernous malformation 1 determines YAP/TAZ signaling dependent metastatic hallmarks of prostate cancer cells

    doi: 10.1101/2020.04.23.057778

    Figure Lengend Snippet: CCM1-mediated regulation of Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) signaling. (A) An androgen receptor (AR) overexpression plasmid was stably transfected in WT PC3 cells, and individual PC3 clones were sorted into AR high and AR low groups according to the expression level of ectopic AR. (B) PC3 AR high (red box) or low (white box) cells were grown in medium supplemented with 10% charcoal-stripped FBS and stimulated with 1 µM DHT for the indicated time points, and ARR reporter activities were measured using luciferase assays. (C) CCM1 was RNAi-silenced in DU145 cells grown in medium containing 10% FBS, and TEAD reporter activity was analyzed. (D) CCM1 was RNAi-silenced in AR low (left two panels) or AR high (right two panels) PC3 cells grown in 10% charcoal-stripped FBS supplemented medium with or without DHT stimulation as indicated, and TEAD reporter activity was analyzed. Reporter values are presented relative to that in each scramble control. (E) YAP1 was suppressed via RNAi in LNCaP cells, and TEAD reporter activity was analyzed. Suppression of YAP1 was confirmed via immunoblotting (Inset). (F) The representative degree of CCM1 suppression in LNCaP, C4-2, and CWR22r cells.

    Article Snippet: Cell culture and reagents Human DU145, PC3, LNCaP, C4-2, and CWR22r cells were grown in RPMI medium (Welgene) supplemented with 10% FBS (Welgene) and 1% penicillin/streptomycin (Invitrogen) at a temperature of 37°C in an atmosphere of 5% CO2 .

    Techniques: Binding Assay, Over Expression, Plasmid Preparation, Stable Transfection, Transfection, Clone Assay, Expressing, Luciferase, Activity Assay

    Effects of tanshinone IIA on phosphorylation and expression levels of STAT-3/5 during 3T3-L1 preadipocyte differentiation. ( A ) 3T3-L1 preadipocytes were differentiated with induction medium containing MDI, insulin and FBS in the presence or absence of tanshinone IIA, and harvested at D2, D5, and D8, respectively. Total cellular protein at the indicated time point were extracted and analyzed by Western blot analysis; ( B ) Western blot analysis in triplicate experiments on D8; ( C ) Western blot analysis in triplicate experiments on D2; ( D , E ) the densitometry data of ( B , C ), respectively.

    Journal: International Journal of Molecular Sciences

    Article Title: Anti-Adipogenic Effects on 3T3-L1 Cells and Zebrafish by Tanshinone IIA

    doi: 10.3390/ijms18102065

    Figure Lengend Snippet: Effects of tanshinone IIA on phosphorylation and expression levels of STAT-3/5 during 3T3-L1 preadipocyte differentiation. ( A ) 3T3-L1 preadipocytes were differentiated with induction medium containing MDI, insulin and FBS in the presence or absence of tanshinone IIA, and harvested at D2, D5, and D8, respectively. Total cellular protein at the indicated time point were extracted and analyzed by Western blot analysis; ( B ) Western blot analysis in triplicate experiments on D8; ( C ) Western blot analysis in triplicate experiments on D2; ( D , E ) the densitometry data of ( B , C ), respectively.

    Article Snippet: Differentiation was then induced by changing the medium to DMEM supplemented with 10% FBS (Welgene) plus a cocktail of hormones (MDI) that include 0.5 mM IBMX (M) (Sigma, St. Louis, MO, USA), 0.5 μM dexamethasone (D) (Sigma) and 5 μg/mL insulin (I) (Sigma) in the presence or absence of tanshinone IIA at the indicated concentrations.

    Techniques: Expressing, Western Blot

    Effects of tanshinone IIA on protein and/or mRNA expression levels of FAS, perilipin A, leptin, and resistin during 3T3-L1 preadipocyte differentiation. ( A , B ) 3T3-L1 preadipocytes were induced to differentiate with induction medium containing MDI, insulin, and FBS in the presence or absence of tanshinone IIA, and harvested at D2, D5, and D8, respectively. Total cellular protein and mRNA at the indicated time point were extracted and analyzed by Western blot ( A ) and RT-PCR ( B ) analysis, respectively; ( C , D ) Western blot ( C ) and RT-PCR ( D ) analysis in triplicate experiments on D8, respectively; ( E , F ) The densitometry data of ( C , D ), respectively. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Anti-Adipogenic Effects on 3T3-L1 Cells and Zebrafish by Tanshinone IIA

    doi: 10.3390/ijms18102065

    Figure Lengend Snippet: Effects of tanshinone IIA on protein and/or mRNA expression levels of FAS, perilipin A, leptin, and resistin during 3T3-L1 preadipocyte differentiation. ( A , B ) 3T3-L1 preadipocytes were induced to differentiate with induction medium containing MDI, insulin, and FBS in the presence or absence of tanshinone IIA, and harvested at D2, D5, and D8, respectively. Total cellular protein and mRNA at the indicated time point were extracted and analyzed by Western blot ( A ) and RT-PCR ( B ) analysis, respectively; ( C , D ) Western blot ( C ) and RT-PCR ( D ) analysis in triplicate experiments on D8, respectively; ( E , F ) The densitometry data of ( C , D ), respectively. * p

    Article Snippet: Differentiation was then induced by changing the medium to DMEM supplemented with 10% FBS (Welgene) plus a cocktail of hormones (MDI) that include 0.5 mM IBMX (M) (Sigma, St. Louis, MO, USA), 0.5 μM dexamethasone (D) (Sigma) and 5 μg/mL insulin (I) (Sigma) in the presence or absence of tanshinone IIA at the indicated concentrations.

    Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0210482

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0210482

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

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

    Techniques: Transfection, Incubation, Fluorescence, Microscopy

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0210482

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

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

    Techniques: Transfection