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Gemini Bio fetal bovine serum fbs
Arhalofenate acid activated AMPK downstream targets involved in regulation of mitochondrial function and maintained mitochondrial cristae area. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulation with monosodium urate (MSU) crystals (0.2 mg/mL) for 1 h or 18 h in <t>RPMI</t> containing 1% <t>FBS.</t> Cell lysates prepared from 18-h treatment samples were subjected to Western blot analysis of phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α, expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM), and expression of thioredoxin (TRX)1, TRX2, and thioredoxin-interacting protein (TXNIP) ( a ). Cells from 1-h treatment were then stained with MitoSOX Red and MitoTracker Green and visualized by fluorescence microscopy ( b , magnification 20×). TEM analysis was performed to examine mitochondrial cristae (the folds of inner mitochondrial membrane indicated by white arrows in c ), and the cristae volume density are presented ( d ). Data in a and b are representative of three individual experiments. Data in c are representative of 30 cells examined for each condition. Data in d are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid
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1) Product Images from "Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling"

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-018-1699-4

Arhalofenate acid activated AMPK downstream targets involved in regulation of mitochondrial function and maintained mitochondrial cristae area. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulation with monosodium urate (MSU) crystals (0.2 mg/mL) for 1 h or 18 h in RPMI containing 1% FBS. Cell lysates prepared from 18-h treatment samples were subjected to Western blot analysis of phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α, expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM), and expression of thioredoxin (TRX)1, TRX2, and thioredoxin-interacting protein (TXNIP) ( a ). Cells from 1-h treatment were then stained with MitoSOX Red and MitoTracker Green and visualized by fluorescence microscopy ( b , magnification 20×). TEM analysis was performed to examine mitochondrial cristae (the folds of inner mitochondrial membrane indicated by white arrows in c ), and the cristae volume density are presented ( d ). Data in a and b are representative of three individual experiments. Data in c are representative of 30 cells examined for each condition. Data in d are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid
Figure Legend Snippet: Arhalofenate acid activated AMPK downstream targets involved in regulation of mitochondrial function and maintained mitochondrial cristae area. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulation with monosodium urate (MSU) crystals (0.2 mg/mL) for 1 h or 18 h in RPMI containing 1% FBS. Cell lysates prepared from 18-h treatment samples were subjected to Western blot analysis of phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α, expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM), and expression of thioredoxin (TRX)1, TRX2, and thioredoxin-interacting protein (TXNIP) ( a ). Cells from 1-h treatment were then stained with MitoSOX Red and MitoTracker Green and visualized by fluorescence microscopy ( b , magnification 20×). TEM analysis was performed to examine mitochondrial cristae (the folds of inner mitochondrial membrane indicated by white arrows in c ), and the cristae volume density are presented ( d ). Data in a and b are representative of three individual experiments. Data in c are representative of 30 cells examined for each condition. Data in d are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid

Techniques Used: Western Blot, Expressing, Pyrolysis Gas Chromatography, Staining, Fluorescence, Microscopy, Transmission Electron Microscopy

Arhalofenate acid prevented prolonged accumulation of p62 by promoting autophagy flux in response to MSU crystals. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 2 h in the presence or absence of bafilomycin (Baf; 100 nM) and for 6 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis of LC3 and p62 ( a ). TEM was performed to examine autophagosomes (indicated by black arrows in b ), and the numbers of autophagosomes containing electron dense material per μm 2 are presented ( c ). Data in a and b are representative of three individual experiments. Data in c are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid
Figure Legend Snippet: Arhalofenate acid prevented prolonged accumulation of p62 by promoting autophagy flux in response to MSU crystals. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 2 h in the presence or absence of bafilomycin (Baf; 100 nM) and for 6 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis of LC3 and p62 ( a ). TEM was performed to examine autophagosomes (indicated by black arrows in b ), and the numbers of autophagosomes containing electron dense material per μm 2 are presented ( c ). Data in a and b are representative of three individual experiments. Data in c are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid

Techniques Used: Western Blot, Transmission Electron Microscopy

Arhalofenate acid attenuates MSU crystal-induced IL-1β release by inhibiting NLRP3 inflammasome activation in BMDMs in vitro. BMDMs were pretreated with arhalofenate acid at a concentration of 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) in RPMI containing 1% FBS for 18 h. The conditioned media was used for ELISA for interleukin (IL)-1β ( a ), and the cell lysates were subjected to Western blot analysis ( b ) for expression of NLRP3, pro-caspase 1, and cleaved caspase 1 (p10). Data in a are the mean ± SD of three individual experiments, and p values represent comparisons between none and MSU crystals alone, or between MSU crystal alone and MSU crystals plus arhalofenate acid. Data in b are representative of three individual experiments
Figure Legend Snippet: Arhalofenate acid attenuates MSU crystal-induced IL-1β release by inhibiting NLRP3 inflammasome activation in BMDMs in vitro. BMDMs were pretreated with arhalofenate acid at a concentration of 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) in RPMI containing 1% FBS for 18 h. The conditioned media was used for ELISA for interleukin (IL)-1β ( a ), and the cell lysates were subjected to Western blot analysis ( b ) for expression of NLRP3, pro-caspase 1, and cleaved caspase 1 (p10). Data in a are the mean ± SD of three individual experiments, and p values represent comparisons between none and MSU crystals alone, or between MSU crystal alone and MSU crystals plus arhalofenate acid. Data in b are representative of three individual experiments

Techniques Used: Activation Assay, In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Arhalofenate acid inhibited MSU crystal-induced IL-1β via AMPK in BMDMs in vitro. BMDMs were treated with arhalofenate acid at 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 18 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α ( a ). The conditioned medium was used for ELISA analysis of interleukin (IL)-1β release ( b ). Data in a are representative of three individual experiments. Data in b are the mean ± SD of three individual experiments. The p values in b represent comparisons between none and MSU crystals alone in the presence or absence of arhalofenate acid in either wild-type (WT) or AMPKα1 knockout (KO) BMDMs. ns not significant
Figure Legend Snippet: Arhalofenate acid inhibited MSU crystal-induced IL-1β via AMPK in BMDMs in vitro. BMDMs were treated with arhalofenate acid at 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 18 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α ( a ). The conditioned medium was used for ELISA analysis of interleukin (IL)-1β release ( b ). Data in a are representative of three individual experiments. Data in b are the mean ± SD of three individual experiments. The p values in b represent comparisons between none and MSU crystals alone in the presence or absence of arhalofenate acid in either wild-type (WT) or AMPKα1 knockout (KO) BMDMs. ns not significant

Techniques Used: In Vitro, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Knock-Out

Arhalofenate acid induced phosphorylation of AMPKα and expression of SIRT1 in BMDMs in vitro. BMDMs prepared from wild-type (WT) and AMPKα1 knockout (KO) mice were pretreated with arhalofenate acid at the concentrations indicated for 18 h ( a ) or at 100 μM for 1 h ( b ) in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α and sirtuin 1 (SIRT1). Data in both a and b are representative of three individual experiments
Figure Legend Snippet: Arhalofenate acid induced phosphorylation of AMPKα and expression of SIRT1 in BMDMs in vitro. BMDMs prepared from wild-type (WT) and AMPKα1 knockout (KO) mice were pretreated with arhalofenate acid at the concentrations indicated for 18 h ( a ) or at 100 μM for 1 h ( b ) in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α and sirtuin 1 (SIRT1). Data in both a and b are representative of three individual experiments

Techniques Used: Expressing, In Vitro, Knock-Out, Mouse Assay, Western Blot

2) Product Images from "Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling"

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-018-1699-4

Arhalofenate acid activated AMPK downstream targets involved in regulation of mitochondrial function and maintained mitochondrial cristae area. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulation with monosodium urate (MSU) crystals (0.2 mg/mL) for 1 h or 18 h in RPMI containing 1% FBS. Cell lysates prepared from 18-h treatment samples were subjected to Western blot analysis of phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α, expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM), and expression of thioredoxin (TRX)1, TRX2, and thioredoxin-interacting protein (TXNIP) ( a ). Cells from 1-h treatment were then stained with MitoSOX Red and MitoTracker Green and visualized by fluorescence microscopy ( b , magnification 20×). TEM analysis was performed to examine mitochondrial cristae (the folds of inner mitochondrial membrane indicated by white arrows in c ), and the cristae volume density are presented ( d ). Data in a and b are representative of three individual experiments. Data in c are representative of 30 cells examined for each condition. Data in d are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid
Figure Legend Snippet: Arhalofenate acid activated AMPK downstream targets involved in regulation of mitochondrial function and maintained mitochondrial cristae area. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulation with monosodium urate (MSU) crystals (0.2 mg/mL) for 1 h or 18 h in RPMI containing 1% FBS. Cell lysates prepared from 18-h treatment samples were subjected to Western blot analysis of phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α, expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM), and expression of thioredoxin (TRX)1, TRX2, and thioredoxin-interacting protein (TXNIP) ( a ). Cells from 1-h treatment were then stained with MitoSOX Red and MitoTracker Green and visualized by fluorescence microscopy ( b , magnification 20×). TEM analysis was performed to examine mitochondrial cristae (the folds of inner mitochondrial membrane indicated by white arrows in c ), and the cristae volume density are presented ( d ). Data in a and b are representative of three individual experiments. Data in c are representative of 30 cells examined for each condition. Data in d are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid

Techniques Used: Western Blot, Expressing, Pyrolysis Gas Chromatography, Staining, Fluorescence, Microscopy, Transmission Electron Microscopy

Arhalofenate acid prevented prolonged accumulation of p62 by promoting autophagy flux in response to MSU crystals. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 2 h in the presence or absence of bafilomycin (Baf; 100 nM) and for 6 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis of LC3 and p62 ( a ). TEM was performed to examine autophagosomes (indicated by black arrows in b ), and the numbers of autophagosomes containing electron dense material per μm 2 are presented ( c ). Data in a and b are representative of three individual experiments. Data in c are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid
Figure Legend Snippet: Arhalofenate acid prevented prolonged accumulation of p62 by promoting autophagy flux in response to MSU crystals. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 2 h in the presence or absence of bafilomycin (Baf; 100 nM) and for 6 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis of LC3 and p62 ( a ). TEM was performed to examine autophagosomes (indicated by black arrows in b ), and the numbers of autophagosomes containing electron dense material per μm 2 are presented ( c ). Data in a and b are representative of three individual experiments. Data in c are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid

Techniques Used: Western Blot, Transmission Electron Microscopy

Arhalofenate acid attenuates MSU crystal-induced IL-1β release by inhibiting NLRP3 inflammasome activation in BMDMs in vitro. BMDMs were pretreated with arhalofenate acid at a concentration of 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) in RPMI containing 1% FBS for 18 h. The conditioned media was used for ELISA for interleukin (IL)-1β ( a ), and the cell lysates were subjected to Western blot analysis ( b ) for expression of NLRP3, pro-caspase 1, and cleaved caspase 1 (p10). Data in a are the mean ± SD of three individual experiments, and p values represent comparisons between none and MSU crystals alone, or between MSU crystal alone and MSU crystals plus arhalofenate acid. Data in b are representative of three individual experiments
Figure Legend Snippet: Arhalofenate acid attenuates MSU crystal-induced IL-1β release by inhibiting NLRP3 inflammasome activation in BMDMs in vitro. BMDMs were pretreated with arhalofenate acid at a concentration of 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) in RPMI containing 1% FBS for 18 h. The conditioned media was used for ELISA for interleukin (IL)-1β ( a ), and the cell lysates were subjected to Western blot analysis ( b ) for expression of NLRP3, pro-caspase 1, and cleaved caspase 1 (p10). Data in a are the mean ± SD of three individual experiments, and p values represent comparisons between none and MSU crystals alone, or between MSU crystal alone and MSU crystals plus arhalofenate acid. Data in b are representative of three individual experiments

Techniques Used: Activation Assay, In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Arhalofenate acid inhibited MSU crystal-induced IL-1β via AMPK in BMDMs in vitro. BMDMs were treated with arhalofenate acid at 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 18 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α ( a ). The conditioned medium was used for ELISA analysis of interleukin (IL)-1β release ( b ). Data in a are representative of three individual experiments. Data in b are the mean ± SD of three individual experiments. The p values in b represent comparisons between none and MSU crystals alone in the presence or absence of arhalofenate acid in either wild-type (WT) or AMPKα1 knockout (KO) BMDMs. ns not significant
Figure Legend Snippet: Arhalofenate acid inhibited MSU crystal-induced IL-1β via AMPK in BMDMs in vitro. BMDMs were treated with arhalofenate acid at 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 18 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α ( a ). The conditioned medium was used for ELISA analysis of interleukin (IL)-1β release ( b ). Data in a are representative of three individual experiments. Data in b are the mean ± SD of three individual experiments. The p values in b represent comparisons between none and MSU crystals alone in the presence or absence of arhalofenate acid in either wild-type (WT) or AMPKα1 knockout (KO) BMDMs. ns not significant

Techniques Used: In Vitro, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Knock-Out

Arhalofenate acid induced phosphorylation of AMPKα and expression of SIRT1 in BMDMs in vitro. BMDMs prepared from wild-type (WT) and AMPKα1 knockout (KO) mice were pretreated with arhalofenate acid at the concentrations indicated for 18 h ( a ) or at 100 μM for 1 h ( b ) in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α and sirtuin 1 (SIRT1). Data in both a and b are representative of three individual experiments
Figure Legend Snippet: Arhalofenate acid induced phosphorylation of AMPKα and expression of SIRT1 in BMDMs in vitro. BMDMs prepared from wild-type (WT) and AMPKα1 knockout (KO) mice were pretreated with arhalofenate acid at the concentrations indicated for 18 h ( a ) or at 100 μM for 1 h ( b ) in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α and sirtuin 1 (SIRT1). Data in both a and b are representative of three individual experiments

Techniques Used: Expressing, In Vitro, Knock-Out, Mouse Assay, Western Blot

3) Product Images from "A dual regulatory circuit consisting of S-adenosylmethionine decarboxylase protein and its reaction product controls expression of the paralogous activator prozyme in Trypanosoma brucei"

Article Title: A dual regulatory circuit consisting of S-adenosylmethionine decarboxylase protein and its reaction product controls expression of the paralogous activator prozyme in Trypanosoma brucei

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007404

Methionine starvation upregulates prozyme. (A) Cell viability of BSF 427 cells grown for 48 h in methionine-free HMI-19 media with 10% FBS supplemented with varying levels of methionine (Met) as indicated. Viable cells were quantitated by CellTiter Glo assay. Colored bars mark methionine levels used in Fig 6B, 6C, 6D, 6E and 6F. The dotted line marks the fitted EC 50 (mean ± standard error of the fit = 9.0 ± 0.85 μM). Error bars represent SD for three biological replicates. (B) BSF 427 cells cultured for 48 h with varying levels of methionine and then treated ±Genz-644131 (15 nM) 24 h prior to harvest. (C) Quantitation of the western blot in Fig 6A normalized to Met 200 μM –Genz. (D) RT-qPCR analysis of prozyme mRNA from cultures used in A normalized to Met 200 μM –Genz. (E) Protein:mRNA ratio. (F) Fold change of dcAdoMet and AdoMet metabolite levels measured by LC-MS normalized to Met 200 μM –Genz. For Fig 6C, 6D, 6E and 6F, error bars represent SEM for three biological replicates, n = 3. For F, significance was determined by multiple T test analysis in GraphPad Prism comparing samples to Met 200 μM –Genz. * P
Figure Legend Snippet: Methionine starvation upregulates prozyme. (A) Cell viability of BSF 427 cells grown for 48 h in methionine-free HMI-19 media with 10% FBS supplemented with varying levels of methionine (Met) as indicated. Viable cells were quantitated by CellTiter Glo assay. Colored bars mark methionine levels used in Fig 6B, 6C, 6D, 6E and 6F. The dotted line marks the fitted EC 50 (mean ± standard error of the fit = 9.0 ± 0.85 μM). Error bars represent SD for three biological replicates. (B) BSF 427 cells cultured for 48 h with varying levels of methionine and then treated ±Genz-644131 (15 nM) 24 h prior to harvest. (C) Quantitation of the western blot in Fig 6A normalized to Met 200 μM –Genz. (D) RT-qPCR analysis of prozyme mRNA from cultures used in A normalized to Met 200 μM –Genz. (E) Protein:mRNA ratio. (F) Fold change of dcAdoMet and AdoMet metabolite levels measured by LC-MS normalized to Met 200 μM –Genz. For Fig 6C, 6D, 6E and 6F, error bars represent SEM for three biological replicates, n = 3. For F, significance was determined by multiple T test analysis in GraphPad Prism comparing samples to Met 200 μM –Genz. * P

Techniques Used: Glo Assay, Cell Culture, Quantitation Assay, Western Blot, Quantitative RT-PCR, Liquid Chromatography with Mass Spectroscopy

4) Product Images from "Hypoxic Reactive Oxygen Species Regulate the Integrated Stress Response and Cell Survival *Hypoxic Reactive Oxygen Species Regulate the Integrated Stress Response and Cell Survival * S⃞"

Article Title: Hypoxic Reactive Oxygen Species Regulate the Integrated Stress Response and Cell Survival *Hypoxic Reactive Oxygen Species Regulate the Integrated Stress Response and Cell Survival * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M805056200

The PERK/eIF2 α pathway is critical for adaptation to low O 2 and growth factor conditions. A , eIF2α S51S and S51A MEFs were exposed to 20 h of 0.5% O 2 ( H ), 1 h 20 μ m H 2 O 2 ( R ), or 4 h of 0.8 μ m thapsigargin ( T ). eIF2α phosphorylation in total lysates was determined. B , eIF2α S51S or S51A MEFs were exposed to 21 or 0.5% O 2 for 48 h in medium containing full (10%) or reduced (0.5%) FBS and full (4.5 g/liter) or reduced glucose ( Gluc ) (0.2 g/liter). Cell survival was examined by colony formation in regular medium (10% FBS/4.5 g/liter glucose) under normoxia for an additional 7 days. Colonies were stained using 4% crystal violet. C , eIF2α phosphorylation in S51S MEFs after growing for 48 h in normoxia in regular medium ( 10/4.5 ), or medium containing 0.2 g/liter glucose ( 10/0.2 ) or 0.5% FBS ( 0.5/4.5 ). D and E , survival for S51S or S51A MEFs exposed to 21 or 0.5% O 2 for 24 h in serum-reduced medium containing 0.5% FBS. Shown are representative assays ( D ) and quantification of colonies ( E ) ( n = 4). ** , p
Figure Legend Snippet: The PERK/eIF2 α pathway is critical for adaptation to low O 2 and growth factor conditions. A , eIF2α S51S and S51A MEFs were exposed to 20 h of 0.5% O 2 ( H ), 1 h 20 μ m H 2 O 2 ( R ), or 4 h of 0.8 μ m thapsigargin ( T ). eIF2α phosphorylation in total lysates was determined. B , eIF2α S51S or S51A MEFs were exposed to 21 or 0.5% O 2 for 48 h in medium containing full (10%) or reduced (0.5%) FBS and full (4.5 g/liter) or reduced glucose ( Gluc ) (0.2 g/liter). Cell survival was examined by colony formation in regular medium (10% FBS/4.5 g/liter glucose) under normoxia for an additional 7 days. Colonies were stained using 4% crystal violet. C , eIF2α phosphorylation in S51S MEFs after growing for 48 h in normoxia in regular medium ( 10/4.5 ), or medium containing 0.2 g/liter glucose ( 10/0.2 ) or 0.5% FBS ( 0.5/4.5 ). D and E , survival for S51S or S51A MEFs exposed to 21 or 0.5% O 2 for 24 h in serum-reduced medium containing 0.5% FBS. Shown are representative assays ( D ) and quantification of colonies ( E ) ( n = 4). ** , p

Techniques Used: Staining

5) Product Images from "A dual regulatory circuit consisting of S-adenosylmethionine decarboxylase protein and its reaction product controls expression of the paralogous activator prozyme in Trypanosoma brucei"

Article Title: A dual regulatory circuit consisting of S-adenosylmethionine decarboxylase protein and its reaction product controls expression of the paralogous activator prozyme in Trypanosoma brucei

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007404

Methionine starvation upregulates prozyme. (A) Cell viability of BSF 427 cells grown for 48 h in methionine-free HMI-19 media with 10% FBS supplemented with varying levels of methionine (Met) as indicated. Viable cells were quantitated by CellTiter Glo assay. Colored bars mark methionine levels used in Fig 6B, 6C, 6D, 6E and 6F. The dotted line marks the fitted EC 50 (mean ± standard error of the fit = 9.0 ± 0.85 μM). Error bars represent SD for three biological replicates. (B) BSF 427 cells cultured for 48 h with varying levels of methionine and then treated ±Genz-644131 (15 nM) 24 h prior to harvest. (C) Quantitation of the western blot in Fig 6A normalized to Met 200 μM –Genz. (D) RT-qPCR analysis of prozyme mRNA from cultures used in A normalized to Met 200 μM –Genz. (E) Protein:mRNA ratio. (F) Fold change of dcAdoMet and AdoMet metabolite levels measured by LC-MS normalized to Met 200 μM –Genz. For Fig 6C, 6D, 6E and 6F, error bars represent SEM for three biological replicates, n = 3. For F, significance was determined by multiple T test analysis in GraphPad Prism comparing samples to Met 200 μM –Genz. * P
Figure Legend Snippet: Methionine starvation upregulates prozyme. (A) Cell viability of BSF 427 cells grown for 48 h in methionine-free HMI-19 media with 10% FBS supplemented with varying levels of methionine (Met) as indicated. Viable cells were quantitated by CellTiter Glo assay. Colored bars mark methionine levels used in Fig 6B, 6C, 6D, 6E and 6F. The dotted line marks the fitted EC 50 (mean ± standard error of the fit = 9.0 ± 0.85 μM). Error bars represent SD for three biological replicates. (B) BSF 427 cells cultured for 48 h with varying levels of methionine and then treated ±Genz-644131 (15 nM) 24 h prior to harvest. (C) Quantitation of the western blot in Fig 6A normalized to Met 200 μM –Genz. (D) RT-qPCR analysis of prozyme mRNA from cultures used in A normalized to Met 200 μM –Genz. (E) Protein:mRNA ratio. (F) Fold change of dcAdoMet and AdoMet metabolite levels measured by LC-MS normalized to Met 200 μM –Genz. For Fig 6C, 6D, 6E and 6F, error bars represent SEM for three biological replicates, n = 3. For F, significance was determined by multiple T test analysis in GraphPad Prism comparing samples to Met 200 μM –Genz. * P

Techniques Used: Glo Assay, Cell Culture, Quantitation Assay, Western Blot, Quantitative RT-PCR, Liquid Chromatography with Mass Spectroscopy

6) Product Images from "Quantitative Techniques for Assessing and Controlling the Dispersion and Biological Effects of Multi-walled Carbon Nanotubes in Mammalian Tissue Culture Cells"

Article Title: Quantitative Techniques for Assessing and Controlling the Dispersion and Biological Effects of Multi-walled Carbon Nanotubes in Mammalian Tissue Culture Cells

Journal: ACS nano

doi: 10.1021/nn102112b

Assessment of the suspension stability index of AP-MWCNT, PD-MWCNT and COOH-MWCNT in DMEM, with or without the addition of dispersing agents [BSA (0.6 mg/mL), DPPC (0.01 mg/mL) and 10 % FBS]. The suspension stability index of the MWCNT in DMEM was determined
Figure Legend Snippet: Assessment of the suspension stability index of AP-MWCNT, PD-MWCNT and COOH-MWCNT in DMEM, with or without the addition of dispersing agents [BSA (0.6 mg/mL), DPPC (0.01 mg/mL) and 10 % FBS]. The suspension stability index of the MWCNT in DMEM was determined

Techniques Used:

7) Product Images from "Sirt1-Deficient Mice Have Hypogonadotropic Hypogonadism due to Defective GnRH Neuronal Migration"

Article Title: Sirt1-Deficient Mice Have Hypogonadotropic Hypogonadism due to Defective GnRH Neuronal Migration

Journal: Molecular Endocrinology

doi: 10.1210/me.2014-1228

Sirt1-cortactin association is regulated by FGFR1/FGF8. A, Subcellular localization of Sirt1 and cortactin in GN11 cells. Cells were assessed at the basal state (10% FBS) in the presence of either FGF8 (+FGF8) or vehicle (−FGF8) at different time points (0, 24, and 48 h). Immunostaining was conducted for Sirt1 (red) and cortactin (green), and 4′,6′-diamino-2-phenylindole (DAPI; (blue) was used as nuclear marker. Colocalized pixels between Sirt1 and cortactin were automatically highlighted as yellow dots by an imaging analysis software (ImageJ; National Institutes of Health). B, Quantitation of cytoplasmic Sirt1 immunoreactivity in GN11 cells. C, Acetylated-cortactin in GN11 cells treated for 24 hours with either FGF8 or vehicle. D, Cortactin acetylation fold enrichment in GN11 cells. Data are shown as mean ± SEM for n = 3 separate experiments. ***, P
Figure Legend Snippet: Sirt1-cortactin association is regulated by FGFR1/FGF8. A, Subcellular localization of Sirt1 and cortactin in GN11 cells. Cells were assessed at the basal state (10% FBS) in the presence of either FGF8 (+FGF8) or vehicle (−FGF8) at different time points (0, 24, and 48 h). Immunostaining was conducted for Sirt1 (red) and cortactin (green), and 4′,6′-diamino-2-phenylindole (DAPI; (blue) was used as nuclear marker. Colocalized pixels between Sirt1 and cortactin were automatically highlighted as yellow dots by an imaging analysis software (ImageJ; National Institutes of Health). B, Quantitation of cytoplasmic Sirt1 immunoreactivity in GN11 cells. C, Acetylated-cortactin in GN11 cells treated for 24 hours with either FGF8 or vehicle. D, Cortactin acetylation fold enrichment in GN11 cells. Data are shown as mean ± SEM for n = 3 separate experiments. ***, P

Techniques Used: Immunostaining, Marker, Imaging, Software, Quantitation Assay

8) Product Images from "Cross Talk between Insulin and Bone Morphogenetic Protein Signaling Systems in Brown Adipogenesis ▿Cross Talk between Insulin and Bone Morphogenetic Protein Signaling Systems in Brown Adipogenesis ▿ †"

Article Title: Cross Talk between Insulin and Bone Morphogenetic Protein Signaling Systems in Brown Adipogenesis ▿Cross Talk between Insulin and Bone Morphogenetic Protein Signaling Systems in Brown Adipogenesis ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00363-10

Overexpression of BMP7 induced spontaneous differentiation of wild-type brown preadipocytes and rescued brown adipocyte differentiation of IRS-1KO preadipocytes. WT and IRS-1KO brown preadipocytes were stably transfected with an hBMP7 construct driven by the aP2 promoter or with empty vector (as a control). Pools of transfected clones were grown to confluence (defined as day 0) and proceeded to continuous culture in DMEM plus 10% FBS without any inducers (for WT cells) or with Dex-IBMX-insulin-indomethacinn-T3 (for IRS-1KO cells) (see Materials and Methods). On day 10, the analyses were performed. (A) hBMP7 levels in the medium supernatants from WT and IRS-1KO cells were determined by ELISA. (B and C) Oil Red O staining and microscopic views of WT cells (B) and IRS-1KO cells (C). (D and E) Expression of adipogenic markers and brown-fat-selective genes in WT cells (D) and IRS-1KO cells (E) was analyzed by Q-RT-PCR. The data are presented as the mean plus SEM from a representative of 3 independent experiments, with each performed in duplicate or triplicate. The values for vector are set as 100 arbitrarily. Statistically significant differences between vector- and hBMP7-transfected cells are indicated above the bars: *, P
Figure Legend Snippet: Overexpression of BMP7 induced spontaneous differentiation of wild-type brown preadipocytes and rescued brown adipocyte differentiation of IRS-1KO preadipocytes. WT and IRS-1KO brown preadipocytes were stably transfected with an hBMP7 construct driven by the aP2 promoter or with empty vector (as a control). Pools of transfected clones were grown to confluence (defined as day 0) and proceeded to continuous culture in DMEM plus 10% FBS without any inducers (for WT cells) or with Dex-IBMX-insulin-indomethacinn-T3 (for IRS-1KO cells) (see Materials and Methods). On day 10, the analyses were performed. (A) hBMP7 levels in the medium supernatants from WT and IRS-1KO cells were determined by ELISA. (B and C) Oil Red O staining and microscopic views of WT cells (B) and IRS-1KO cells (C). (D and E) Expression of adipogenic markers and brown-fat-selective genes in WT cells (D) and IRS-1KO cells (E) was analyzed by Q-RT-PCR. The data are presented as the mean plus SEM from a representative of 3 independent experiments, with each performed in duplicate or triplicate. The values for vector are set as 100 arbitrarily. Statistically significant differences between vector- and hBMP7-transfected cells are indicated above the bars: *, P

Techniques Used: Over Expression, Stable Transfection, Transfection, Construct, Plasmid Preparation, Clone Assay, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction

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Article Snippet: .. HEK293 cells and MEFs were propagated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) (Gemini Bioproducts) and 4.5 g/liter glucose, as described previously ( ). ..

Article Title: Cross Talk between Insulin and Bone Morphogenetic Protein Signaling Systems in Brown Adipogenesis ▿Cross Talk between Insulin and Bone Morphogenetic Protein Signaling Systems in Brown Adipogenesis ▿ †
Article Snippet: .. Briefly, brown preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products). ..

Cell Culture:

Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling
Article Snippet: .. Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA). .. After 5–7 days, the M-CSF-derived macrophages were re-plated onto 24-well (5 × 105 /well) or six-well (2 × 106 /well) plates and primed with 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Gemini Bio-products, West Sacramento, CA) for 24 h in complete RPMI medium before treatment with the indicated reagents in fresh RPMI containing only 1% FBS.

Article Title: A dual regulatory circuit consisting of S-adenosylmethionine decarboxylase protein and its reaction product controls expression of the paralogous activator prozyme in Trypanosoma brucei
Article Snippet: .. T . brucei cells were cultured in HMI-19 medium [ ] with 10% fetal bovine serum (FBS) (Tet-free, heat-inactivated; Gemini Bio-Products) or dialyzed FBS (Tet-free, heat-inactivated; Gemini Bio-Products) at 37°C and 5% CO2 [ , ]. .. Parasite transfections were performed as previously described [ ].

Article Title: Cross Talk between Insulin and Bone Morphogenetic Protein Signaling Systems in Brown Adipogenesis ▿Cross Talk between Insulin and Bone Morphogenetic Protein Signaling Systems in Brown Adipogenesis ▿ †
Article Snippet: .. Briefly, brown preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products). ..

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    Arhalofenate acid activated AMPK downstream targets involved in regulation of mitochondrial function and maintained mitochondrial cristae area. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulation with monosodium urate (MSU) crystals (0.2 mg/mL) for 1 h or 18 h in <t>RPMI</t> containing 1% <t>FBS.</t> Cell lysates prepared from 18-h treatment samples were subjected to Western blot analysis of phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α, expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM), and expression of thioredoxin (TRX)1, TRX2, and thioredoxin-interacting protein (TXNIP) ( a ). Cells from 1-h treatment were then stained with MitoSOX Red and MitoTracker Green and visualized by fluorescence microscopy ( b , magnification 20×). TEM analysis was performed to examine mitochondrial cristae (the folds of inner mitochondrial membrane indicated by white arrows in c ), and the cristae volume density are presented ( d ). Data in a and b are representative of three individual experiments. Data in c are representative of 30 cells examined for each condition. Data in d are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid
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    Characterization of SERT in JAR cells with glucose at different concentrations. (a) JAR cells were cultured in RPMI supplemented with 10% <t>FBS</t> with/without insulin and D-glucose at different concentrations, 5.5, 10, 15, 25 <t>mmol/L.</t> The [ 3 H]-5HT uptake rate
    Insulin Free Fbs, supplied by Gemini Bio, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of methyl-β-cyclodextrin <t>(MβCD)</t> and cholesterol (chol)-MβCD treatments on membrane cholesterol level and cell viability of MSCs. a Cholesterol level of MSCs after treatment with MβCD for 40 min at the concentrations indicated. Membrane cholesterol levels were measured and compared (untreated cells = 100%). b Cholesterol level of MSCs after treatment with 10 mM MβCD or 100 μM chol-MβCD for the time indicated. c Time course of cholesterol level of MSCs after initial treatment with 10 mM MβCD for 60 min. After treatment, cells were washed with PBS and incubated in fresh PM containing <t>CS-FBS</t> for the time indicated. d Viability of cells, treated as described in Fig. 1b, evaluated by MTS assay. Results are presented as mean ± SD; n ≥ 3 independent experiments per cell line per time point. The results showed the effectiveness of MβCD and chol-MβCD in altering MSC membrane cholesterol level without compromising cell viability. * p
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    Arhalofenate acid activated AMPK downstream targets involved in regulation of mitochondrial function and maintained mitochondrial cristae area. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulation with monosodium urate (MSU) crystals (0.2 mg/mL) for 1 h or 18 h in RPMI containing 1% FBS. Cell lysates prepared from 18-h treatment samples were subjected to Western blot analysis of phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α, expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM), and expression of thioredoxin (TRX)1, TRX2, and thioredoxin-interacting protein (TXNIP) ( a ). Cells from 1-h treatment were then stained with MitoSOX Red and MitoTracker Green and visualized by fluorescence microscopy ( b , magnification 20×). TEM analysis was performed to examine mitochondrial cristae (the folds of inner mitochondrial membrane indicated by white arrows in c ), and the cristae volume density are presented ( d ). Data in a and b are representative of three individual experiments. Data in c are representative of 30 cells examined for each condition. Data in d are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid

    Journal: Arthritis Research & Therapy

    Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

    doi: 10.1186/s13075-018-1699-4

    Figure Lengend Snippet: Arhalofenate acid activated AMPK downstream targets involved in regulation of mitochondrial function and maintained mitochondrial cristae area. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulation with monosodium urate (MSU) crystals (0.2 mg/mL) for 1 h or 18 h in RPMI containing 1% FBS. Cell lysates prepared from 18-h treatment samples were subjected to Western blot analysis of phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α, expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM), and expression of thioredoxin (TRX)1, TRX2, and thioredoxin-interacting protein (TXNIP) ( a ). Cells from 1-h treatment were then stained with MitoSOX Red and MitoTracker Green and visualized by fluorescence microscopy ( b , magnification 20×). TEM analysis was performed to examine mitochondrial cristae (the folds of inner mitochondrial membrane indicated by white arrows in c ), and the cristae volume density are presented ( d ). Data in a and b are representative of three individual experiments. Data in c are representative of 30 cells examined for each condition. Data in d are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid

    Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

    Techniques: Western Blot, Expressing, Pyrolysis Gas Chromatography, Staining, Fluorescence, Microscopy, Transmission Electron Microscopy

    Arhalofenate acid prevented prolonged accumulation of p62 by promoting autophagy flux in response to MSU crystals. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 2 h in the presence or absence of bafilomycin (Baf; 100 nM) and for 6 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis of LC3 and p62 ( a ). TEM was performed to examine autophagosomes (indicated by black arrows in b ), and the numbers of autophagosomes containing electron dense material per μm 2 are presented ( c ). Data in a and b are representative of three individual experiments. Data in c are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid

    Journal: Arthritis Research & Therapy

    Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

    doi: 10.1186/s13075-018-1699-4

    Figure Lengend Snippet: Arhalofenate acid prevented prolonged accumulation of p62 by promoting autophagy flux in response to MSU crystals. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 2 h in the presence or absence of bafilomycin (Baf; 100 nM) and for 6 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis of LC3 and p62 ( a ). TEM was performed to examine autophagosomes (indicated by black arrows in b ), and the numbers of autophagosomes containing electron dense material per μm 2 are presented ( c ). Data in a and b are representative of three individual experiments. Data in c are the mean ± SEM of 30 cells. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid

    Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

    Techniques: Western Blot, Transmission Electron Microscopy

    Arhalofenate acid attenuates MSU crystal-induced IL-1β release by inhibiting NLRP3 inflammasome activation in BMDMs in vitro. BMDMs were pretreated with arhalofenate acid at a concentration of 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) in RPMI containing 1% FBS for 18 h. The conditioned media was used for ELISA for interleukin (IL)-1β ( a ), and the cell lysates were subjected to Western blot analysis ( b ) for expression of NLRP3, pro-caspase 1, and cleaved caspase 1 (p10). Data in a are the mean ± SD of three individual experiments, and p values represent comparisons between none and MSU crystals alone, or between MSU crystal alone and MSU crystals plus arhalofenate acid. Data in b are representative of three individual experiments

    Journal: Arthritis Research & Therapy

    Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

    doi: 10.1186/s13075-018-1699-4

    Figure Lengend Snippet: Arhalofenate acid attenuates MSU crystal-induced IL-1β release by inhibiting NLRP3 inflammasome activation in BMDMs in vitro. BMDMs were pretreated with arhalofenate acid at a concentration of 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) in RPMI containing 1% FBS for 18 h. The conditioned media was used for ELISA for interleukin (IL)-1β ( a ), and the cell lysates were subjected to Western blot analysis ( b ) for expression of NLRP3, pro-caspase 1, and cleaved caspase 1 (p10). Data in a are the mean ± SD of three individual experiments, and p values represent comparisons between none and MSU crystals alone, or between MSU crystal alone and MSU crystals plus arhalofenate acid. Data in b are representative of three individual experiments

    Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

    Techniques: Activation Assay, In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

    Arhalofenate acid inhibited MSU crystal-induced IL-1β via AMPK in BMDMs in vitro. BMDMs were treated with arhalofenate acid at 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 18 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α ( a ). The conditioned medium was used for ELISA analysis of interleukin (IL)-1β release ( b ). Data in a are representative of three individual experiments. Data in b are the mean ± SD of three individual experiments. The p values in b represent comparisons between none and MSU crystals alone in the presence or absence of arhalofenate acid in either wild-type (WT) or AMPKα1 knockout (KO) BMDMs. ns not significant

    Journal: Arthritis Research & Therapy

    Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

    doi: 10.1186/s13075-018-1699-4

    Figure Lengend Snippet: Arhalofenate acid inhibited MSU crystal-induced IL-1β via AMPK in BMDMs in vitro. BMDMs were treated with arhalofenate acid at 100 μM for 1 h before being stimulated with monosodium urate (MSU) crystals (0.2 mg/mL) for 18 h in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α ( a ). The conditioned medium was used for ELISA analysis of interleukin (IL)-1β release ( b ). Data in a are representative of three individual experiments. Data in b are the mean ± SD of three individual experiments. The p values in b represent comparisons between none and MSU crystals alone in the presence or absence of arhalofenate acid in either wild-type (WT) or AMPKα1 knockout (KO) BMDMs. ns not significant

    Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

    Techniques: In Vitro, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Knock-Out

    Arhalofenate acid induced phosphorylation of AMPKα and expression of SIRT1 in BMDMs in vitro. BMDMs prepared from wild-type (WT) and AMPKα1 knockout (KO) mice were pretreated with arhalofenate acid at the concentrations indicated for 18 h ( a ) or at 100 μM for 1 h ( b ) in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α and sirtuin 1 (SIRT1). Data in both a and b are representative of three individual experiments

    Journal: Arthritis Research & Therapy

    Article Title: Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

    doi: 10.1186/s13075-018-1699-4

    Figure Lengend Snippet: Arhalofenate acid induced phosphorylation of AMPKα and expression of SIRT1 in BMDMs in vitro. BMDMs prepared from wild-type (WT) and AMPKα1 knockout (KO) mice were pretreated with arhalofenate acid at the concentrations indicated for 18 h ( a ) or at 100 μM for 1 h ( b ) in RPMI containing 1% FBS. Cell lysates were subjected to Western blot analysis for phosphorylation (p) and expression of AMP-activated protein kinase (AMPK)α and sirtuin 1 (SIRT1). Data in both a and b are representative of three individual experiments

    Article Snippet: Briefly, bone marrow cells were cultured in complete RPMI media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence of macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Gemini Bio-products, West Sacramento, CA).

    Techniques: Expressing, In Vitro, Knock-Out, Mouse Assay, Western Blot

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

    Journal: Cell

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

    doi: 10.1016/j.cell.2018.11.011

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

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

    Techniques: Cell Culture, Expressing, Quantitative RT-PCR

    Characterization of SERT in JAR cells with glucose at different concentrations. (a) JAR cells were cultured in RPMI supplemented with 10% FBS with/without insulin and D-glucose at different concentrations, 5.5, 10, 15, 25 mmol/L. The [ 3 H]-5HT uptake rate

    Journal:

    Article Title: At diabetes-like concentration, glucose down-regulates the placental serotonin transport system in a cell-cycle-dependent manner

    doi: 10.1111/j.1471-4159.2007.04469.x

    Figure Lengend Snippet: Characterization of SERT in JAR cells with glucose at different concentrations. (a) JAR cells were cultured in RPMI supplemented with 10% FBS with/without insulin and D-glucose at different concentrations, 5.5, 10, 15, 25 mmol/L. The [ 3 H]-5HT uptake rate

    Article Snippet: Also, these data demonstrate that there is a 60% increase in overall DNA synthesis of JAR cells if they enter the cell cycle in media supplemented with 25 mmol/L D-glucose and insulin-free FBS ( ).

    Techniques: Cell Culture

    Concentration-dependent effect of exogenous D-glucose on relative SERT mRNA in JAR cells. JAR cells were cultured in RPMI supplemented with 10% FBS without insulin and D-glucose at different concentrations, 5.5, 10, 15, 25 mmol/L (lanes 2–5).

    Journal:

    Article Title: At diabetes-like concentration, glucose down-regulates the placental serotonin transport system in a cell-cycle-dependent manner

    doi: 10.1111/j.1471-4159.2007.04469.x

    Figure Lengend Snippet: Concentration-dependent effect of exogenous D-glucose on relative SERT mRNA in JAR cells. JAR cells were cultured in RPMI supplemented with 10% FBS without insulin and D-glucose at different concentrations, 5.5, 10, 15, 25 mmol/L (lanes 2–5).

    Article Snippet: Also, these data demonstrate that there is a 60% increase in overall DNA synthesis of JAR cells if they enter the cell cycle in media supplemented with 25 mmol/L D-glucose and insulin-free FBS ( ).

    Techniques: Concentration Assay, Cell Culture

    Effects of methyl-β-cyclodextrin (MβCD) and cholesterol (chol)-MβCD treatments on membrane cholesterol level and cell viability of MSCs. a Cholesterol level of MSCs after treatment with MβCD for 40 min at the concentrations indicated. Membrane cholesterol levels were measured and compared (untreated cells = 100%). b Cholesterol level of MSCs after treatment with 10 mM MβCD or 100 μM chol-MβCD for the time indicated. c Time course of cholesterol level of MSCs after initial treatment with 10 mM MβCD for 60 min. After treatment, cells were washed with PBS and incubated in fresh PM containing CS-FBS for the time indicated. d Viability of cells, treated as described in Fig. 1b, evaluated by MTS assay. Results are presented as mean ± SD; n ≥ 3 independent experiments per cell line per time point. The results showed the effectiveness of MβCD and chol-MβCD in altering MSC membrane cholesterol level without compromising cell viability. * p

    Journal: Stem Cell Research & Therapy

    Article Title: Influence of cholesterol/caveolin-1/caveolae homeostasis on membrane properties and substrate adhesion characteristics of adult human mesenchymal stem cells

    doi: 10.1186/s13287-018-0830-4

    Figure Lengend Snippet: Effects of methyl-β-cyclodextrin (MβCD) and cholesterol (chol)-MβCD treatments on membrane cholesterol level and cell viability of MSCs. a Cholesterol level of MSCs after treatment with MβCD for 40 min at the concentrations indicated. Membrane cholesterol levels were measured and compared (untreated cells = 100%). b Cholesterol level of MSCs after treatment with 10 mM MβCD or 100 μM chol-MβCD for the time indicated. c Time course of cholesterol level of MSCs after initial treatment with 10 mM MβCD for 60 min. After treatment, cells were washed with PBS and incubated in fresh PM containing CS-FBS for the time indicated. d Viability of cells, treated as described in Fig. 1b, evaluated by MTS assay. Results are presented as mean ± SD; n ≥ 3 independent experiments per cell line per time point. The results showed the effectiveness of MβCD and chol-MβCD in altering MSC membrane cholesterol level without compromising cell viability. * p

    Article Snippet: After MβCD pretreatment, cholesterol-depleted cells were cultured in fresh medium containing charcoal stripped FBS (CS-FBS; Gemini Bio-Products, West Sacramento, CA, USA).

    Techniques: Incubation, MTS Assay