fatty acid free bsa  (Millipore)


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    Name:
    Bovine Serum Albumin
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    Catalog Number:
    a9306
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    Millipore fatty acid free bsa
    Bovine Serum Albumin

    https://www.bioz.com/result/fatty acid free bsa/product/Millipore
    Average 99 stars, based on 507 article reviews
    Price from $9.99 to $1999.99
    fatty acid free bsa - by Bioz Stars, 2020-10
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    1) Product Images from "Involvement of Band3 in the efflux of sphingosine 1-phosphate from erythrocytes"

    Article Title: Involvement of Band3 in the efflux of sphingosine 1-phosphate from erythrocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177543

    Modulation of S1P homeostasis of K562 cells by sodium butyrate-induced differentiation into erythroblast-like cells. K562 cells were treated with 2 mM sodium butyrate (NaB); 72 hours later, we investigated the modulation of S1P homeostasis. (A) The mRNA levels of GYPA and ALAS were determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (B, C) The expression of CD235a was investigated with a flow cytometer (n = 3/group). (D) C 17 S1P formation assay. NaB-treated or vehicle-treated K562 cells were treated with 10 μM of C 17 sphingosine for 20 minutes. Then, we replaced the supernatant with PBS containing 0.5% BSA and incubated the cells at 37°C for another 20 minutes. Then, the supernatants and cells were collected and used for the C 17 S1P measurements (n = 6/group). (E) The expression of key enzymes in S1P metabolism was determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (F) SK activity assay. The SK activity assay was performed using NaB-treated or vehicle-treated K562 cells (n = 6/group). (G) Reverse transcription PCR was performed using cDNAs prepared from NaB-treated K562 cells (K), HepG2 cells (He), and HUVECs (Hu). (H) The expression of possible S1P transporters was determined using real-time PCR. GAPDH was utilized as an internal control (n = 4/group). (I) Real-time PCR of Band3. GAPDH was utilized as an internal control (n = 8/group). (J) Western blot of Band3 with membranous protein. The whole cell lysate of RBCs (2 μg) was placed as a positive control. Pan-cadherin was utilized as an internal control (n = 3/group).
    Figure Legend Snippet: Modulation of S1P homeostasis of K562 cells by sodium butyrate-induced differentiation into erythroblast-like cells. K562 cells were treated with 2 mM sodium butyrate (NaB); 72 hours later, we investigated the modulation of S1P homeostasis. (A) The mRNA levels of GYPA and ALAS were determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (B, C) The expression of CD235a was investigated with a flow cytometer (n = 3/group). (D) C 17 S1P formation assay. NaB-treated or vehicle-treated K562 cells were treated with 10 μM of C 17 sphingosine for 20 minutes. Then, we replaced the supernatant with PBS containing 0.5% BSA and incubated the cells at 37°C for another 20 minutes. Then, the supernatants and cells were collected and used for the C 17 S1P measurements (n = 6/group). (E) The expression of key enzymes in S1P metabolism was determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (F) SK activity assay. The SK activity assay was performed using NaB-treated or vehicle-treated K562 cells (n = 6/group). (G) Reverse transcription PCR was performed using cDNAs prepared from NaB-treated K562 cells (K), HepG2 cells (He), and HUVECs (Hu). (H) The expression of possible S1P transporters was determined using real-time PCR. GAPDH was utilized as an internal control (n = 4/group). (I) Real-time PCR of Band3. GAPDH was utilized as an internal control (n = 8/group). (J) Western blot of Band3 with membranous protein. The whole cell lysate of RBCs (2 μg) was placed as a positive control. Pan-cadherin was utilized as an internal control (n = 3/group).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Tube Formation Assay, Incubation, Activity Assay, Polymerase Chain Reaction, Western Blot, Positive Control

    2) Product Images from "A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis"

    Article Title: A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09277-9

    HIF-2α selectively enriches polyunsaturated lipids. a Heatmap representing the relative lipid abundances in indicated cell lines. The abundance of each lipid species is normalized to the mean of that in the EPAS1 +/+ 786-O WT cells and the ratios are log2 transformed. The lipids are grouped by classes, and within each class, the lipid species are ordered first with increasing carbon number, then with increasing unsaturation levels. Abbreviations: CE, cholesterol ester; Cer, ceramide; MAG, monoacylglycerol; DAG, diacylglycerol; TAG, triacylglycerol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; ePC, (vinyl ether-linked) PC-plasmalogen; ePE, (vinyl ether-linked) PE-plasmalogen; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin. Blue: down-regulated relative to the WT cells, red: upregulated relative to the WT cells. The wave-like pattern in the TAG class corresponds to the more significant losses in the polyunsaturated fatty acyl (PUFA)-TAGs than saturated/monounsaturated fatty acyl (SFA/MUFA)-TAGs in response to HIF-2α-depletion. S , ferroptosis-sensitive; ( R ), ferroptosis-resistant. b Volcano plots showing the changes in TAGs grouped as PUFA-TAGs (red fill) and SFA/MUFA-TAGs (white fill) between the indicated cell lines. n = 3, two-tailed t-test. c Volcano plots showing changes in PE and ePE lipids grouped as PUFA-PE/ePEs (red fill) and SFA/MUFA-PE/ePEs (white fill) between the indicated cell lines. d Bar graph representing the relative abundances of the indicated PUFA-PE/ePE lipids in the labeled groups. Log2 fold changes relative to 786-O WT cells are presented for each condition. n = 3, error bars: ±s.d. e Bar graph representing the relative abundances of the indicated free fatty acids grouped as PUFAs or SFA/MUFAs in the indicated conditions. nd, not-detectable under the experimental condition. n = 3, error bars: ±s.d. f Viability curves of 786-O-Cas9 and 769-P-Cas9 cells expressing control (sgNC) or EPAS1 -targeting sgRNAs, first treated with BSA or PUFA (arachidonic acid, C20:4) for 3 days, then treated with indicated concentrations of ML210 or RSL3 for 48 h. Representative plot of experiments repeated twice. g The ratios between PUFA-PE/ePE and total PE/ePE (left), and between PUFA-PC/ePC and total PC/ePCs in ccRCC tumor samples ( n = 49; red) and the matched normal tissues ( n = 49; gray) from previously reported lipidomics datasets. Student’s T -test, *** p
    Figure Legend Snippet: HIF-2α selectively enriches polyunsaturated lipids. a Heatmap representing the relative lipid abundances in indicated cell lines. The abundance of each lipid species is normalized to the mean of that in the EPAS1 +/+ 786-O WT cells and the ratios are log2 transformed. The lipids are grouped by classes, and within each class, the lipid species are ordered first with increasing carbon number, then with increasing unsaturation levels. Abbreviations: CE, cholesterol ester; Cer, ceramide; MAG, monoacylglycerol; DAG, diacylglycerol; TAG, triacylglycerol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; ePC, (vinyl ether-linked) PC-plasmalogen; ePE, (vinyl ether-linked) PE-plasmalogen; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin. Blue: down-regulated relative to the WT cells, red: upregulated relative to the WT cells. The wave-like pattern in the TAG class corresponds to the more significant losses in the polyunsaturated fatty acyl (PUFA)-TAGs than saturated/monounsaturated fatty acyl (SFA/MUFA)-TAGs in response to HIF-2α-depletion. S , ferroptosis-sensitive; ( R ), ferroptosis-resistant. b Volcano plots showing the changes in TAGs grouped as PUFA-TAGs (red fill) and SFA/MUFA-TAGs (white fill) between the indicated cell lines. n = 3, two-tailed t-test. c Volcano plots showing changes in PE and ePE lipids grouped as PUFA-PE/ePEs (red fill) and SFA/MUFA-PE/ePEs (white fill) between the indicated cell lines. d Bar graph representing the relative abundances of the indicated PUFA-PE/ePE lipids in the labeled groups. Log2 fold changes relative to 786-O WT cells are presented for each condition. n = 3, error bars: ±s.d. e Bar graph representing the relative abundances of the indicated free fatty acids grouped as PUFAs or SFA/MUFAs in the indicated conditions. nd, not-detectable under the experimental condition. n = 3, error bars: ±s.d. f Viability curves of 786-O-Cas9 and 769-P-Cas9 cells expressing control (sgNC) or EPAS1 -targeting sgRNAs, first treated with BSA or PUFA (arachidonic acid, C20:4) for 3 days, then treated with indicated concentrations of ML210 or RSL3 for 48 h. Representative plot of experiments repeated twice. g The ratios between PUFA-PE/ePE and total PE/ePE (left), and between PUFA-PC/ePC and total PC/ePCs in ccRCC tumor samples ( n = 49; red) and the matched normal tissues ( n = 49; gray) from previously reported lipidomics datasets. Student’s T -test, *** p

    Techniques Used: Transformation Assay, Two Tailed Test, Labeling, Expressing

    3) Product Images from "Thioesterase-mediated control of cellular calcium homeostasis enables hepatic ER stress"

    Article Title: Thioesterase-mediated control of cellular calcium homeostasis enables hepatic ER stress

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI93123

    Them2 –/– and Pctp –/– mouse primary hepatocytes are protected against induction of ER stress by tunicamycin and palmitic acid. ( A and B ) ER stress was induced in mouse primary hepatocytes that were harvested from Them2 +/+ and Them2 –/– mice ( A ) and Pctp +/+ and Pctp –/– mice ( B ) by treatment of cells with tunicamycin (1 μg/ml) for 5 hours. ( C and D ) ER stress was induced in mouse primary hepatocytes harvested from Them2 +/+ and Them2 –/– mice ( C ) and Pctp +/+ and Pctp –/– mice ( D ) by treatment of cells with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours. Rescue of SFA-induced ER stress was performed by treatment of cells with the monounsaturated NEFA oleic acid (0.5 mM) in the presence of palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours. Hepatocytes were serum-starved overnight before treatments. Immunoblots are representative of 3 independent experiments.
    Figure Legend Snippet: Them2 –/– and Pctp –/– mouse primary hepatocytes are protected against induction of ER stress by tunicamycin and palmitic acid. ( A and B ) ER stress was induced in mouse primary hepatocytes that were harvested from Them2 +/+ and Them2 –/– mice ( A ) and Pctp +/+ and Pctp –/– mice ( B ) by treatment of cells with tunicamycin (1 μg/ml) for 5 hours. ( C and D ) ER stress was induced in mouse primary hepatocytes harvested from Them2 +/+ and Them2 –/– mice ( C ) and Pctp +/+ and Pctp –/– mice ( D ) by treatment of cells with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours. Rescue of SFA-induced ER stress was performed by treatment of cells with the monounsaturated NEFA oleic acid (0.5 mM) in the presence of palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours. Hepatocytes were serum-starved overnight before treatments. Immunoblots are representative of 3 independent experiments.

    Techniques Used: Mouse Assay, Western Blot

    Them2 and PC-TP enable palmitic acid–induced calcium efflux from the ER. ( A – C ) Serum-starved HEK 293E cells were treated with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours following knockdown of scrambled control (black/gray) ( A ), Them2 (red/pink) ( B ), or PC-TP (blue/aqua) ( C ). Calcium release into the cytosol was measured as a function of the fluorescence intensity of the cytosolic calcium indicator Fluo4-AM following the induction of ER calcium release by thapsigargin (top panels; Tg, 2 μM) or ionomycin (bottom panels; IO, 5 μM). ( D ) Steady-state cytosolic calcium levels in HEK 293E cells following treatment with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours. Cells were treated with Them2, PC-TP, or scrambled control siRNA for 48 hours and serum-starved overnight before palmitic acid treatment. * P
    Figure Legend Snippet: Them2 and PC-TP enable palmitic acid–induced calcium efflux from the ER. ( A – C ) Serum-starved HEK 293E cells were treated with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours following knockdown of scrambled control (black/gray) ( A ), Them2 (red/pink) ( B ), or PC-TP (blue/aqua) ( C ). Calcium release into the cytosol was measured as a function of the fluorescence intensity of the cytosolic calcium indicator Fluo4-AM following the induction of ER calcium release by thapsigargin (top panels; Tg, 2 μM) or ionomycin (bottom panels; IO, 5 μM). ( D ) Steady-state cytosolic calcium levels in HEK 293E cells following treatment with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours. Cells were treated with Them2, PC-TP, or scrambled control siRNA for 48 hours and serum-starved overnight before palmitic acid treatment. * P

    Techniques Used: Fluorescence

    4) Product Images from "The Pleiotropic Actions of Adiponectin are Initiated via Receptor-Mediated Activation of Ceramidase Activity"

    Article Title: The Pleiotropic Actions of Adiponectin are Initiated via Receptor-Mediated Activation of Ceramidase Activity

    Journal: Nature medicine

    doi: 10.1038/nm.2277

    Adiponectin promotes cardiomyocyte and Heart ATTAC survival ( a ) Female heart ATTAC transgenic mice crossed into indicated adiponectin backgrounds were challenged with AP20187 (0.010 μg/kg, IP) and survival was recorded as a Kaplan-Meier plot (n=12/group). ( b ) Ceramide was quantified from left ventricle or serum and normalized to the average content from adiponectin wildtype mice (63.9 pmol/mg in left ventricle, 9.5 pmol/μL in serum) (n=12/group). ( c ) Sphingosine, dihyrosphingosine, S1P, and dihydroS1P were quantified in left ventricle of WT (+/+), adiponectin (−/+), and adiponectin null mice (n=6/group). ( d ) Male HEART-ATTAC mice were treated with myriocin (0.3 mg/kg, IP), FTY720 (1 mg/kg, IP), S1P (1mg/kg, IP) or PBS immediately prior to injection with AP20187 (0.05 μg/kg, IP) and survival was recorded as a Kaplan-Meier plot (n=10/group). Additionally, treating HEART-ATTAC mice with S1P (1mg/kg, IP) just prior to AP20187 treatment prevented death in 100% of the animals tested (n=15) ( e ) Primary cardiomyocytes were isolated from heart ATTAC transgenic pups. After 72 hours of maintenance, cells were washed and treated with 2% BSA conjugated with: C2-ceramide (10 μM), myriocin (10 μM), palmitate (375 μM), or palmitate plus myriocin. PBS, adiponectin (3 μg/mL), or S1P (1 μM) were immediately added. Apoptosis was initiated by the addition of AP20187 (6.25 ng/mL), and viability was determined after 16 hours (n=6/group from 3 separate experiments). * denotes significant (p
    Figure Legend Snippet: Adiponectin promotes cardiomyocyte and Heart ATTAC survival ( a ) Female heart ATTAC transgenic mice crossed into indicated adiponectin backgrounds were challenged with AP20187 (0.010 μg/kg, IP) and survival was recorded as a Kaplan-Meier plot (n=12/group). ( b ) Ceramide was quantified from left ventricle or serum and normalized to the average content from adiponectin wildtype mice (63.9 pmol/mg in left ventricle, 9.5 pmol/μL in serum) (n=12/group). ( c ) Sphingosine, dihyrosphingosine, S1P, and dihydroS1P were quantified in left ventricle of WT (+/+), adiponectin (−/+), and adiponectin null mice (n=6/group). ( d ) Male HEART-ATTAC mice were treated with myriocin (0.3 mg/kg, IP), FTY720 (1 mg/kg, IP), S1P (1mg/kg, IP) or PBS immediately prior to injection with AP20187 (0.05 μg/kg, IP) and survival was recorded as a Kaplan-Meier plot (n=10/group). Additionally, treating HEART-ATTAC mice with S1P (1mg/kg, IP) just prior to AP20187 treatment prevented death in 100% of the animals tested (n=15) ( e ) Primary cardiomyocytes were isolated from heart ATTAC transgenic pups. After 72 hours of maintenance, cells were washed and treated with 2% BSA conjugated with: C2-ceramide (10 μM), myriocin (10 μM), palmitate (375 μM), or palmitate plus myriocin. PBS, adiponectin (3 μg/mL), or S1P (1 μM) were immediately added. Apoptosis was initiated by the addition of AP20187 (6.25 ng/mL), and viability was determined after 16 hours (n=6/group from 3 separate experiments). * denotes significant (p

    Techniques Used: Transgenic Assay, Mouse Assay, Injection, Isolation

    Adiponectin alters sensitivity to ceramide-induced apoptosis in INS-1 β-cells ( a ) INS-1 cells were washed and removed to 2% BSA, Palmitate (750 μM in 2% BSA), or C2-Ceramide (50 μM in 2% BSA). Adiponectin (3 μg/mL) or PBS was immediately added and cell viability was determined after 18 hours (n=6/group from 3 separate experiments). ( b ) Cell viability was determined on INS-1 cells pretreated with sphingosine kinase inhibitor (2-( p -Hydroxyanilino)-4-( p -chlorophenyl) thiazole, 0.5 μM) or DMSO prior to delivery of adiponectin (3μg/mL) or PBS and maintained for 18 hours in the presence of 2% BSA or Palmitate (750 μM in 2% BSA) ( c ) Ceramidase activity was determined in lysates from cultured INS-1 cells under a range of pH conditions (n=4 from separate experiments) in the presence or absence of adiponectin (0.3 μg/mL, in vitro ). “Fold change over baseline” refers to the change compared to BSA treatment without adiponectin. ( d ) INS-1 cells were challenged with C2-ceramide (50 μM) in the presence or absence of S1P (5 μM) cell death was determined by live/dead staining with cFDA or annexin V (image is representative of 3 separate experiments, bar=50μm). ( e ) Apoptosis of INS-1 cells was determined by FACS analysis of annexin V and propidium iodide stained cells following 18 hours of treatment with BSA, palmitate (750 μM), or coadministered palmitate and S1P (5μM) (representative of 3 independent experiments). * denotes significant (p
    Figure Legend Snippet: Adiponectin alters sensitivity to ceramide-induced apoptosis in INS-1 β-cells ( a ) INS-1 cells were washed and removed to 2% BSA, Palmitate (750 μM in 2% BSA), or C2-Ceramide (50 μM in 2% BSA). Adiponectin (3 μg/mL) or PBS was immediately added and cell viability was determined after 18 hours (n=6/group from 3 separate experiments). ( b ) Cell viability was determined on INS-1 cells pretreated with sphingosine kinase inhibitor (2-( p -Hydroxyanilino)-4-( p -chlorophenyl) thiazole, 0.5 μM) or DMSO prior to delivery of adiponectin (3μg/mL) or PBS and maintained for 18 hours in the presence of 2% BSA or Palmitate (750 μM in 2% BSA) ( c ) Ceramidase activity was determined in lysates from cultured INS-1 cells under a range of pH conditions (n=4 from separate experiments) in the presence or absence of adiponectin (0.3 μg/mL, in vitro ). “Fold change over baseline” refers to the change compared to BSA treatment without adiponectin. ( d ) INS-1 cells were challenged with C2-ceramide (50 μM) in the presence or absence of S1P (5 μM) cell death was determined by live/dead staining with cFDA or annexin V (image is representative of 3 separate experiments, bar=50μm). ( e ) Apoptosis of INS-1 cells was determined by FACS analysis of annexin V and propidium iodide stained cells following 18 hours of treatment with BSA, palmitate (750 μM), or coadministered palmitate and S1P (5μM) (representative of 3 independent experiments). * denotes significant (p

    Techniques Used: Activity Assay, Cell Culture, In Vitro, Staining, FACS

    5) Product Images from "Activation and Reversal of Lipotoxicity in PC12 and Rat Cortical Cells Following Exposure to Palmitic Acid"

    Article Title: Activation and Reversal of Lipotoxicity in PC12 and Rat Cortical Cells Following Exposure to Palmitic Acid

    Journal: Journal of neuroscience research

    doi: 10.1002/jnr.21918

    PA-induced lipotoxicity and DHA neuroprotective action on primary rat cortical cells (RCC). A : Cellular morphology of control and treated (PA/BSA, 2:1, 48 hr) RCC. B : Nuclear morphology of control and treated RCC. Arrows indicate fragmented nuclei. C : Viability of RCC treated for 48 hr with PA/BSA (2:1 ratio; PA) or in cotreatment with DHA (PA + DHA). Viability was determined using the WST-1 assay. Data represent mean ± SEM of three independent experiments. ★ P
    Figure Legend Snippet: PA-induced lipotoxicity and DHA neuroprotective action on primary rat cortical cells (RCC). A : Cellular morphology of control and treated (PA/BSA, 2:1, 48 hr) RCC. B : Nuclear morphology of control and treated RCC. Arrows indicate fragmented nuclei. C : Viability of RCC treated for 48 hr with PA/BSA (2:1 ratio; PA) or in cotreatment with DHA (PA + DHA). Viability was determined using the WST-1 assay. Data represent mean ± SEM of three independent experiments. ★ P

    Techniques Used: WST-1 Assay

    Palmitic acid-induced lipotoxicity in NGFDPC12 cells. A : NGFDPC12 cells were exposed to PA/BSA (2:1 ratio), and viability was determined using WST-1 assay at the indicated times. B : NGFDPC12 cells were exposed to different PA/BSA ratios (0.25:1, 0.5:1, 1:1, and 2:1 with BSA at a concentration of 0.150 mM) for 24 hr. Cell viability was measured by the WST-1 assay. C : NGFDPC12 cells were exposed to 0.150 mM BSA without PA (control) or PA/BSA (2:1; PA) for 12 hr. Cells were stained with Hoechst (10 ng/liter), and nuclei were visualized under fluorescent microscopy. Arrows indicate nuclei showing chromatin condensation and fragmentation. D : Regulation of apoptosis-asociated genes in NGFDPC12 cultures exposed to PA/BSA (2:1). Quantitative RT-PCR experiments show the time-dependent mRNA expression of Bim, Bad, AIF, BNIP-3, 14.3.3, and FAS-R. GAPDH mRNA expression was used as the internal control. Data represent mean 6 SEM of three independent experiments. ★ P
    Figure Legend Snippet: Palmitic acid-induced lipotoxicity in NGFDPC12 cells. A : NGFDPC12 cells were exposed to PA/BSA (2:1 ratio), and viability was determined using WST-1 assay at the indicated times. B : NGFDPC12 cells were exposed to different PA/BSA ratios (0.25:1, 0.5:1, 1:1, and 2:1 with BSA at a concentration of 0.150 mM) for 24 hr. Cell viability was measured by the WST-1 assay. C : NGFDPC12 cells were exposed to 0.150 mM BSA without PA (control) or PA/BSA (2:1; PA) for 12 hr. Cells were stained with Hoechst (10 ng/liter), and nuclei were visualized under fluorescent microscopy. Arrows indicate nuclei showing chromatin condensation and fragmentation. D : Regulation of apoptosis-asociated genes in NGFDPC12 cultures exposed to PA/BSA (2:1). Quantitative RT-PCR experiments show the time-dependent mRNA expression of Bim, Bad, AIF, BNIP-3, 14.3.3, and FAS-R. GAPDH mRNA expression was used as the internal control. Data represent mean 6 SEM of three independent experiments. ★ P

    Techniques Used: WST-1 Assay, Concentration Assay, Staining, Microscopy, Quantitative RT-PCR, Expressing

    DNA fragmentation and caspase-3 activity of NGFDPC12 cells exposed to PA. A : Representative experimental results of BRDU-FITC plots (apoptotic cells) vs. PI (total DNA marker) for controls and cells exposed to PA/BSA (2:1) for 12 hr. The R2 quadrant shows cells exhibiting increased BrdU-FITC fluorescence (green). Nonapoptotic cells are indicated in red. B : Representative distribution diagram of positive apoptotic NGFDPC12 cells. Cells were treated with BSA for negative control (green), PA/BSA (2:1) for 12 hr (red) or 24 hr (blue), or staurosporine for positive control (orange). C : Quantitative analysis of positive apoptotic NGFDPC12 cells as shown in B. The data represent the mean ± SEM of three independent experiments done in triplicate. D : Caspase-3 activity in cell extracts was determined in control or NGFDPC12 cells treated with PA/BSA for 12 hr. E : Early posttreatment with BSA protects NGFDPC12 cells from PA-induced lipotoxicity. Additional BSA (0.6 mM final concentration) was added to NGFDPC-12 cells at 2 or 6 hr after initial PA/BSA (2:1 ratio) treatment. Percentage of viable cells was measured by the WST-1 assay at the end of a 24-hr incubation period. Nuclei in bottom panel were visualized with Hoechst staining. Data represent mean ± SEM of three independent experiments. ★ P
    Figure Legend Snippet: DNA fragmentation and caspase-3 activity of NGFDPC12 cells exposed to PA. A : Representative experimental results of BRDU-FITC plots (apoptotic cells) vs. PI (total DNA marker) for controls and cells exposed to PA/BSA (2:1) for 12 hr. The R2 quadrant shows cells exhibiting increased BrdU-FITC fluorescence (green). Nonapoptotic cells are indicated in red. B : Representative distribution diagram of positive apoptotic NGFDPC12 cells. Cells were treated with BSA for negative control (green), PA/BSA (2:1) for 12 hr (red) or 24 hr (blue), or staurosporine for positive control (orange). C : Quantitative analysis of positive apoptotic NGFDPC12 cells as shown in B. The data represent the mean ± SEM of three independent experiments done in triplicate. D : Caspase-3 activity in cell extracts was determined in control or NGFDPC12 cells treated with PA/BSA for 12 hr. E : Early posttreatment with BSA protects NGFDPC12 cells from PA-induced lipotoxicity. Additional BSA (0.6 mM final concentration) was added to NGFDPC-12 cells at 2 or 6 hr after initial PA/BSA (2:1 ratio) treatment. Percentage of viable cells was measured by the WST-1 assay at the end of a 24-hr incubation period. Nuclei in bottom panel were visualized with Hoechst staining. Data represent mean ± SEM of three independent experiments. ★ P

    Techniques Used: Activity Assay, Marker, Fluorescence, Negative Control, Positive Control, Concentration Assay, WST-1 Assay, Incubation, Staining

    ROS generation and changes in mitochondrial membrane permeability in NGFDPC12 cells undergoing PA-induced lipotoxicity. A : ROS generation in NGFDPC12 cells was detected by the 2′,7′ -dichlorofluorescein diacetate (DCF) method at 12 hr after the initial exposure to PA using flow cytometry. NGFDPC12 cells were treated with no DCF (light green), DCF (dark green), PA + DCF (blue), or hydrogen peroxide + DCF (red). B : Quantification of ROS production in NGFDPC12 cells at 3, 6, 9, and 12 hr after PA/BSA treatment. C : ROS modulation after addition of DHA, MCI-186, or BSA to NCGDPC12 cells treated with PA for 6 hr. D: Mitochondrial depolarization in NGFDPC12 cells, control or PA/BSA treated, detected by JC-1 flow cytometric analysis. JC-1 was detected in the FL1 or FL2 channel depending on its aggregation status (see Mqaterials and Methods). The R2 quadrant defines cells with leaky mitochondria showing reduced FL2 readings (green). Cells with intact mitochondria are indicated in R1 (red). Data represent mean ± SEM sent of at least three independent experiments. ★ P
    Figure Legend Snippet: ROS generation and changes in mitochondrial membrane permeability in NGFDPC12 cells undergoing PA-induced lipotoxicity. A : ROS generation in NGFDPC12 cells was detected by the 2′,7′ -dichlorofluorescein diacetate (DCF) method at 12 hr after the initial exposure to PA using flow cytometry. NGFDPC12 cells were treated with no DCF (light green), DCF (dark green), PA + DCF (blue), or hydrogen peroxide + DCF (red). B : Quantification of ROS production in NGFDPC12 cells at 3, 6, 9, and 12 hr after PA/BSA treatment. C : ROS modulation after addition of DHA, MCI-186, or BSA to NCGDPC12 cells treated with PA for 6 hr. D: Mitochondrial depolarization in NGFDPC12 cells, control or PA/BSA treated, detected by JC-1 flow cytometric analysis. JC-1 was detected in the FL1 or FL2 channel depending on its aggregation status (see Mqaterials and Methods). The R2 quadrant defines cells with leaky mitochondria showing reduced FL2 readings (green). Cells with intact mitochondria are indicated in R1 (red). Data represent mean ± SEM sent of at least three independent experiments. ★ P

    Techniques Used: Permeability, Flow Cytometry, Cytometry

    Inhibition of fatty acid-induced lipotoxicity by DHA. A : DHA inhibits induction of cell death by PA in NGFDPC12 cells. Cell viability was determined after treatment for 24 hr as follows: 0.150 mM BSA alone (control), PA/BSA 2:1 ratio (PA), PA/BSA 2:1 cotreated with DHA followed by an increase in BSA final concentration to 0.6 mM at 6 hr (PA + DHA + BSA at 6 hr), and DHA pretreatment for 12 hr followed by treatment with PA/BSA 2:1 (DHA 12 hr pre + PA). B : DHA treatment inhibits apoptotic features of NGFDPC12 cells treated with PA/BSA. Morphology of NGFDPC12 cells treated with BSA alone (control), PA/BSA 2:1 cotreated with DHA, or PA/BSA 2:1 for 24 hr. C : Nuclear morphology of NGFDPC12 cells treated with BSA alone (control), PA/BSA 2:1 cotreated with DHA or PA/BSA 2:1 for 24 hr. Arrows indicate nuclei exhibiting fragmentation. D : DHA cotreatment produced a significant inhibitory effect of caspase-3 activity in cellular extracts of NGFDPC12 cells exposed to PA/BSA (2:1). E : Cleavage of PARP and lamin B in NGFDPC-12 cells exposed to PA/BSA (2:1), as assessed by Western blotting. PA treatment induced the appearance of the signature apoptotic fragments of PARP (85 kDa) or lamin B (46 kDa). DHA inhibited PARP and lamin B cleavage. Data in A and D represent mean ± SEM of three independent experiments. ★ P
    Figure Legend Snippet: Inhibition of fatty acid-induced lipotoxicity by DHA. A : DHA inhibits induction of cell death by PA in NGFDPC12 cells. Cell viability was determined after treatment for 24 hr as follows: 0.150 mM BSA alone (control), PA/BSA 2:1 ratio (PA), PA/BSA 2:1 cotreated with DHA followed by an increase in BSA final concentration to 0.6 mM at 6 hr (PA + DHA + BSA at 6 hr), and DHA pretreatment for 12 hr followed by treatment with PA/BSA 2:1 (DHA 12 hr pre + PA). B : DHA treatment inhibits apoptotic features of NGFDPC12 cells treated with PA/BSA. Morphology of NGFDPC12 cells treated with BSA alone (control), PA/BSA 2:1 cotreated with DHA, or PA/BSA 2:1 for 24 hr. C : Nuclear morphology of NGFDPC12 cells treated with BSA alone (control), PA/BSA 2:1 cotreated with DHA or PA/BSA 2:1 for 24 hr. Arrows indicate nuclei exhibiting fragmentation. D : DHA cotreatment produced a significant inhibitory effect of caspase-3 activity in cellular extracts of NGFDPC12 cells exposed to PA/BSA (2:1). E : Cleavage of PARP and lamin B in NGFDPC-12 cells exposed to PA/BSA (2:1), as assessed by Western blotting. PA treatment induced the appearance of the signature apoptotic fragments of PARP (85 kDa) or lamin B (46 kDa). DHA inhibited PARP and lamin B cleavage. Data in A and D represent mean ± SEM of three independent experiments. ★ P

    Techniques Used: Inhibition, Concentration Assay, Produced, Activity Assay, Western Blot

    DHA reduces mitochondrial depolarization of NGFDPC12 cells undergoing PA-induced lipotoxicity. A : NGFDPC12 cells were treated with PA/BSA alone (PA) or in the presence DHA (PA + DHA) for 9 or 12 hr, and mitochondrial depolarization was determined by JC-1 flow cytometry assays. Representative flow cytometric plots are shown. B : Quantification of mitochondria depolarization (R2 in JC-1 flow cytometry) in NGFDPC12 cells undergoing PA-induced lipotoxicity. Data represent mean ± SEM of three independent experiments. ★ P
    Figure Legend Snippet: DHA reduces mitochondrial depolarization of NGFDPC12 cells undergoing PA-induced lipotoxicity. A : NGFDPC12 cells were treated with PA/BSA alone (PA) or in the presence DHA (PA + DHA) for 9 or 12 hr, and mitochondrial depolarization was determined by JC-1 flow cytometry assays. Representative flow cytometric plots are shown. B : Quantification of mitochondria depolarization (R2 in JC-1 flow cytometry) in NGFDPC12 cells undergoing PA-induced lipotoxicity. Data represent mean ± SEM of three independent experiments. ★ P

    Techniques Used: Flow Cytometry, Cytometry

    6) Product Images from "Characterization of an acyl-coenzyme A binding protein predominantly expressed in human primitive progenitor cells *Characterization of an acyl-coenzyme A binding protein predominantly expressed in human primitive progenitor cells * s⃞"

    Article Title: Characterization of an acyl-coenzyme A binding protein predominantly expressed in human primitive progenitor cells *Characterization of an acyl-coenzyme A binding protein predominantly expressed in human primitive progenitor cells * s⃞

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M800007-JLR200

    Isothermal calibration titration of the binding of fatty acid (A) and acyl-CoA (B). Experiments were performed on a VP-ITC instrument with ligands at a concentration of 5 mM injected into the chamber containing 25 μM ACBD6 (left panels) or BSA
    Figure Legend Snippet: Isothermal calibration titration of the binding of fatty acid (A) and acyl-CoA (B). Experiments were performed on a VP-ITC instrument with ligands at a concentration of 5 mM injected into the chamber containing 25 μM ACBD6 (left panels) or BSA

    Techniques Used: Titration, Binding Assay, Concentration Assay, Injection

    7) Product Images from "Kdo2-Lipid A, a TLR4-specific Agonist, Induces de Novo Sphingolipid Biosynthesis in RAW264.7 Macrophages, Which Is Essential for Induction of Autophagy *"

    Article Title: Kdo2-Lipid A, a TLR4-specific Agonist, Induces de Novo Sphingolipid Biosynthesis in RAW264.7 Macrophages, Which Is Essential for Induction of Autophagy *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.170621

    Analysis of de novo sphingolipid biosynthesis in RAW264.7 cells by [ 13 C]palmitate labeling. RAW264.7 cells were incubated with 0.1 m m [U- 13 C]palmitic acid (as the BSA complex) with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μ m ), or KLA+ ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA for the times shown. The appearance of 13 C in newly synthesized sphingolipids was quantified by mass spectrometry as described under supplemental “Materials and Methods” . A , summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer subspecies. Data represent the means ± S.E. ( n = 3). B , summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species in cells treated with control ( left ) or KLA ( right ), Data represent the means ( n = 3). Because of the difficulties in visualization, the quantities of ceramide subspecies in control treated cells at the 24-h time point are as follows: C16 (7.6 pmol/μg DNA); C18 (0.7 pmol/μg DNA); C20 (0.3 pmol/μg DNA); C22 (3.2 pmol/μg DNA); C24:1 (6 pmol/μg DNA); C24 (6.4 pmol/μg DNA); C26:1 (0.2 pmol/μg DNA); C26 (0.1 pmol/μg DNA). C , summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species ( left ) and 12 C (unlabeled) Cer species ( right ) in cells treated with KLA + ISP1. Data represent the means ( n = 3).
    Figure Legend Snippet: Analysis of de novo sphingolipid biosynthesis in RAW264.7 cells by [ 13 C]palmitate labeling. RAW264.7 cells were incubated with 0.1 m m [U- 13 C]palmitic acid (as the BSA complex) with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μ m ), or KLA+ ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA for the times shown. The appearance of 13 C in newly synthesized sphingolipids was quantified by mass spectrometry as described under supplemental “Materials and Methods” . A , summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer subspecies. Data represent the means ± S.E. ( n = 3). B , summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species in cells treated with control ( left ) or KLA ( right ), Data represent the means ( n = 3). Because of the difficulties in visualization, the quantities of ceramide subspecies in control treated cells at the 24-h time point are as follows: C16 (7.6 pmol/μg DNA); C18 (0.7 pmol/μg DNA); C20 (0.3 pmol/μg DNA); C22 (3.2 pmol/μg DNA); C24:1 (6 pmol/μg DNA); C24 (6.4 pmol/μg DNA); C26:1 (0.2 pmol/μg DNA); C26 (0.1 pmol/μg DNA). C , summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species ( left ) and 12 C (unlabeled) Cer species ( right ) in cells treated with KLA + ISP1. Data represent the means ( n = 3).

    Techniques Used: Labeling, Incubation, Synthesized, Mass Spectrometry

    8) Product Images from "Palmitate Induces mRNA Translation and Increases ER Protein Load in Islet β-Cells via Activation of the Mammalian Target of Rapamycin Pathway"

    Article Title: Palmitate Induces mRNA Translation and Increases ER Protein Load in Islet β-Cells via Activation of the Mammalian Target of Rapamycin Pathway

    Journal: Diabetes

    doi: 10.2337/db14-0105

    Palmitate activates mTOR signaling. A : Representative immunoblot for the indicated proteins after incubation of MIN6 cells under control (CTL) or with varying concentrations of palmitate (PAL) for 1 h. B : Representative PRP analysis of MIN6 cells after incubation under control, palmitate (0.5 mmol/L), or palmitate + rapamycin (Rapa, 50 nmol/L) conditions for 1 h. C : Representative PRP analysis of MIN6 cells after incubation under control, palmitate (0.5 mmol/L), or palmitate + Torin1 (Torin, 250 mmol/L) conditions for 1 h. In all panels, control cells were incubated at the same glucose concentrations as palmitate-treated cells, and BSA was used in place of palmitate-BSA conjugates.
    Figure Legend Snippet: Palmitate activates mTOR signaling. A : Representative immunoblot for the indicated proteins after incubation of MIN6 cells under control (CTL) or with varying concentrations of palmitate (PAL) for 1 h. B : Representative PRP analysis of MIN6 cells after incubation under control, palmitate (0.5 mmol/L), or palmitate + rapamycin (Rapa, 50 nmol/L) conditions for 1 h. C : Representative PRP analysis of MIN6 cells after incubation under control, palmitate (0.5 mmol/L), or palmitate + Torin1 (Torin, 250 mmol/L) conditions for 1 h. In all panels, control cells were incubated at the same glucose concentrations as palmitate-treated cells, and BSA was used in place of palmitate-BSA conjugates.

    Techniques Used: Incubation, CTL Assay

    Acute and chronic effects of palmitate on mRNA translation. A : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control (CTL) or palmitate (PAL, 0.5 mmol/L) conditions for 1 h. Numbers above the peaks indicate numbers of ribosomes. B : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control and palmitate conditions for 24 h. C : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control and palmitate conditions for 72 h. D : Representative immunoblot ( top panel ) and corresponding quantitation (from n = 4) ( bottom panel ) of p-eIF2α and total eIF2α after incubation of MIN6 cells with control or palmitate for the indicated times. E : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control and thapsigargin (Tg, 1 μmol/L) conditions for 4 h. F : Representative immunoblot ( top panel ) and corresponding quantitation (from n = 4) ( bottom panel ) for p-eIF2α and total eIF2α after incubation of MIN6 cells with control or thapsigargin for 4 h. In all panels, control cells were incubated at the same glucose concentrations as palmitate-treated cells, and BSA was added in place of palmitate-BSA conjugates. Quantitative data represent mean ± SEM of at least three independent determinations. * P
    Figure Legend Snippet: Acute and chronic effects of palmitate on mRNA translation. A : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control (CTL) or palmitate (PAL, 0.5 mmol/L) conditions for 1 h. Numbers above the peaks indicate numbers of ribosomes. B : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control and palmitate conditions for 24 h. C : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control and palmitate conditions for 72 h. D : Representative immunoblot ( top panel ) and corresponding quantitation (from n = 4) ( bottom panel ) of p-eIF2α and total eIF2α after incubation of MIN6 cells with control or palmitate for the indicated times. E : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control and thapsigargin (Tg, 1 μmol/L) conditions for 4 h. F : Representative immunoblot ( top panel ) and corresponding quantitation (from n = 4) ( bottom panel ) for p-eIF2α and total eIF2α after incubation of MIN6 cells with control or thapsigargin for 4 h. In all panels, control cells were incubated at the same glucose concentrations as palmitate-treated cells, and BSA was added in place of palmitate-BSA conjugates. Quantitative data represent mean ± SEM of at least three independent determinations. * P

    Techniques Used: Incubation, CTL Assay, Quantitation Assay

    9) Product Images from "Type 2 Iodothyronine Deiodinase Activity Is Required for Rapid Stimulation of PI3K by Thyroxine in Human Umbilical Vein Endothelial Cells"

    Article Title: Type 2 Iodothyronine Deiodinase Activity Is Required for Rapid Stimulation of PI3K by Thyroxine in Human Umbilical Vein Endothelial Cells

    Journal: Endocrinology

    doi: 10.1210/en.2014-1988

    The effects of wortmannin and IOP on TH-induced Akt phosphorylation and migration in HUVECs. Before performing the experiment described below, HUVECs were treated for 4 h at 37°C with RPMI 1640 medium containing 0.1% fatty acid–free BSA.
    Figure Legend Snippet: The effects of wortmannin and IOP on TH-induced Akt phosphorylation and migration in HUVECs. Before performing the experiment described below, HUVECs were treated for 4 h at 37°C with RPMI 1640 medium containing 0.1% fatty acid–free BSA.

    Techniques Used: Migration

    10) Product Images from "Palmitate Induces mRNA Translation and Increases ER Protein Load in Islet β-Cells via Activation of the Mammalian Target of Rapamycin Pathway"

    Article Title: Palmitate Induces mRNA Translation and Increases ER Protein Load in Islet β-Cells via Activation of the Mammalian Target of Rapamycin Pathway

    Journal: Diabetes

    doi: 10.2337/db14-0105

    Palmitate activates mTOR signaling. A : Representative immunoblot for the indicated proteins after incubation of MIN6 cells under control (CTL) or with varying concentrations of palmitate (PAL) for 1 h. B : Representative PRP analysis of MIN6 cells after incubation under control, palmitate (0.5 mmol/L), or palmitate + rapamycin (Rapa, 50 nmol/L) conditions for 1 h. C : Representative PRP analysis of MIN6 cells after incubation under control, palmitate (0.5 mmol/L), or palmitate + Torin1 (Torin, 250 mmol/L) conditions for 1 h. In all panels, control cells were incubated at the same glucose concentrations as palmitate-treated cells, and BSA was used in place of palmitate-BSA conjugates.
    Figure Legend Snippet: Palmitate activates mTOR signaling. A : Representative immunoblot for the indicated proteins after incubation of MIN6 cells under control (CTL) or with varying concentrations of palmitate (PAL) for 1 h. B : Representative PRP analysis of MIN6 cells after incubation under control, palmitate (0.5 mmol/L), or palmitate + rapamycin (Rapa, 50 nmol/L) conditions for 1 h. C : Representative PRP analysis of MIN6 cells after incubation under control, palmitate (0.5 mmol/L), or palmitate + Torin1 (Torin, 250 mmol/L) conditions for 1 h. In all panels, control cells were incubated at the same glucose concentrations as palmitate-treated cells, and BSA was used in place of palmitate-BSA conjugates.

    Techniques Used: Incubation, CTL Assay

    Acute and chronic effects of palmitate on mRNA translation. A : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control (CTL) or palmitate (PAL, 0.5 mmol/L) conditions for 1 h. Numbers above the peaks indicate numbers of ribosomes. B : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control and palmitate conditions for 24 h. C : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control and palmitate conditions for 72 h. D : Representative immunoblot ( top panel ) and corresponding quantitation (from n = 4) ( bottom panel ) of p-eIF2α and total eIF2α after incubation of MIN6 cells with control or palmitate for the indicated times. E : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control and thapsigargin (Tg, 1 μmol/L) conditions for 4 h. F : Representative immunoblot ( top panel ) and corresponding quantitation (from n = 4) ( bottom panel ) for p-eIF2α and total eIF2α after incubation of MIN6 cells with control or thapsigargin for 4 h. In all panels, control cells were incubated at the same glucose concentrations as palmitate-treated cells, and BSA was added in place of palmitate-BSA conjugates. Quantitative data represent mean ± SEM of at least three independent determinations. * P
    Figure Legend Snippet: Acute and chronic effects of palmitate on mRNA translation. A : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control (CTL) or palmitate (PAL, 0.5 mmol/L) conditions for 1 h. Numbers above the peaks indicate numbers of ribosomes. B : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control and palmitate conditions for 24 h. C : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control and palmitate conditions for 72 h. D : Representative immunoblot ( top panel ) and corresponding quantitation (from n = 4) ( bottom panel ) of p-eIF2α and total eIF2α after incubation of MIN6 cells with control or palmitate for the indicated times. E : PRP analysis ( left panel ) and associated P/M ratio ( right panel ) of MIN6 cells after incubation under control and thapsigargin (Tg, 1 μmol/L) conditions for 4 h. F : Representative immunoblot ( top panel ) and corresponding quantitation (from n = 4) ( bottom panel ) for p-eIF2α and total eIF2α after incubation of MIN6 cells with control or thapsigargin for 4 h. In all panels, control cells were incubated at the same glucose concentrations as palmitate-treated cells, and BSA was added in place of palmitate-BSA conjugates. Quantitative data represent mean ± SEM of at least three independent determinations. * P

    Techniques Used: Incubation, CTL Assay, Quantitation Assay

    11) Product Images from "IRE1 α RNase–dependent lipid homeostasis promotes survival in Myc-transformed cancers"

    Article Title: IRE1 α RNase–dependent lipid homeostasis promotes survival in Myc-transformed cancers

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI95864

    B-I09 treatment results in phenotypes dependent on SCD1 loss in P493 cells. ( A ) Heatmap shows relative expression of lipid metabolism genes in P493 cells with different c-Myc levels upon 10 μM B-I09 treatment for 48 hours ( n = 3). Expression signals are depicted using pseudocoloring, in which expression for each gene is shown as high (red) or low (blue). ( B ) U- 13 C-glucose tracing and fatty acid labeling in P493 High Myc and No Myc cells with 10 μM B-I09 treatment for 24 hours ( n = 3). Labeled/total ratios were calculated for palmitate (C16:0), stearate (C:18:0), and oleate (C18:1). ( C ) Immunoblot analysis for P493 cells treated with DMSO or B-I09 for 48 hours. ( D ) Mean enrichment of C18:1/C18:0 was calculated in P493 High Myc cells from data in B ( n = 3, 2-tailed Student’s t test). ( E ) ChIP-qPCR assay performed using anti-XBP1s antibody to detect enriched gene-promoter fragments in 3 conditions: control (Ct); tunicamycin (5 μg/ml) treatment for 6 hours (Tm); and tunicamycin+B-I09 (10 μM) treatment for 6 hours (Tm+B-I09). IgG was used as mock ChIP control. ERdj4 serves as a positive control for XBP1s binding. Values represent relative increase of real-time PCR signals compared with the signal of IgG ChIP under control conditions. Three technical triplicates are presented. ( F ) Cell growth of P493 High Myc cells treated with 10 μM B-I09 and rescued with BSA control or OA ( n = 3). ( G ) Relative viability of P493 High Myc cells treated with 10 μM B-I09 and rescued with BSA control, OA, or POA for 48 hours. For viability assays, results are representative of 3 independent experiments. P values were determined by 2-way ANOVA with Bonferroni’s correction, if not specified elsewhere. *** P
    Figure Legend Snippet: B-I09 treatment results in phenotypes dependent on SCD1 loss in P493 cells. ( A ) Heatmap shows relative expression of lipid metabolism genes in P493 cells with different c-Myc levels upon 10 μM B-I09 treatment for 48 hours ( n = 3). Expression signals are depicted using pseudocoloring, in which expression for each gene is shown as high (red) or low (blue). ( B ) U- 13 C-glucose tracing and fatty acid labeling in P493 High Myc and No Myc cells with 10 μM B-I09 treatment for 24 hours ( n = 3). Labeled/total ratios were calculated for palmitate (C16:0), stearate (C:18:0), and oleate (C18:1). ( C ) Immunoblot analysis for P493 cells treated with DMSO or B-I09 for 48 hours. ( D ) Mean enrichment of C18:1/C18:0 was calculated in P493 High Myc cells from data in B ( n = 3, 2-tailed Student’s t test). ( E ) ChIP-qPCR assay performed using anti-XBP1s antibody to detect enriched gene-promoter fragments in 3 conditions: control (Ct); tunicamycin (5 μg/ml) treatment for 6 hours (Tm); and tunicamycin+B-I09 (10 μM) treatment for 6 hours (Tm+B-I09). IgG was used as mock ChIP control. ERdj4 serves as a positive control for XBP1s binding. Values represent relative increase of real-time PCR signals compared with the signal of IgG ChIP under control conditions. Three technical triplicates are presented. ( F ) Cell growth of P493 High Myc cells treated with 10 μM B-I09 and rescued with BSA control or OA ( n = 3). ( G ) Relative viability of P493 High Myc cells treated with 10 μM B-I09 and rescued with BSA control, OA, or POA for 48 hours. For viability assays, results are representative of 3 independent experiments. P values were determined by 2-way ANOVA with Bonferroni’s correction, if not specified elsewhere. *** P

    Techniques Used: Expressing, Labeling, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Binding Assay

    N-Myc activates the IRE1α/XBP1 pathway, rendering cells vulnerable to XBP1s loss. ( A – D ) SHEP N-MycER cells were treated with 4-OHT (200 nM) to activate N-Myc nuclear translocation ( A ). XBP1s/XBP1t ratios examined with qRT-PCR ( B ) ( n = 3). XBP1 splicing analyzed by RT-PCR ( C ) and XBP1s protein accumulation determined by immunoblots ( D ). ( E ) SHEP cells cultured in vehicle (control) or 4-OHT containing medium for 48 hours before treatment with DMSO or B-I09. WST-1 assay was used to examine cell growth. Relative absorbance was determined by normalizing to absorbance at time 0 hours ( n = 6). IC 50 was then determined ( n = 3, 2-tailed Student’s t test). ( F ) Representative contour plots of control and 4-OHT SHEP cells treated with 30 μM B-I09 for 96 hours. ( G ) SHEP cells pretreated with CHX (0.5 μg/ml) for 2 hours and then cultured with DMSO or 30 μM B-I09 for 72 hours. Relative viability was determined by normalizing to viability upon DMSO treatment or DMSO+CHX treatment, respectively. ( H ) Immunoblot for control and 4-OHT SHEP cells with B-I09 treatment for 72 hours. ( I ) Control or N-Myc SHEP cells treated with DMSO or B-I09 and rescued with BSA or OA for 72 hours. Viability was examined and relative viability was determined by normalizing to viability upon DMSO treatment. For viability assays, results are representative of 3 independent experiments. P values were determined by 2-way ANOVA with Bonferroni’s correction, if not specified elsewhere. * P
    Figure Legend Snippet: N-Myc activates the IRE1α/XBP1 pathway, rendering cells vulnerable to XBP1s loss. ( A – D ) SHEP N-MycER cells were treated with 4-OHT (200 nM) to activate N-Myc nuclear translocation ( A ). XBP1s/XBP1t ratios examined with qRT-PCR ( B ) ( n = 3). XBP1 splicing analyzed by RT-PCR ( C ) and XBP1s protein accumulation determined by immunoblots ( D ). ( E ) SHEP cells cultured in vehicle (control) or 4-OHT containing medium for 48 hours before treatment with DMSO or B-I09. WST-1 assay was used to examine cell growth. Relative absorbance was determined by normalizing to absorbance at time 0 hours ( n = 6). IC 50 was then determined ( n = 3, 2-tailed Student’s t test). ( F ) Representative contour plots of control and 4-OHT SHEP cells treated with 30 μM B-I09 for 96 hours. ( G ) SHEP cells pretreated with CHX (0.5 μg/ml) for 2 hours and then cultured with DMSO or 30 μM B-I09 for 72 hours. Relative viability was determined by normalizing to viability upon DMSO treatment or DMSO+CHX treatment, respectively. ( H ) Immunoblot for control and 4-OHT SHEP cells with B-I09 treatment for 72 hours. ( I ) Control or N-Myc SHEP cells treated with DMSO or B-I09 and rescued with BSA or OA for 72 hours. Viability was examined and relative viability was determined by normalizing to viability upon DMSO treatment. For viability assays, results are representative of 3 independent experiments. P values were determined by 2-way ANOVA with Bonferroni’s correction, if not specified elsewhere. * P

    Techniques Used: Translocation Assay, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, WST-1 Assay

    B-I09 treatment results in phenotypes dependent on SCD1 loss in BL cells. ( A ) Immunoblot analysis of Ramos and Daudi cells treated with indicated concentrations of B-I09 for 48 hours. ( B ) Cell growth ( n = 3) of Ramos and Daudi cells treated with B-I09 (20 μM for Ramos, 10 μM for Daudi) and rescued with BSA control or OA. ( C ) Relative viability of Ramos and Daudi cells treated with 10 μM B-I09 and rescued with BSA control, OA, or POA for 48 hours. ( D ) Relative viability of Ramos and Daudi cells treated with 0.5 μM SCDi and rescued with BSA control or OA for 48 hours. ( E ) Normalized reads of SCD in human BL and CB from healthy donors. Microarray data were obtained from the Oncomine database. Whiskers denote the minimal to maximal values. ( F ) Tumor growth and weight of xenografted Ramos tumors treated with control or SCDi (5 mg/kg, orally twice daily). For all viability assays, results are representative of 3 independent experiments. ** P
    Figure Legend Snippet: B-I09 treatment results in phenotypes dependent on SCD1 loss in BL cells. ( A ) Immunoblot analysis of Ramos and Daudi cells treated with indicated concentrations of B-I09 for 48 hours. ( B ) Cell growth ( n = 3) of Ramos and Daudi cells treated with B-I09 (20 μM for Ramos, 10 μM for Daudi) and rescued with BSA control or OA. ( C ) Relative viability of Ramos and Daudi cells treated with 10 μM B-I09 and rescued with BSA control, OA, or POA for 48 hours. ( D ) Relative viability of Ramos and Daudi cells treated with 0.5 μM SCDi and rescued with BSA control or OA for 48 hours. ( E ) Normalized reads of SCD in human BL and CB from healthy donors. Microarray data were obtained from the Oncomine database. Whiskers denote the minimal to maximal values. ( F ) Tumor growth and weight of xenografted Ramos tumors treated with control or SCDi (5 mg/kg, orally twice daily). For all viability assays, results are representative of 3 independent experiments. ** P

    Techniques Used: Microarray

    12) Product Images from "Palmitate induces ER calcium depletion and apoptosis in mouse podocytes subsequent to mitochondrial oxidative stress"

    Article Title: Palmitate induces ER calcium depletion and apoptosis in mouse podocytes subsequent to mitochondrial oxidative stress

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.331

    Palmitate-induced ER Ca 2+ depletion and cytotoxicity were attenuated by a FAT/CD36 inhibitor and a mitochondrial antioxidant. ( a ) Sulfosuccinimidyl oleate (SSO), a FAT/CD36 inhibitor, was used to pretreat podocytes for 30 min before BSA or palmitate (PA, 300 μ M) incubation (for 24 h), and cytotoxicity was estimated by using an MTT assay ( N =5). ( b , c ) Luminal Ca 2+ content in ER ([Ca 2+ ] ER ) was measured in D1ER-transfected podocytes incubated with BSA or PA in Ca 2+ -free KRB solution for 90 min ( N =5). ( d – j ) A mitochondrial antioxidant, mitoTEMPO (mTEMPO; 100 nM), was pretreated to podocytes for 30 min before BSA or PA incubation. The mRNA ( d ) and protein ( e – g ) levels of ER stress-related proteins were analyzed, and cytotoxicity induced by palmitate (( h ), N =11) were estimated. Effects of mTEMPO pretreatment on [Ca 2+ ] ER levels were analyzed in D1ER-transfected podocytes incubated with BSA or PA upon cyclopiazonic acid (CPA) application (( i , j ), N =13–15). Data are presented as mean ± S.E.M. *, ** and *** denote P
    Figure Legend Snippet: Palmitate-induced ER Ca 2+ depletion and cytotoxicity were attenuated by a FAT/CD36 inhibitor and a mitochondrial antioxidant. ( a ) Sulfosuccinimidyl oleate (SSO), a FAT/CD36 inhibitor, was used to pretreat podocytes for 30 min before BSA or palmitate (PA, 300 μ M) incubation (for 24 h), and cytotoxicity was estimated by using an MTT assay ( N =5). ( b , c ) Luminal Ca 2+ content in ER ([Ca 2+ ] ER ) was measured in D1ER-transfected podocytes incubated with BSA or PA in Ca 2+ -free KRB solution for 90 min ( N =5). ( d – j ) A mitochondrial antioxidant, mitoTEMPO (mTEMPO; 100 nM), was pretreated to podocytes for 30 min before BSA or PA incubation. The mRNA ( d ) and protein ( e – g ) levels of ER stress-related proteins were analyzed, and cytotoxicity induced by palmitate (( h ), N =11) were estimated. Effects of mTEMPO pretreatment on [Ca 2+ ] ER levels were analyzed in D1ER-transfected podocytes incubated with BSA or PA upon cyclopiazonic acid (CPA) application (( i , j ), N =13–15). Data are presented as mean ± S.E.M. *, ** and *** denote P

    Techniques Used: Incubation, MTT Assay, Transfection

    Diacylglycerol and protein kinase C are involved in ER Ca 2+ depletion in palmitate-treated podocytes. Changes in luminal Ca 2+ content in ER ([Ca 2+ ] ER ) upon cyclopiazonic acid (CPA) application were measured in D1ER-transfected podocytes incubated with BSA or PA (300 μ M) in Ca 2+ -free KRB solution for 90 min. ( a – c ) A diacylglycerol (DAG) kinase inhibitor, R59022 (DKI) was used to pretreat cells for 30 min before 50 μ M PA incubation, and CPA-induced reduction of [Ca 2+ ] ER ( b ) and ER Ca 2+ refilling by 1.8 mM extracellular Ca 2+ ( c ) were compared among different groups ( N =13–24). ( d ) An inhibitor of protein kinase C, GF109203X was used to pretreat cells for 30 min before BSA or PA incubation, and CPA-induced reduction of [Ca 2+ ] ER was analyzed ( N =19). Data are presented as mean±S.E.M. ** and *** denote P
    Figure Legend Snippet: Diacylglycerol and protein kinase C are involved in ER Ca 2+ depletion in palmitate-treated podocytes. Changes in luminal Ca 2+ content in ER ([Ca 2+ ] ER ) upon cyclopiazonic acid (CPA) application were measured in D1ER-transfected podocytes incubated with BSA or PA (300 μ M) in Ca 2+ -free KRB solution for 90 min. ( a – c ) A diacylglycerol (DAG) kinase inhibitor, R59022 (DKI) was used to pretreat cells for 30 min before 50 μ M PA incubation, and CPA-induced reduction of [Ca 2+ ] ER ( b ) and ER Ca 2+ refilling by 1.8 mM extracellular Ca 2+ ( c ) were compared among different groups ( N =13–24). ( d ) An inhibitor of protein kinase C, GF109203X was used to pretreat cells for 30 min before BSA or PA incubation, and CPA-induced reduction of [Ca 2+ ] ER was analyzed ( N =19). Data are presented as mean±S.E.M. ** and *** denote P

    Techniques Used: Transfection, Incubation

    Alterations in the actin cytoskeleton and albumin permeability in palmitate-treated podocytes. Differentiated podocytes cultured on coverslips were incubated in Ca 2+ -free KRB solution containing BSA ( a ) or palmitate (PA; 300 μ M) ( c ) for 90 min, or cyclopiazonic acid (CPA; 15 μ M) for 15 min ( b ). A mitochondrial antioxidant, mitoTEMPO (mTEMPO; 100 nM) ( d ) or a phospholipase C (PLC) inhibitor, edelfosine (edelf; 10 μ M) ( e ), were used to pretreat podocytes for 15 min before PA incubation. Actin filaments were stained with phalloidin (red) and focal adhesions were detected by paxillin (green) staining. ( f , g ) The effects of BSA or PA on albumin permeability were estimated using transwell chambers with FITC-labeled BSA. ( h ) Proposed hypothetical model for palmitate-induced toxicity
    Figure Legend Snippet: Alterations in the actin cytoskeleton and albumin permeability in palmitate-treated podocytes. Differentiated podocytes cultured on coverslips were incubated in Ca 2+ -free KRB solution containing BSA ( a ) or palmitate (PA; 300 μ M) ( c ) for 90 min, or cyclopiazonic acid (CPA; 15 μ M) for 15 min ( b ). A mitochondrial antioxidant, mitoTEMPO (mTEMPO; 100 nM) ( d ) or a phospholipase C (PLC) inhibitor, edelfosine (edelf; 10 μ M) ( e ), were used to pretreat podocytes for 15 min before PA incubation. Actin filaments were stained with phalloidin (red) and focal adhesions were detected by paxillin (green) staining. ( f , g ) The effects of BSA or PA on albumin permeability were estimated using transwell chambers with FITC-labeled BSA. ( h ) Proposed hypothetical model for palmitate-induced toxicity

    Techniques Used: Permeability, Cell Culture, Incubation, Planar Chromatography, Staining, Labeling

    Palmitate, but not oleate, increased cytosolic and mitochondrial ROS production and induced cytotoxicity in mouse podocytes. Palmitate (PA) or bovine serum albumin (BSA) was applied to differentiated mouse podocytes with different concentrations (( a ), N =10) and durations (( b ), N =7). The effect of oleate (OA) on PA-induced cytotoxicity was also analyzed (( c ), N =3). Cytotoxicity by palmitate treatment (24 h) was evaluated by MTT assay ( a – c ) and quantitative determination of apoptotic DNA fragments (( d ), N =5). Condensed and fragmented apoptotic nuclei induced by palmitate were detected by DAPI staining ( e ) and are expressed as the percent of apoptotic nuclei (( f ), N =8). Podocytes treated with BSA or palmitate (PA) were loaded with DCF-DA ( g – i ) or mitoSox ( j – l ) and the fluorescence intensity was measured using a fluorescence microscope imaging system. Average florescence intensities of DCF (19–29 images from ≥5 independent experiments; ( b ) and ( c )) or mitoSox (7–10 images from ≥3 independent experiments; e and f ) were analyzed. Data are presented as mean±S.E.M., and *, **, *** denote P
    Figure Legend Snippet: Palmitate, but not oleate, increased cytosolic and mitochondrial ROS production and induced cytotoxicity in mouse podocytes. Palmitate (PA) or bovine serum albumin (BSA) was applied to differentiated mouse podocytes with different concentrations (( a ), N =10) and durations (( b ), N =7). The effect of oleate (OA) on PA-induced cytotoxicity was also analyzed (( c ), N =3). Cytotoxicity by palmitate treatment (24 h) was evaluated by MTT assay ( a – c ) and quantitative determination of apoptotic DNA fragments (( d ), N =5). Condensed and fragmented apoptotic nuclei induced by palmitate were detected by DAPI staining ( e ) and are expressed as the percent of apoptotic nuclei (( f ), N =8). Podocytes treated with BSA or palmitate (PA) were loaded with DCF-DA ( g – i ) or mitoSox ( j – l ) and the fluorescence intensity was measured using a fluorescence microscope imaging system. Average florescence intensities of DCF (19–29 images from ≥5 independent experiments; ( b ) and ( c )) or mitoSox (7–10 images from ≥3 independent experiments; e and f ) were analyzed. Data are presented as mean±S.E.M., and *, **, *** denote P

    Techniques Used: MTT Assay, Staining, Fluorescence, Microscopy, Imaging

    Inhibition of phospholipase C prevented intracellular IP 3 generation and loss of the ER Ca 2+ pool in palmitate-treated podocytes. ( a , b ) Changes in luminal Ca 2+ content in ER ([Ca 2+ ] ER ) upon cyclopiazonic acid (CPA) application were measured in D1ER-transfected podocytes. Edelfosine (edelf; 10 μ M), a phospholipase C (PLC) inhibitor, was used to pretreat cells for 30 min before BSA or palmitate (PA, 300 μ M) incubation in Ca 2+ -free KRB solution for 90 min ( N =4). ( c – h ) To observe IP 3 generation from PIP 2 , a plasmid encoding the pleckstrin homology domain of phospholipase C δ (PLC δ -PH)-green fluorescence protein (GFP) was transfected into podocytes. ( c – g ) Podocytes were incubated with BSA, PA or H 2 O 2 in Ca 2+ -free KRB solution for 90 min and GFP distribution was observed using a confocal microscope. Edelfosine ( f ) or a mitochondrial antioxidant, mitoTEMPO (mTEMPO; 100 nM) ( g ) was used to pretreat cells for 30 min before BSA or PA incubation. Line analyses of the fluorescence intensity for each picture are shown on the right. The percentage of cells showing PLCδ-PH-GFP translocation from the plasma membrane to the cytosol reflects PLC activation and IP 3 synthesis. ( h ) Data are presented as mean±S.E.M. *** denote P
    Figure Legend Snippet: Inhibition of phospholipase C prevented intracellular IP 3 generation and loss of the ER Ca 2+ pool in palmitate-treated podocytes. ( a , b ) Changes in luminal Ca 2+ content in ER ([Ca 2+ ] ER ) upon cyclopiazonic acid (CPA) application were measured in D1ER-transfected podocytes. Edelfosine (edelf; 10 μ M), a phospholipase C (PLC) inhibitor, was used to pretreat cells for 30 min before BSA or palmitate (PA, 300 μ M) incubation in Ca 2+ -free KRB solution for 90 min ( N =4). ( c – h ) To observe IP 3 generation from PIP 2 , a plasmid encoding the pleckstrin homology domain of phospholipase C δ (PLC δ -PH)-green fluorescence protein (GFP) was transfected into podocytes. ( c – g ) Podocytes were incubated with BSA, PA or H 2 O 2 in Ca 2+ -free KRB solution for 90 min and GFP distribution was observed using a confocal microscope. Edelfosine ( f ) or a mitochondrial antioxidant, mitoTEMPO (mTEMPO; 100 nM) ( g ) was used to pretreat cells for 30 min before BSA or PA incubation. Line analyses of the fluorescence intensity for each picture are shown on the right. The percentage of cells showing PLCδ-PH-GFP translocation from the plasma membrane to the cytosol reflects PLC activation and IP 3 synthesis. ( h ) Data are presented as mean±S.E.M. *** denote P

    Techniques Used: Inhibition, Transfection, Planar Chromatography, Incubation, Plasmid Preparation, Fluorescence, Microscopy, Translocation Assay, Activation Assay

    13) Product Images from "Adaptor Protein 1A Facilitates Dengue Virus Replication"

    Article Title: Adaptor Protein 1A Facilitates Dengue Virus Replication

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0130065

    Exogenous fatty acid increased DENV production after A5 treatment. DENV-infected Huh7 cells were incubated with A5 in the presence of oleic acid-BSA or fatty acid free-BSA for 24 h. The culture supernatants were collected for FFU assay. Statistical significance was analyzed using the unpaired t test. *P
    Figure Legend Snippet: Exogenous fatty acid increased DENV production after A5 treatment. DENV-infected Huh7 cells were incubated with A5 in the presence of oleic acid-BSA or fatty acid free-BSA for 24 h. The culture supernatants were collected for FFU assay. Statistical significance was analyzed using the unpaired t test. *P

    Techniques Used: Infection, Incubation

    14) Product Images from "Enriching Islet Phospholipids With Eicosapentaenoic Acid Reduces Prostaglandin E2 Signaling and Enhances Diabetic β-Cell Function"

    Article Title: Enriching Islet Phospholipids With Eicosapentaenoic Acid Reduces Prostaglandin E2 Signaling and Enhances Diabetic β-Cell Function

    Journal: Diabetes

    doi: 10.2337/db16-1362

    IL-1β expression is correlated with Ptgs2 expression and PGE 2 production in BTBR-Ob islets and can promote PGE 2 /PGE 3 production from BTBR-WT islets. A: BTBR-WT and BTBR-Ob islets were incubated with BSA control (Ctrl) or 100 μmol/L EPA for 48 h, and islets were then snap frozen for gene expression analysis. Data were compared by one-way ANOVA with a Tukey posttest. N = 4 to 5; *** P
    Figure Legend Snippet: IL-1β expression is correlated with Ptgs2 expression and PGE 2 production in BTBR-Ob islets and can promote PGE 2 /PGE 3 production from BTBR-WT islets. A: BTBR-WT and BTBR-Ob islets were incubated with BSA control (Ctrl) or 100 μmol/L EPA for 48 h, and islets were then snap frozen for gene expression analysis. Data were compared by one-way ANOVA with a Tukey posttest. N = 4 to 5; *** P

    Techniques Used: Expressing, Incubation

    Islet phospholipid AA and EPA composition can be altered ex vivo and impacts the GSIS response. A and B: Islets were incubated with BSA control (Ctrl) or 100 μmol/L EPA medium for 48 h before lipid extraction. AA and EPA are displayed as a percentage of total fatty acids ( A ) or the ratio between the two ( B ). Data were compared by unpaired t test within each species ( n = 5 – 7). **** P
    Figure Legend Snippet: Islet phospholipid AA and EPA composition can be altered ex vivo and impacts the GSIS response. A and B: Islets were incubated with BSA control (Ctrl) or 100 μmol/L EPA medium for 48 h before lipid extraction. AA and EPA are displayed as a percentage of total fatty acids ( A ) or the ratio between the two ( B ). Data were compared by unpaired t test within each species ( n = 5 – 7). **** P

    Techniques Used: Ex Vivo, Incubation

    PGE 2 production is reduced and PGE 3 is increased in BTBR-Ob islets treated with EPA. A : BTBR-WT and BTBR-Ob islets were incubated with BSA control (Ctrl) or EPA-enriched medium for 48 h, and islets were then snap frozen for gene expression analysis. Data were compared by one-way ANOVA with a Tukey posttest ( n = 4 to 5). **** P
    Figure Legend Snippet: PGE 2 production is reduced and PGE 3 is increased in BTBR-Ob islets treated with EPA. A : BTBR-WT and BTBR-Ob islets were incubated with BSA control (Ctrl) or EPA-enriched medium for 48 h, and islets were then snap frozen for gene expression analysis. Data were compared by one-way ANOVA with a Tukey posttest ( n = 4 to 5). **** P

    Techniques Used: Incubation, Expressing

    EPA reduces EP3 mRNA expression in BTBR-Ob islets. BTBR-WT and Ob islets were incubated with BSA control (Ctrl) or EPA-enriched medium for 48 h, and islets were then snap frozen for gene expression analysis. Data were compared by one-way ANOVA with a Tukey posttest ( n = 4 to 5). * P
    Figure Legend Snippet: EPA reduces EP3 mRNA expression in BTBR-Ob islets. BTBR-WT and Ob islets were incubated with BSA control (Ctrl) or EPA-enriched medium for 48 h, and islets were then snap frozen for gene expression analysis. Data were compared by one-way ANOVA with a Tukey posttest ( n = 4 to 5). * P

    Techniques Used: Expressing, Incubation

    15) Product Images from "Decreased lipid efflux and increased susceptibility to cholesterol-induced apoptosis in macrophages lacking phosphatidylcholine transfer protein"

    Article Title: Decreased lipid efflux and increased susceptibility to cholesterol-induced apoptosis in macrophages lacking phosphatidylcholine transfer protein

    Journal: Biochemical Journal

    doi: 10.1042/BJ20041899

    PC-TP expression does not influence the responses of apoAI-mediated lipid efflux to LXR/RXR ligands and Brefeldin A in CE-loaded macrophages ( A ) Western-blot analysis demonstrating ABCA1 expression in CE-loaded wild-type and Pctp −/− macrophages before and after activation of LXR using 22( R )-hydroxycholesterol (10 μM) and RXR using 9- cis retinoic acid (10 μM) in the absence or presence of Brefeldin A (BFA; 0.5 μg/ml) during CE loading. (Blots are representative of two independent experiments). ( B ) Phospholipid and ( C ) cholesterol efflux from CE-loaded peritoneal macrophages from wild-type (open bars) and Pctp −/− (hatched bars) mice that were incubated for 24 h with 22( R )-hydroxycholesterol (10 μM) and 9- cis retinoic acid (10 μM) in DMEM/BSA in the absence (LXR/RXR) or presence of 0.5 μg/ml BFA (LXR/RXR+BFA) during CE loading. Lipid efflux was measured after 4 h incubation with 20 μg/ml apoAI in DMEM/BSA plus 10 μM each of 22( R )-hydroxycholesterol and 9- cis retinoic acid in the absence (LXR/RXR) or presence of 0.5 μg/ml BFA (LXR/RXR+BFA). Results are the means±S.D. for triplicate determinations obtained from one of two independent experiments.
    Figure Legend Snippet: PC-TP expression does not influence the responses of apoAI-mediated lipid efflux to LXR/RXR ligands and Brefeldin A in CE-loaded macrophages ( A ) Western-blot analysis demonstrating ABCA1 expression in CE-loaded wild-type and Pctp −/− macrophages before and after activation of LXR using 22( R )-hydroxycholesterol (10 μM) and RXR using 9- cis retinoic acid (10 μM) in the absence or presence of Brefeldin A (BFA; 0.5 μg/ml) during CE loading. (Blots are representative of two independent experiments). ( B ) Phospholipid and ( C ) cholesterol efflux from CE-loaded peritoneal macrophages from wild-type (open bars) and Pctp −/− (hatched bars) mice that were incubated for 24 h with 22( R )-hydroxycholesterol (10 μM) and 9- cis retinoic acid (10 μM) in DMEM/BSA in the absence (LXR/RXR) or presence of 0.5 μg/ml BFA (LXR/RXR+BFA) during CE loading. Lipid efflux was measured after 4 h incubation with 20 μg/ml apoAI in DMEM/BSA plus 10 μM each of 22( R )-hydroxycholesterol and 9- cis retinoic acid in the absence (LXR/RXR) or presence of 0.5 μg/ml BFA (LXR/RXR+BFA). Results are the means±S.D. for triplicate determinations obtained from one of two independent experiments.

    Techniques Used: Expressing, Western Blot, Activation Assay, Mouse Assay, Incubation

    16) Product Images from "Platelet Glycoprotein Ib?/IX Mediates Glycoprotein Ib? Localization to Membrane Lipid Domain Critical for von Willebrand Factor Interaction at High Shear *"

    Article Title: Platelet Glycoprotein Ib?/IX Mediates Glycoprotein Ib? Localization to Membrane Lipid Domain Critical for von Willebrand Factor Interaction at High Shear *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.202549

    Partial dissociation of GP Ibα from the GEMs inhibits the interaction of the GP Ib-IX complex with vWf under high shear stresses. A , mutant GP Ibα (α SS ) was transfected into CHO cells expressing wild-type Ibβ and IX (CHOα SS βIX). The sorted stable cell lines expressed comparable levels of either wild-type or mutant GP Ibα, detected by WM23 staining. B , ristocetin-induced vWf binding was determined as described under “Experimental Procedures.” In the presence of 1.5 mg/ml ristocetin, wild-type or mutant GP Ibα-expressing CHO cells bound to vWf equally at all concentrations of vWf tested. C , the cells expressing wild-type or mutant GP Ibα were incubated on immobilized vWf for 1 min in a parallel-plate flow chamber and perfused with TBS/0.5% BSA at flow rates that generated wall shear stresses of 2.5, 10 or 20 dynes/cm 2 . The apparent difference in the rolling velocity between CHOα WT βIX and CHOα SS βIX was only seen under high shear stresses (10 and 20 dynes/cm 2 ), indicating that the association with the GEMs is critical for GP Ibα interaction with vWf at high shear.
    Figure Legend Snippet: Partial dissociation of GP Ibα from the GEMs inhibits the interaction of the GP Ib-IX complex with vWf under high shear stresses. A , mutant GP Ibα (α SS ) was transfected into CHO cells expressing wild-type Ibβ and IX (CHOα SS βIX). The sorted stable cell lines expressed comparable levels of either wild-type or mutant GP Ibα, detected by WM23 staining. B , ristocetin-induced vWf binding was determined as described under “Experimental Procedures.” In the presence of 1.5 mg/ml ristocetin, wild-type or mutant GP Ibα-expressing CHO cells bound to vWf equally at all concentrations of vWf tested. C , the cells expressing wild-type or mutant GP Ibα were incubated on immobilized vWf for 1 min in a parallel-plate flow chamber and perfused with TBS/0.5% BSA at flow rates that generated wall shear stresses of 2.5, 10 or 20 dynes/cm 2 . The apparent difference in the rolling velocity between CHOα WT βIX and CHOα SS βIX was only seen under high shear stresses (10 and 20 dynes/cm 2 ), indicating that the association with the GEMs is critical for GP Ibα interaction with vWf at high shear.

    Techniques Used: Mutagenesis, Transfection, Expressing, Stable Transfection, Staining, Binding Assay, Incubation, Flow Cytometry, Generated

    17) Product Images from "?B-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid ?-peptide and ?2-microglobulin"

    Article Title: ?B-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid ?-peptide and ?2-microglobulin

    Journal: Biochemical Journal

    doi: 10.1042/BJ20050339

    Effect of αB-crystallin (A), αA-crystallin (B), BSA (C) and HEWL (D) on the amyloid fibril growth of Aβ-(1–40)
    Figure Legend Snippet: Effect of αB-crystallin (A), αA-crystallin (B), BSA (C) and HEWL (D) on the amyloid fibril growth of Aβ-(1–40)

    Techniques Used:

    18) Product Images from "Contribution of myo-inositol oxygenase in AGE:RAGE-mediated renal tubulointerstitial injury in the context of diabetic nephropathy"

    Article Title: Contribution of myo-inositol oxygenase in AGE:RAGE-mediated renal tubulointerstitial injury in the context of diabetic nephropathy

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00434.2017

    Glycation and advanced glycation end products (AGE):receptor of advanced glycation end products (RAGE) interaction enhances cellular adhesiveness and myo-inositol oxygenase (MIOX) activity. A : solid-phase binding assays revealed a dose-dependent increase in the binding of glycated proteins with RAGE (0.5–4.0 μg/ml). The control S-100B exhibited a strong binding among various proteins. However, the glycated albumin had higher binding with RAGE, and it was comparable to glycated laminin. B : cell adhesion assays revealed that cellular adherence of HK-2 renal cells was glycation-dependent, and results with AGE-BSA and AGE-laminin were comparable. C : no significant differences were observed in viability of cells plated onto glycated vs. nonglycated substrata. D : interestingly, the cells maintained on glycated proteins had higher levels of MIOX activity and the highest being with AGE-BSA. E: adherence of cells plated on AGE-BSA substratum was time-dependent, and it increased over a period of 24 h. RAGE-small interfering (si) RNA treatment significantly reduced the adherence of cells maintained on AGE-BSA, suggesting AGE:RAGE interaction facilitates cellular adhesiveness. Lam, laminin; Col. IV, collagen IV. # P
    Figure Legend Snippet: Glycation and advanced glycation end products (AGE):receptor of advanced glycation end products (RAGE) interaction enhances cellular adhesiveness and myo-inositol oxygenase (MIOX) activity. A : solid-phase binding assays revealed a dose-dependent increase in the binding of glycated proteins with RAGE (0.5–4.0 μg/ml). The control S-100B exhibited a strong binding among various proteins. However, the glycated albumin had higher binding with RAGE, and it was comparable to glycated laminin. B : cell adhesion assays revealed that cellular adherence of HK-2 renal cells was glycation-dependent, and results with AGE-BSA and AGE-laminin were comparable. C : no significant differences were observed in viability of cells plated onto glycated vs. nonglycated substrata. D : interestingly, the cells maintained on glycated proteins had higher levels of MIOX activity and the highest being with AGE-BSA. E: adherence of cells plated on AGE-BSA substratum was time-dependent, and it increased over a period of 24 h. RAGE-small interfering (si) RNA treatment significantly reduced the adherence of cells maintained on AGE-BSA, suggesting AGE:RAGE interaction facilitates cellular adhesiveness. Lam, laminin; Col. IV, collagen IV. # P

    Techniques Used: Activity Assay, Binding Assay, Laser Capture Microdissection

    19) Product Images from "Stimulatory effects of advanced glycation endproducts (AGEs) on fibronectin matrix assembly"

    Article Title: Stimulatory effects of advanced glycation endproducts (AGEs) on fibronectin matrix assembly

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2016.07.003

    Stimulation of FN matrix assembly by AGE-modified BSA Mesangial cells were grown for 2 days on tissue culture plastic in the presence of AGE-BSA or BSA (20 µg/mL) which were replenished after 1 day. 10 µg/mL rat plasma FN was added for the final 24 hours. (A) Left, phase: Phase images of cells were captured after 2 days of culture. Right, rat FN: Immunofluorescence images of cells stained with IC3 anti-rat FN antibody. Relative average fluorescence intensities are indicated on the images (± standard error, n = 3, p
    Figure Legend Snippet: Stimulation of FN matrix assembly by AGE-modified BSA Mesangial cells were grown for 2 days on tissue culture plastic in the presence of AGE-BSA or BSA (20 µg/mL) which were replenished after 1 day. 10 µg/mL rat plasma FN was added for the final 24 hours. (A) Left, phase: Phase images of cells were captured after 2 days of culture. Right, rat FN: Immunofluorescence images of cells stained with IC3 anti-rat FN antibody. Relative average fluorescence intensities are indicated on the images (± standard error, n = 3, p

    Techniques Used: Modification, Immunofluorescence, Staining, Fluorescence

    20) Product Images from "Autocrine Function of Aldehyde Dehydrogenase 1 as a Determinant of Diet- and Sex-Specific Differences in Visceral Adiposity"

    Article Title: Autocrine Function of Aldehyde Dehydrogenase 1 as a Determinant of Diet- and Sex-Specific Differences in Visceral Adiposity

    Journal: Diabetes

    doi: 10.2337/db11-1779

    Sex-specific proteome and Atgl protein levels in VF of WT and Aldh1a1 −/− mice. A : Representative images of the DIGE proteomic gels comparing cy2-, cy3-, and cy5-stained proteins from VF isolated from WT and Aldh1a1 −/− males (180-day HF groups). Four gels from four pairs of different WT and Aldh1a1 −/− VF pads were run in each experiment. Spots, 176 in male ( left panel ) and 167 in female ( right panel ) groups, were different between WT and Aldh1a1 −/− mice without false discovery rate correction (FDRC) and 13 proteins after FDRC analysis. The identified 13 spots are shown on the gel image as a square around a spot that was significantly changed by at least 1.5-fold between groups with P ≤ 0.05 after FDRC using an independent t test. Atgl mRNA expression levels were measured by TaqMan ( B , VF group), and Atgl protein levels ( C ) were analyzed by Western blot in VF from WT and Aldh1a1 −/− male and female mice (all mean ± SD; n = 4) on an HF diet as well as in SVF fraction isolated from three age-matched females on a regular diet ( B , SVF VF group for mRNA expression). Inset in B confirms negligible Atgl protein levels in SVF compared with mature (M) adipocytes from same WT female using Western blot. Inset in C shows Atgl protein levels in two animals from each group. gapdh, glyceraldehyde-3-phosphate dehydrogenase. *Significant difference between WT vs. KO groups. D : Release of NEFA from VF explants (0.1 g WT males and females, 0.08 g knockout [KO] males, and 0.06 g KO females using 5 mL medium/g fat) stimulated with isoproterenol for 1.5 h in DMEM containing 2% delipidated BSA. VF was isolated from three mice per group. Data were normalized to the levels seen in WT male mice (100%). FFA, free fatty acid. E : Area of unilocular adipocytes was quantified from the images (original magnification ×10) shown in F . Total of 300 adipocytes were quantified per group. F : Representative Atgl staining of paraffin-embedded VF from WT and Aldh1a1 −/− male and female mice (all n = 4; mean ± SD) on an HF diet (180 days). In Aldh1a1 −/− female mice, Atgl protein was found in multilocular adipocytes. 10×, 40×, and 100× indicates magnification. Arrows in ×100 original magnification group show cytosolic and perilipid droplet Atgl staining in a multilocular adipocyte. G : Representative Ucp1 staining of same sections from Aldh1a1 −/− male and female mice. Brown Ucp1 staining was associated with multilocular adipocytes. P S , significantly different between male and female Aldh1a1 −/− mice. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: Sex-specific proteome and Atgl protein levels in VF of WT and Aldh1a1 −/− mice. A : Representative images of the DIGE proteomic gels comparing cy2-, cy3-, and cy5-stained proteins from VF isolated from WT and Aldh1a1 −/− males (180-day HF groups). Four gels from four pairs of different WT and Aldh1a1 −/− VF pads were run in each experiment. Spots, 176 in male ( left panel ) and 167 in female ( right panel ) groups, were different between WT and Aldh1a1 −/− mice without false discovery rate correction (FDRC) and 13 proteins after FDRC analysis. The identified 13 spots are shown on the gel image as a square around a spot that was significantly changed by at least 1.5-fold between groups with P ≤ 0.05 after FDRC using an independent t test. Atgl mRNA expression levels were measured by TaqMan ( B , VF group), and Atgl protein levels ( C ) were analyzed by Western blot in VF from WT and Aldh1a1 −/− male and female mice (all mean ± SD; n = 4) on an HF diet as well as in SVF fraction isolated from three age-matched females on a regular diet ( B , SVF VF group for mRNA expression). Inset in B confirms negligible Atgl protein levels in SVF compared with mature (M) adipocytes from same WT female using Western blot. Inset in C shows Atgl protein levels in two animals from each group. gapdh, glyceraldehyde-3-phosphate dehydrogenase. *Significant difference between WT vs. KO groups. D : Release of NEFA from VF explants (0.1 g WT males and females, 0.08 g knockout [KO] males, and 0.06 g KO females using 5 mL medium/g fat) stimulated with isoproterenol for 1.5 h in DMEM containing 2% delipidated BSA. VF was isolated from three mice per group. Data were normalized to the levels seen in WT male mice (100%). FFA, free fatty acid. E : Area of unilocular adipocytes was quantified from the images (original magnification ×10) shown in F . Total of 300 adipocytes were quantified per group. F : Representative Atgl staining of paraffin-embedded VF from WT and Aldh1a1 −/− male and female mice (all n = 4; mean ± SD) on an HF diet (180 days). In Aldh1a1 −/− female mice, Atgl protein was found in multilocular adipocytes. 10×, 40×, and 100× indicates magnification. Arrows in ×100 original magnification group show cytosolic and perilipid droplet Atgl staining in a multilocular adipocyte. G : Representative Ucp1 staining of same sections from Aldh1a1 −/− male and female mice. Brown Ucp1 staining was associated with multilocular adipocytes. P S , significantly different between male and female Aldh1a1 −/− mice. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Mouse Assay, Staining, Isolation, Expressing, Western Blot, Knock-Out

    21) Product Images from "Reactive Species Mediate Inhibition of Alveolar Type II Sodium Transport during Mycoplasma Infection"

    Article Title: Reactive Species Mediate Inhibition of Alveolar Type II Sodium Transport during Mycoplasma Infection

    Journal: American Journal of Respiratory and Critical Care Medicine

    doi: 10.1164/rccm.200501-155OC

    Contribution of myeloperoxidase (MPO) to M. pulmonis –mediated alterations of AFC in vivo . MPO +/+ and littermate MPO −/− mice were infected with 10 7 cfu M. pulmonis , and AFC was measured 48 h later. AFC measurements were performed on ventilated mice, and percentage of clearance was calculated from changes in instilled BSA concentration during a 30-min period. Data are means ± SE; n = 5–9 animals for each group. In all cases, the instillate contained 5% fat-free BSA in NaCl with the osmolality adjusted to that of murine plasma (322 mOsm/kg H 2 O). Amiloride (1.5 mM) was added to the instillate as indicated. Black bars , uninfected; hatched bars , amiloride; gray bars , 48-h postinfection; cross-hatched bars , 48-h postinfection amiloride. * Significant difference from uninfected same strain control; † significant difference from 48-h infected group (p = 0.04).
    Figure Legend Snippet: Contribution of myeloperoxidase (MPO) to M. pulmonis –mediated alterations of AFC in vivo . MPO +/+ and littermate MPO −/− mice were infected with 10 7 cfu M. pulmonis , and AFC was measured 48 h later. AFC measurements were performed on ventilated mice, and percentage of clearance was calculated from changes in instilled BSA concentration during a 30-min period. Data are means ± SE; n = 5–9 animals for each group. In all cases, the instillate contained 5% fat-free BSA in NaCl with the osmolality adjusted to that of murine plasma (322 mOsm/kg H 2 O). Amiloride (1.5 mM) was added to the instillate as indicated. Black bars , uninfected; hatched bars , amiloride; gray bars , 48-h postinfection; cross-hatched bars , 48-h postinfection amiloride. * Significant difference from uninfected same strain control; † significant difference from 48-h infected group (p = 0.04).

    Techniques Used: In Vivo, Mouse Assay, Infection, Concentration Assay

    Effect of forskolin on M. pulmonis –mediated alterations of AFC in vivo . B6 mice were inoculated with sterile broth or mycoplasmas (10 7 cfu), and AFC was measured 48 h later. AFC measurements were performed on ventilated mice, and percentage of clearance was calculated from changes in instilled BSA concentration in the presence or absence of forskolin (100 μM) during a 30-min period. Data are means ± SE; n = 5–9 animals for each group. In all cases, the instillate contained 5% fat-free BSA in NaCl with the osmolality adjusted to that of murine plasma (322 mOsm/l H 2 O). Amiloride (1.5 mM) was added to the instillate as indicated. * Significant difference from uninfected; † significant difference from 48-h infected (p
    Figure Legend Snippet: Effect of forskolin on M. pulmonis –mediated alterations of AFC in vivo . B6 mice were inoculated with sterile broth or mycoplasmas (10 7 cfu), and AFC was measured 48 h later. AFC measurements were performed on ventilated mice, and percentage of clearance was calculated from changes in instilled BSA concentration in the presence or absence of forskolin (100 μM) during a 30-min period. Data are means ± SE; n = 5–9 animals for each group. In all cases, the instillate contained 5% fat-free BSA in NaCl with the osmolality adjusted to that of murine plasma (322 mOsm/l H 2 O). Amiloride (1.5 mM) was added to the instillate as indicated. * Significant difference from uninfected; † significant difference from 48-h infected (p

    Techniques Used: In Vivo, Mouse Assay, Concentration Assay, Infection

    Effect of M. pulmonis on alveolar fluid clearance (AFC) and amiloride sensitivity in anesthetized, ventilated B6 mice. Mice were inoculated with sterile broth or mycoplasmas (10 7 cfu), and AFC was performed 24, 48, or 72 h later. AFC measurements were performed on ventilated mice, and percent clearance was calculated from changes in instilled bovine serum albumin (BSA) concentration during a 30-min period. In all cases, the instillate contained 5% fat-free BSA in NaCl with the osmolality adjusted to that of plasma (321 mOsm/l H 2 O). Amiloride (1.5 mM) was added to the instillate as indicated. Data are means ± SE; n = 5–8 mice for each group. * Denotes statistical significance at p
    Figure Legend Snippet: Effect of M. pulmonis on alveolar fluid clearance (AFC) and amiloride sensitivity in anesthetized, ventilated B6 mice. Mice were inoculated with sterile broth or mycoplasmas (10 7 cfu), and AFC was performed 24, 48, or 72 h later. AFC measurements were performed on ventilated mice, and percent clearance was calculated from changes in instilled bovine serum albumin (BSA) concentration during a 30-min period. In all cases, the instillate contained 5% fat-free BSA in NaCl with the osmolality adjusted to that of plasma (321 mOsm/l H 2 O). Amiloride (1.5 mM) was added to the instillate as indicated. Data are means ± SE; n = 5–8 mice for each group. * Denotes statistical significance at p

    Techniques Used: Mouse Assay, Concentration Assay

    22) Product Images from "Human umbilical cord-derived mesenchymal stem cells ameliorate insulin resistance by suppressing NLRP3 inflammasome-mediated inflammation in type 2 diabetes rats"

    Article Title: Human umbilical cord-derived mesenchymal stem cells ameliorate insulin resistance by suppressing NLRP3 inflammasome-mediated inflammation in type 2 diabetes rats

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0668-1

    PA–LPS stimulated inflammatory cytokine release in insulin-resistant HepG2 cells. HepG2 cells were incubated with BSA, PA-loaded BSA, LPS, and PA–LPS and then stimulated with insulin (100 nm) for 3 h. ( a ) Glycogen content in cells determined using a glycogen assay kit. ( b ) A quantity of 300 μl media in all groups was measured before and after the inducer by glucose assay kit, whose difference values were further normalized with cellular protein concentration. ( c–e ) Levels of IL-1β, IL-18, and TNF-α quantified by a commercial ELISA kit. * P
    Figure Legend Snippet: PA–LPS stimulated inflammatory cytokine release in insulin-resistant HepG2 cells. HepG2 cells were incubated with BSA, PA-loaded BSA, LPS, and PA–LPS and then stimulated with insulin (100 nm) for 3 h. ( a ) Glycogen content in cells determined using a glycogen assay kit. ( b ) A quantity of 300 μl media in all groups was measured before and after the inducer by glucose assay kit, whose difference values were further normalized with cellular protein concentration. ( c–e ) Levels of IL-1β, IL-18, and TNF-α quantified by a commercial ELISA kit. * P

    Techniques Used: Incubation, Glucose Assay, Protein Concentration, Enzyme-linked Immunosorbent Assay

    23) Product Images from "Entry of muscle satellite cells into the cell cycle requires sphingolipid signaling"

    Article Title: Entry of muscle satellite cells into the cell cycle requires sphingolipid signaling

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200605028

    S1P is required for inducing entry of satellite cells into the cell cycle. Isolated myofibers were cultured in DME/BSA (a–c) or DME/BSA/ 1 μM S1P (d–f) in the presence of BrdU. Myofibers were fixed 48 h later, and their associated satellite cells were coimmunostained for Pax7 (red) and BrdU (green; arrowheads in a–f). Similarly, immunostaining for MyoD (red) and PCNA (green; arrowheads in g–i) showed that there were significantly more satellite cells entering the cell cycle in the presence of S1P than in DME/BSA (j). All nuclei present were identified with DAPI (c, f, and i). Bar, 50 μm. To determine whether S1P is generated in response to mitogen stimulation during satellite cell activation, isolated myofibers were treated with 10 μM DMS in DME for 45 min before their associated satellite cells were stimulated with CEE/serum and analyzed for the expression of PCNA (k). In the absence of DMS, satellite cells were readily activated and practically all entered the cell cycle, but after exposure to the inhibitor, significantly less did so (k). To determine whether sphingomyelin was the source of S1P, isolated myofibers were incubated with 10 μM GW4869 to inhibit N-SMase activity before mitogen stimulation and then immunostained. Again, there was a significant reduction in the number of satellite cells entering the cell cycle (k), showing that S1P is derived from sphingomyelin. Data represent the mean ± SEM of immunostained cells per myofiber from three independent experiments (20 myofibers per experiment). Asterisks indicate that data are statistically significant using a t test (P
    Figure Legend Snippet: S1P is required for inducing entry of satellite cells into the cell cycle. Isolated myofibers were cultured in DME/BSA (a–c) or DME/BSA/ 1 μM S1P (d–f) in the presence of BrdU. Myofibers were fixed 48 h later, and their associated satellite cells were coimmunostained for Pax7 (red) and BrdU (green; arrowheads in a–f). Similarly, immunostaining for MyoD (red) and PCNA (green; arrowheads in g–i) showed that there were significantly more satellite cells entering the cell cycle in the presence of S1P than in DME/BSA (j). All nuclei present were identified with DAPI (c, f, and i). Bar, 50 μm. To determine whether S1P is generated in response to mitogen stimulation during satellite cell activation, isolated myofibers were treated with 10 μM DMS in DME for 45 min before their associated satellite cells were stimulated with CEE/serum and analyzed for the expression of PCNA (k). In the absence of DMS, satellite cells were readily activated and practically all entered the cell cycle, but after exposure to the inhibitor, significantly less did so (k). To determine whether sphingomyelin was the source of S1P, isolated myofibers were incubated with 10 μM GW4869 to inhibit N-SMase activity before mitogen stimulation and then immunostained. Again, there was a significant reduction in the number of satellite cells entering the cell cycle (k), showing that S1P is derived from sphingomyelin. Data represent the mean ± SEM of immunostained cells per myofiber from three independent experiments (20 myofibers per experiment). Asterisks indicate that data are statistically significant using a t test (P

    Techniques Used: Isolation, Cell Culture, Immunostaining, Generated, Activation Assay, Expressing, Incubation, Activity Assay, Derivative Assay

    24) Product Images from "Albumin induces upregulation of matrix metalloproteinase-9 in astrocytes via MAPK and reactive oxygen species-dependent pathways"

    Article Title: Albumin induces upregulation of matrix metalloproteinase-9 in astrocytes via MAPK and reactive oxygen species-dependent pathways

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-9-68

    Albumin-induced increase in matrix metalloproteinase (MMP)-9 involves reactive oxygen species. (a) Representative images of 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H 2 DCF-DA) fluorescence alone (green) or merged with DAPI nuclear stain (blue) in astrocytes treated with BSA, PBS (inset) or BSA with polyethylene glycol–superoxide dismutase (PEG-SOD, 200U) or polyethylene glycol–catalase (PEG-CAT) or the positive control tert -butyl hydroperoxide (TBHP). Treatment with albumin induced an increase in the DCF-DA fluorescence in astrocytes that was suppressed by the presence of PEG-SOD and PEG-CAT. Bars represent 50 μm. The data represent two independent experiments, each performed in duplicate. Representative zymogram and corresponding quantification of the level of MMP-9 release by astrocytes treated with albumin in the presence (+) or absence (−) of (b) PEG-SOD and PEG-CAT, and (c) the NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI). Inhibition of albumin-induced increase in MMP-9 by antioxidant enzymes SOD and CAT and the NADPH oxidase inhibitor, DPI suggest that the increase in MMP-9 involves reactive oxygen species. Data are representative of mean ± SEM of three independent experiments. *** P
    Figure Legend Snippet: Albumin-induced increase in matrix metalloproteinase (MMP)-9 involves reactive oxygen species. (a) Representative images of 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H 2 DCF-DA) fluorescence alone (green) or merged with DAPI nuclear stain (blue) in astrocytes treated with BSA, PBS (inset) or BSA with polyethylene glycol–superoxide dismutase (PEG-SOD, 200U) or polyethylene glycol–catalase (PEG-CAT) or the positive control tert -butyl hydroperoxide (TBHP). Treatment with albumin induced an increase in the DCF-DA fluorescence in astrocytes that was suppressed by the presence of PEG-SOD and PEG-CAT. Bars represent 50 μm. The data represent two independent experiments, each performed in duplicate. Representative zymogram and corresponding quantification of the level of MMP-9 release by astrocytes treated with albumin in the presence (+) or absence (−) of (b) PEG-SOD and PEG-CAT, and (c) the NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI). Inhibition of albumin-induced increase in MMP-9 by antioxidant enzymes SOD and CAT and the NADPH oxidase inhibitor, DPI suggest that the increase in MMP-9 involves reactive oxygen species. Data are representative of mean ± SEM of three independent experiments. *** P

    Techniques Used: Fluorescence, Staining, Positive Control, Inhibition

    Albumin stimulated an increase in the release of MMP-9 released in the conditioned media by astrocytes. Astrocytes were exposed to control PBS (−) or BSA (+). The conditioned media were collected after 0.5, 1.5, 6 and 24 hours and analyzed by gelatin zymography. (a) Representative zymogram showing a time-dependent increase in matrix metalloproteinase (MMP)-9; (b) there was a significant increase in the level of MMP-9 released 24 hours after BSA treatment compared with the control group; (c) the level of MMP-2 measured after BSA treatment was similar to that of the control group at all time points; (d ) treatment with fraction V (FrV) BSA also induced an increase in the level of MMP-9 released from astrocytes. Human serum albumin (HSA) and rat serum albumin (RSA) induced a similar increase in MMP-9 level to that seen with BSA. Treatment with dextran (0.1 mmol/L) did not induce any change in the level of MMP-9 in astrocytes. Data are representative of mean ± SEM of three independent experiments. *** P
    Figure Legend Snippet: Albumin stimulated an increase in the release of MMP-9 released in the conditioned media by astrocytes. Astrocytes were exposed to control PBS (−) or BSA (+). The conditioned media were collected after 0.5, 1.5, 6 and 24 hours and analyzed by gelatin zymography. (a) Representative zymogram showing a time-dependent increase in matrix metalloproteinase (MMP)-9; (b) there was a significant increase in the level of MMP-9 released 24 hours after BSA treatment compared with the control group; (c) the level of MMP-2 measured after BSA treatment was similar to that of the control group at all time points; (d ) treatment with fraction V (FrV) BSA also induced an increase in the level of MMP-9 released from astrocytes. Human serum albumin (HSA) and rat serum albumin (RSA) induced a similar increase in MMP-9 level to that seen with BSA. Treatment with dextran (0.1 mmol/L) did not induce any change in the level of MMP-9 in astrocytes. Data are representative of mean ± SEM of three independent experiments. *** P

    Techniques Used: Zymography

    25) Product Images from "Parental metabolic syndrome epigenetically reprograms offspring hepatic lipid metabolism in mice"

    Article Title: Parental metabolic syndrome epigenetically reprograms offspring hepatic lipid metabolism in mice

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI127502

    NREP is downregulated by palmitate-induced steatosis in HepG2, and NREP regulates hepatic lipid metabolism. ( A ) NREP protein levels in HepG2 cells treated with BSA or palmitate for 24 hours ( n = 3 independent experiments). ( B ) Quantification of NREP protein levels. ( C ) NREP knockdown (KD) in HepG2 cells at protein levels ( n = 3). ( D ) Representative oil red staining in HepG2 cells with NREP KD challenged with palmitate for 24 hours ( n = 3 independent experiments; original magnification, ×400; scale bar: 50 μm). ( E and F ) Triglyceride ( E ) and cholesterol ( F ) content quantification in HepG2 cell lysates after stimulation for 24 hours with 500 μM palmitate ( n = 3 independent experiments). ( G and H ) RNA sequencing selected enriched pathway analyses of upregulated ( G ) and downregulated genes ( H ) in HepG2 cells with NREP KD compared with scramble ( n = 3 per group). ( I ) Heatmap representation of differently expressed genes involved in cholesterol biosynthesis, AKT signaling, apoptosis, fibrosis, and cell cycle. ( J ) Basal signaling analyses in lysates from HepG2 cells treated with scramble (left) or NREP KD (right) ( n = 3 independent experiments). ( K ) Protein levels of indicated proteins in HepG2-scramble and NREP KD treated with BSA or palmitate for 24 hours in the presence of AKT inhibitor (MK-2206) or DMSO ( n = 2 independent experiments). Significance was determined by 2-tailed unpaired t test in B , and 1-way ANOVA with Holm-Šidák multiple-comparisons test in E and F . ** P
    Figure Legend Snippet: NREP is downregulated by palmitate-induced steatosis in HepG2, and NREP regulates hepatic lipid metabolism. ( A ) NREP protein levels in HepG2 cells treated with BSA or palmitate for 24 hours ( n = 3 independent experiments). ( B ) Quantification of NREP protein levels. ( C ) NREP knockdown (KD) in HepG2 cells at protein levels ( n = 3). ( D ) Representative oil red staining in HepG2 cells with NREP KD challenged with palmitate for 24 hours ( n = 3 independent experiments; original magnification, ×400; scale bar: 50 μm). ( E and F ) Triglyceride ( E ) and cholesterol ( F ) content quantification in HepG2 cell lysates after stimulation for 24 hours with 500 μM palmitate ( n = 3 independent experiments). ( G and H ) RNA sequencing selected enriched pathway analyses of upregulated ( G ) and downregulated genes ( H ) in HepG2 cells with NREP KD compared with scramble ( n = 3 per group). ( I ) Heatmap representation of differently expressed genes involved in cholesterol biosynthesis, AKT signaling, apoptosis, fibrosis, and cell cycle. ( J ) Basal signaling analyses in lysates from HepG2 cells treated with scramble (left) or NREP KD (right) ( n = 3 independent experiments). ( K ) Protein levels of indicated proteins in HepG2-scramble and NREP KD treated with BSA or palmitate for 24 hours in the presence of AKT inhibitor (MK-2206) or DMSO ( n = 2 independent experiments). Significance was determined by 2-tailed unpaired t test in B , and 1-way ANOVA with Holm-Šidák multiple-comparisons test in E and F . ** P

    Techniques Used: Staining, RNA Sequencing Assay

    26) Product Images from "Compared Binding Properties between Resveratrol and Other Polyphenols to Plasmatic Albumin: Consequences for the Health Protecting Effect of Dietary Plant Microcomponents"

    Article Title: Compared Binding Properties between Resveratrol and Other Polyphenols to Plasmatic Albumin: Consequences for the Health Protecting Effect of Dietary Plant Microcomponents

    Journal: Molecules

    doi: 10.3390/molecules191117066

    Infrared differential spectrum of resveratrol with BSA (the spectrum of trans resveratrol with BSA minus the spectrum of cis resveratrol with BSA) obtained after photo-isomerization in the same infrared cell.
    Figure Legend Snippet: Infrared differential spectrum of resveratrol with BSA (the spectrum of trans resveratrol with BSA minus the spectrum of cis resveratrol with BSA) obtained after photo-isomerization in the same infrared cell.

    Techniques Used:

    27) Product Images from "Host HDL Biogenesis Machinery is Recruited to the Inclusion of C. trachomatis-Infected Cells and Regulates Chlamydial Growth"

    Article Title: Host HDL Biogenesis Machinery is Recruited to the Inclusion of C. trachomatis-Infected Cells and Regulates Chlamydial Growth

    Journal: Cellular microbiology

    doi: 10.1111/j.1462-5822.2012.01823.x

    HeLa cells infected with Chlamydia trachomatis serovar D were incubated in the presence of 1.5μM β-BODIPY FL C 5 -HPC in fatty acid-free BSA for 1 hour prior to fixation in 4% paraformaldehyde in PBS at 48 (A) or 24 (B) hours PI. The labeled cells were then permeabilized by a brief incubation in methanol and incubated with a monoclonal antibody directed against IncA and a rabbit polyclonal antibody directed against apoA-1. Following incubation with appropriate secondary antibodies, the cells were analyzed by confocal microscopy. The arrow in B indicates the perinuclear pool of β-BODIPY FL C 5 -HPC in an infected cell. The white bars are 5μm.
    Figure Legend Snippet: HeLa cells infected with Chlamydia trachomatis serovar D were incubated in the presence of 1.5μM β-BODIPY FL C 5 -HPC in fatty acid-free BSA for 1 hour prior to fixation in 4% paraformaldehyde in PBS at 48 (A) or 24 (B) hours PI. The labeled cells were then permeabilized by a brief incubation in methanol and incubated with a monoclonal antibody directed against IncA and a rabbit polyclonal antibody directed against apoA-1. Following incubation with appropriate secondary antibodies, the cells were analyzed by confocal microscopy. The arrow in B indicates the perinuclear pool of β-BODIPY FL C 5 -HPC in an infected cell. The white bars are 5μm.

    Techniques Used: Infection, Incubation, Labeling, Confocal Microscopy

    HeLa cells infected with C. trachomatis serovar D were incubated at 37°C for 28 hours. At this time, the cells were incubated in the absence or presence of 300μM glyburide for 3 hours at 37°C. β-BODIPY FL C 5 -HPC in fatty acid-free BSA was then added to the media of control and drug-treated cells at a final concentration of 1.5μM and the cells were incubated an additional hour at 37°C. The cells were then fixed in 4% paraformaldehyde in PBS and imaged by confocal microscopy (A). The levels of total and inclusion-associated β-BODIPY FL C 5 -HPC fluorescence in control and glyburide-treated cells were quantified using the imaging software for the Zeiss LSM 510 confocal microscope as described in the Methods (B). The average diameter of the inclusions in control and glyburide-treated cells was also quantified using the Zeiss imaging software (C). The values shown in the histograms in B and C represent the average values obtained from the analysis of at least 75 cells from two independent experiments. *** p
    Figure Legend Snippet: HeLa cells infected with C. trachomatis serovar D were incubated at 37°C for 28 hours. At this time, the cells were incubated in the absence or presence of 300μM glyburide for 3 hours at 37°C. β-BODIPY FL C 5 -HPC in fatty acid-free BSA was then added to the media of control and drug-treated cells at a final concentration of 1.5μM and the cells were incubated an additional hour at 37°C. The cells were then fixed in 4% paraformaldehyde in PBS and imaged by confocal microscopy (A). The levels of total and inclusion-associated β-BODIPY FL C 5 -HPC fluorescence in control and glyburide-treated cells were quantified using the imaging software for the Zeiss LSM 510 confocal microscope as described in the Methods (B). The average diameter of the inclusions in control and glyburide-treated cells was also quantified using the Zeiss imaging software (C). The values shown in the histograms in B and C represent the average values obtained from the analysis of at least 75 cells from two independent experiments. *** p

    Techniques Used: Infection, Incubation, Concentration Assay, Confocal Microscopy, Fluorescence, Imaging, Software, Microscopy

    HeLa cells infected with Chlamydia trachomatis serovar D were fixed with 4% paraformaldehyde in PBS at 48 hours PI. The cells were then permeabilized in PBS containing 0.1% saponin and incubated with antibodies directed against Inc A and apoA-1. The cells were then washed and incubated with the appropriate secondary antibodies. Following washing, the cells were stained with the dye Bodipy 493 and imaged by confocal microscopy (A). Alternatively, infected cells were incubated in the presence of β-BODIPY FL C 5 -HPC in fatty acid-free BSA at a final concentration of 1.5μM for 1 hour prior to fixation in 4% paraformaldehyde in PBS at 48 hours PI. These labeled cells were either directly imaged by confocal microscopy (B) or permeabilized by a brief incubation in methanol. Permeabilized cells were incubated with monoclonal antibodies directed against MOMP (C) or IncA (D) followed by the appropriate secondary antibody and imaged by confocal microscopy. The arrowhead in A denotes a lipid droplet that lies adjacent to a foci of apoA-1 within an inclusion of an infected cell. The arrow in B indicates the perinuclear pool of β-BODIPY FL C 5 -HPC that accumulates in uninfected cells, while the arrowhead in B points to an infected cell where β-BODIPY FL C 5- HPC accumulates in membranes that surround the inclusion and within the inclusion. The arrowheads in C indicate β-BODIPY FL C 5 -HPC that accumulates at sites within the inclusion that do not stain with MOMP antibodies. The asterisks in B indicate nuclei. The white bars are 5μm.
    Figure Legend Snippet: HeLa cells infected with Chlamydia trachomatis serovar D were fixed with 4% paraformaldehyde in PBS at 48 hours PI. The cells were then permeabilized in PBS containing 0.1% saponin and incubated with antibodies directed against Inc A and apoA-1. The cells were then washed and incubated with the appropriate secondary antibodies. Following washing, the cells were stained with the dye Bodipy 493 and imaged by confocal microscopy (A). Alternatively, infected cells were incubated in the presence of β-BODIPY FL C 5 -HPC in fatty acid-free BSA at a final concentration of 1.5μM for 1 hour prior to fixation in 4% paraformaldehyde in PBS at 48 hours PI. These labeled cells were either directly imaged by confocal microscopy (B) or permeabilized by a brief incubation in methanol. Permeabilized cells were incubated with monoclonal antibodies directed against MOMP (C) or IncA (D) followed by the appropriate secondary antibody and imaged by confocal microscopy. The arrowhead in A denotes a lipid droplet that lies adjacent to a foci of apoA-1 within an inclusion of an infected cell. The arrow in B indicates the perinuclear pool of β-BODIPY FL C 5 -HPC that accumulates in uninfected cells, while the arrowhead in B points to an infected cell where β-BODIPY FL C 5- HPC accumulates in membranes that surround the inclusion and within the inclusion. The arrowheads in C indicate β-BODIPY FL C 5 -HPC that accumulates at sites within the inclusion that do not stain with MOMP antibodies. The asterisks in B indicate nuclei. The white bars are 5μm.

    Techniques Used: Infection, Incubation, Staining, Confocal Microscopy, Concentration Assay, Labeling

    28) Product Images from "Susceptibility of podocytes to palmitic acid is regulated by fatty acid oxidation and inversely depends on acetyl-CoA carboxylases 1 and 2"

    Article Title: Susceptibility of podocytes to palmitic acid is regulated by fatty acid oxidation and inversely depends on acetyl-CoA carboxylases 1 and 2

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00454.2013

    AICAR increases and etomoxir decreases oxidation of palmitic acid in podocytes. A : AICAR upregulated oxidation of palmitic acid. Podocytes were treated with 0.5% free fatty acid (FFA)-free BSA with 200 μM palmitic acid in the presence of 0.5 μCi/ml
    Figure Legend Snippet: AICAR increases and etomoxir decreases oxidation of palmitic acid in podocytes. A : AICAR upregulated oxidation of palmitic acid. Podocytes were treated with 0.5% free fatty acid (FFA)-free BSA with 200 μM palmitic acid in the presence of 0.5 μCi/ml

    Techniques Used:

    29) Product Images from "Autotaxin expression and its connection with the TNF-alpha-NF-?B axis in human hepatocellular carcinoma"

    Article Title: Autotaxin expression and its connection with the TNF-alpha-NF-?B axis in human hepatocellular carcinoma

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-71

    Up-regulation of ATX induced by TNF-α is associated with increased lysophospholipase D (lyso-PLD) activity by conversion of LPC into LPA in Hep3B and Huh7 cells . Serum starved Hep3B or Huh7 cells were treated with TNF-α (10 ng/ml) or vehicle (0.1%BSA/PBS) for 20 hours. A . Conditioned media (CM) or control media (DMEM or EMEM) were concentrated (40-fold) and assayed for ATX activity using the FS-3 compound. The results are shown as the average of relative fluorescence activity ± SD from three experiments. B . CM were incubated with 15 μM LPC (18:1) for 3 hours at 37°C. Lipids were analyzed by liquid chromatography-mass spectrometry (LC-MS). Results are level of LPA (18:1) from three experiments and presented as mean ± SD. *, P
    Figure Legend Snippet: Up-regulation of ATX induced by TNF-α is associated with increased lysophospholipase D (lyso-PLD) activity by conversion of LPC into LPA in Hep3B and Huh7 cells . Serum starved Hep3B or Huh7 cells were treated with TNF-α (10 ng/ml) or vehicle (0.1%BSA/PBS) for 20 hours. A . Conditioned media (CM) or control media (DMEM or EMEM) were concentrated (40-fold) and assayed for ATX activity using the FS-3 compound. The results are shown as the average of relative fluorescence activity ± SD from three experiments. B . CM were incubated with 15 μM LPC (18:1) for 3 hours at 37°C. Lipids were analyzed by liquid chromatography-mass spectrometry (LC-MS). Results are level of LPA (18:1) from three experiments and presented as mean ± SD. *, P

    Techniques Used: Activity Assay, Fluorescence, Incubation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    30) Product Images from "Design of Group IIA Secreted/Synovial Phospholipase A2 Inhibitors: An Oxadiazolone Derivative Suppresses Chondrocyte Prostaglandin E2 Secretion"

    Article Title: Design of Group IIA Secreted/Synovial Phospholipase A2 Inhibitors: An Oxadiazolone Derivative Suppresses Chondrocyte Prostaglandin E2 Secretion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010914

    Effect of IL-1β and C8 on viability of articular chondrocytes. Chondrocytes were untreated (white bars) or treated for 20 h with IL-1β alone (black bars) or 1 h after the addition of C8 (grey bars) in DMEM alone (A) or supplemented with 0.1% BSA (B) or 2% FCS (C). Cell viability was evaluated by an MTT-based test. Data represent the absorbance 570nm - absorbance 690nm and are expressed as relative arbitrary units, where the IL-1β-untreated group represents 100%. Values are means ± SEM (n = 3 to 8 independent determinations). ‡ P
    Figure Legend Snippet: Effect of IL-1β and C8 on viability of articular chondrocytes. Chondrocytes were untreated (white bars) or treated for 20 h with IL-1β alone (black bars) or 1 h after the addition of C8 (grey bars) in DMEM alone (A) or supplemented with 0.1% BSA (B) or 2% FCS (C). Cell viability was evaluated by an MTT-based test. Data represent the absorbance 570nm - absorbance 690nm and are expressed as relative arbitrary units, where the IL-1β-untreated group represents 100%. Values are means ± SEM (n = 3 to 8 independent determinations). ‡ P

    Techniques Used: MTT Assay

    Effect of IL-1β and C8 on nitrite secretion by articular chondrocytes. Chondrocytes were untreated (white bars) or treated for 20 h with IL-1β alone (black bars) or 1 h after the addition of C8 (grey bars) in DMEM alone (A) or supplemented with 0.1% BSA (B) or 2% FCS (C). NO secretion was indirectly evaluated by determination of nitrite concentration in conditioned culture medium. Protein concentration was determined in whole-cell protein extracts. Data represent the ratio of nitrite concentrations relative to whole-cell protein concentration (pmol nitrites/µg protein). Values are means ± SEM (n = 4 to 7 independent determinations). ‡ P
    Figure Legend Snippet: Effect of IL-1β and C8 on nitrite secretion by articular chondrocytes. Chondrocytes were untreated (white bars) or treated for 20 h with IL-1β alone (black bars) or 1 h after the addition of C8 (grey bars) in DMEM alone (A) or supplemented with 0.1% BSA (B) or 2% FCS (C). NO secretion was indirectly evaluated by determination of nitrite concentration in conditioned culture medium. Protein concentration was determined in whole-cell protein extracts. Data represent the ratio of nitrite concentrations relative to whole-cell protein concentration (pmol nitrites/µg protein). Values are means ± SEM (n = 4 to 7 independent determinations). ‡ P

    Techniques Used: Concentration Assay, Protein Concentration

    Effect of IL-1β and C8 on PGE 2 secretion by articular chondrocytes. Chondrocytes were untreated (white bars) or treated for 20 h with IL-1β alone (black bars) or 1 h after the addition of C8 (grey bars) in DMEM alone (A) or with 0.1% BSA (B) or with 2% FCS (C). PGE 2 concentration was determined in conditioned culture medium, and protein concentration was determined in whole-cell protein extracts. Data represent the ratio of PGE 2 concentration relative to whole cell protein concentration (pg PGE 2 /µg proteins). Values are means ± SEM (n = 3 to 7 independent determinations). ‡ P
    Figure Legend Snippet: Effect of IL-1β and C8 on PGE 2 secretion by articular chondrocytes. Chondrocytes were untreated (white bars) or treated for 20 h with IL-1β alone (black bars) or 1 h after the addition of C8 (grey bars) in DMEM alone (A) or with 0.1% BSA (B) or with 2% FCS (C). PGE 2 concentration was determined in conditioned culture medium, and protein concentration was determined in whole-cell protein extracts. Data represent the ratio of PGE 2 concentration relative to whole cell protein concentration (pg PGE 2 /µg proteins). Values are means ± SEM (n = 3 to 7 independent determinations). ‡ P

    Techniques Used: Concentration Assay, Protein Concentration

    31) Product Images from "Genetic Evidence for ATP-dependent Endoplasmic Reticulum-to-Golgi Apparatus Trafficking of Ceramide for Sphingomyelin Synthesis in Chinese Hamster Ovary Cells "

    Article Title: Genetic Evidence for ATP-dependent Endoplasmic Reticulum-to-Golgi Apparatus Trafficking of Ceramide for Sphingomyelin Synthesis in Chinese Hamster Ovary Cells

    Journal: The Journal of Cell Biology

    doi:

    Metabolism of C 5 -DMB-Cer in wild-type and LY-A cells in the presence or absence of brefeldin A. Wild-type CHO (open circles) and mutant LY-A (closed circles) cells were preincubated in Nutridoma medium without (A) or with (B) 1 μg/ml brefeldin A for 30 min at 33°C, then incubated with 1.25 μM C 5 -DMB-Cer for 30 min at 4°C. After washing, cell monolayers were further incubated in Nutridoma medium containing 0.34 mg/ml BSA without or with 1 μg/ml brefeldin A for the indicated times at 33°C. Cellular lipids were extracted and analyzed by TLC. The formation of the DMB derivative of GlcCer was too low (
    Figure Legend Snippet: Metabolism of C 5 -DMB-Cer in wild-type and LY-A cells in the presence or absence of brefeldin A. Wild-type CHO (open circles) and mutant LY-A (closed circles) cells were preincubated in Nutridoma medium without (A) or with (B) 1 μg/ml brefeldin A for 30 min at 33°C, then incubated with 1.25 μM C 5 -DMB-Cer for 30 min at 4°C. After washing, cell monolayers were further incubated in Nutridoma medium containing 0.34 mg/ml BSA without or with 1 μg/ml brefeldin A for the indicated times at 33°C. Cellular lipids were extracted and analyzed by TLC. The formation of the DMB derivative of GlcCer was too low (

    Techniques Used: Mutagenesis, Incubation, Thin Layer Chromatography

    32) Product Images from "The Antioxidant 3H-1,2-Dithiole-3-Thione Potentiates Advanced Glycation End-Product-Induced Oxidative Stress in SH-SY5Y Cells"

    Article Title: The Antioxidant 3H-1,2-Dithiole-3-Thione Potentiates Advanced Glycation End-Product-Induced Oxidative Stress in SH-SY5Y Cells

    Journal: Experimental Diabetes Research

    doi: 10.1155/2012/137607

    D3T potentiates AGE-induced cytotoxicity. (a) SH-SY5Y cells were pretreated for 24 hours with 0–50 μ M D3T followed by a 24 hr treatment with BSA or AGE-BSA. MTT was performed as indicated in the Materials and Methods section and absorbances were standardized to 0.1% DMSO treatment. Results are from three independent experiments ( n = 8–14); bar values are expressed as mean ± SD. (b) GSH was measured by HPLC and standardized to total protein. Results are from four independent experiments ( n = 4); bar values are expressed as mean ± SD (* P
    Figure Legend Snippet: D3T potentiates AGE-induced cytotoxicity. (a) SH-SY5Y cells were pretreated for 24 hours with 0–50 μ M D3T followed by a 24 hr treatment with BSA or AGE-BSA. MTT was performed as indicated in the Materials and Methods section and absorbances were standardized to 0.1% DMSO treatment. Results are from three independent experiments ( n = 8–14); bar values are expressed as mean ± SD. (b) GSH was measured by HPLC and standardized to total protein. Results are from four independent experiments ( n = 4); bar values are expressed as mean ± SD (* P

    Techniques Used: MTT Assay, High Performance Liquid Chromatography

    D3T potentiates AGE-induced ROS generation. SH-SY5Y cells were pretreated for 24 hours with DMSO or 50 μ M D3T. Cells were treated with or without the NADPH oxidase inhibitor DPI (20 nM) or the PKC inhibitor rottlerin (2 μ M) for 30 min and then exposed to 4 mg/mL BSA or AGE-BSA for 16 hours. ROS generation is determined through DCF fluorescence. Results are from three independent experiments ( n = 9). Bar values are expressed as mean ± SD. Letters that are not the same are significantly different ( P
    Figure Legend Snippet: D3T potentiates AGE-induced ROS generation. SH-SY5Y cells were pretreated for 24 hours with DMSO or 50 μ M D3T. Cells were treated with or without the NADPH oxidase inhibitor DPI (20 nM) or the PKC inhibitor rottlerin (2 μ M) for 30 min and then exposed to 4 mg/mL BSA or AGE-BSA for 16 hours. ROS generation is determined through DCF fluorescence. Results are from three independent experiments ( n = 9). Bar values are expressed as mean ± SD. Letters that are not the same are significantly different ( P

    Techniques Used: Fluorescence

    33) Product Images from "Advanced glycation end products facilitate bacterial adherence in urinary tract infection in diabetic mice"

    Article Title: Advanced glycation end products facilitate bacterial adherence in urinary tract infection in diabetic mice

    Journal: Pathogens and Disease

    doi: 10.1093/femspd/ftu004

    Lectins bind to AGEs. AGE-modified and sham-modified BSA and collagen were adsorbed to wells of 96-well plates and incubated with various biotinylated lectins in triplicate as described in the section ‘Materials and Methods’, and absorbance
    Figure Legend Snippet: Lectins bind to AGEs. AGE-modified and sham-modified BSA and collagen were adsorbed to wells of 96-well plates and incubated with various biotinylated lectins in triplicate as described in the section ‘Materials and Methods’, and absorbance

    Techniques Used: Modification, Incubation

    Type 1 fimbriae bind to AGEs in a specific manner in vitro . AGE-modified or sham-modified BSA (Sh-BSA) was adsorbed to wells of 96-well plates and incubated in the presence or absence of biotinylated type 1 fimbriae that had been preincubated with (i)
    Figure Legend Snippet: Type 1 fimbriae bind to AGEs in a specific manner in vitro . AGE-modified or sham-modified BSA (Sh-BSA) was adsorbed to wells of 96-well plates and incubated in the presence or absence of biotinylated type 1 fimbriae that had been preincubated with (i)

    Techniques Used: In Vitro, Modification, Incubation

    34) Product Images from "Lysophosphatidic acid stimulates hyaluronan production by mouse cumulus–oocyte complexes"

    Article Title: Lysophosphatidic acid stimulates hyaluronan production by mouse cumulus–oocyte complexes

    Journal: Reproductive Medicine and Biology

    doi: 10.1007/s12522-013-0169-6

    LPA induces HA production through transactivation of EGF receptor followed by activation of two MAP kinase pathways. In 0.1 ml of 0.2 % BSA‐containing mHTF, COC were incubated with 10 nM LPA, 1 ng/ml EGF, or 100 ng/ml of FSH in the presence or absence of 200 nM AG1478 ( a ), 15 μM SB203580 ( b ) or 15 μM PD98059 ( c ). Increases in HA productions induced by LPA, EGF, and FSH were calculated by subtraction the control value in the absence of the stimulant from the value in the presence of the stimulant. Black and white columns with a bar show mean values and SE for experiments in the presence and absence of AG1478, SB203580 or PD98059, respectively. * P
    Figure Legend Snippet: LPA induces HA production through transactivation of EGF receptor followed by activation of two MAP kinase pathways. In 0.1 ml of 0.2 % BSA‐containing mHTF, COC were incubated with 10 nM LPA, 1 ng/ml EGF, or 100 ng/ml of FSH in the presence or absence of 200 nM AG1478 ( a ), 15 μM SB203580 ( b ) or 15 μM PD98059 ( c ). Increases in HA productions induced by LPA, EGF, and FSH were calculated by subtraction the control value in the absence of the stimulant from the value in the presence of the stimulant. Black and white columns with a bar show mean values and SE for experiments in the presence and absence of AG1478, SB203580 or PD98059, respectively. * P

    Techniques Used: Activation Assay, Incubation

    Role of LPA receptor coupled to Gi in LPA‐induced HA production. a COC were incubated in 0.1 ml of 0.2 % BSA‐containing mHTF with 10 nM LPA, 1 ng/ml EGF, or 100 ng/ml of FSH in the presence or absence of 10 μM Ki 16425. Induction of increased HA productions by LPA, EGF, and FSH were calculated by subtracting the control value in the absence of the stimulant from the value in the presence of the stimulant. Black and white columns show the mean values obtained by the experiment in the presence and absence of Ki16425, respectively. * P
    Figure Legend Snippet: Role of LPA receptor coupled to Gi in LPA‐induced HA production. a COC were incubated in 0.1 ml of 0.2 % BSA‐containing mHTF with 10 nM LPA, 1 ng/ml EGF, or 100 ng/ml of FSH in the presence or absence of 10 μM Ki 16425. Induction of increased HA productions by LPA, EGF, and FSH were calculated by subtracting the control value in the absence of the stimulant from the value in the presence of the stimulant. Black and white columns show the mean values obtained by the experiment in the presence and absence of Ki16425, respectively. * P

    Techniques Used: Incubation

    LPA‐induced HA production in mouse COC in vitro. a Mouse COC were incubated in 0.1 ml of 0.2 % BSA‐containing mHTF with or without 10 nM LPA at 37 °C for 15 h. HA concentrations in the culture medium (released HA) and COC suspension (retained HA) were measured after pronase treatment. HA concentrations were expressed as nanograms per milliliter (ng/ml). Numbers in brackets show the numbers of separate experiments where measurements were done in triplicate. * P
    Figure Legend Snippet: LPA‐induced HA production in mouse COC in vitro. a Mouse COC were incubated in 0.1 ml of 0.2 % BSA‐containing mHTF with or without 10 nM LPA at 37 °C for 15 h. HA concentrations in the culture medium (released HA) and COC suspension (retained HA) were measured after pronase treatment. HA concentrations were expressed as nanograms per milliliter (ng/ml). Numbers in brackets show the numbers of separate experiments where measurements were done in triplicate. * P

    Techniques Used: In Vitro, Incubation

    35) Product Images from "An Early Stage of Membrane Fusion Mediated by the Low pH Conformation of Influenza Hemagglutinin Depends upon Membrane Lipids"

    Article Title: An Early Stage of Membrane Fusion Mediated by the Low pH Conformation of Influenza Hemagglutinin Depends upon Membrane Lipids

    Journal: The Journal of Cell Biology

    doi:

    LPC incorporated into either one or both of two fusing membranes reversibly inhibits fusion. Fusion was triggered by a 10-min application of pH 4.9 medium and assayed by fluorescence microscopy as R18 redistribution. Each bar is mean ± SEM, n = 3. ( a ) Fusion inhibition is caused by membrane-incorporated rather than unbound LPC. □, fusion of HAb2 cells with prebound RBCs, no lipids added; ▒⃞ , fusion in the presence of 36 μM stearoyl LPC; ▪, unbound LPC was removed before low pH application. Cells were incubated with LPC (same concentration as above) for 10 min and then washed out with LPC-free PBS; ▨ , LPC was extracted from membranes by BSA before low pH application. Cells were incubated with LPC as above and then washed out with LPC-free PBS containing BSA. Each point is mean ± SEM, n = 3. ( b ) LPC inhibits fusion when present either in HA-expressing membrane or in RBC membrane. □, fusion of HAb2 cells with RBCs, no lipids added, ▒⃞ , only HAb2 cells were treated by stearoyl LPC (42 μM); ▪, only RBCs were treated by stearoyl LPC (1 μM/5 × 10 6 RBCs); ▨ , both RBCs and HAb2 cells were separately treated with stearoyl LPC (same concentrations as above).
    Figure Legend Snippet: LPC incorporated into either one or both of two fusing membranes reversibly inhibits fusion. Fusion was triggered by a 10-min application of pH 4.9 medium and assayed by fluorescence microscopy as R18 redistribution. Each bar is mean ± SEM, n = 3. ( a ) Fusion inhibition is caused by membrane-incorporated rather than unbound LPC. □, fusion of HAb2 cells with prebound RBCs, no lipids added; ▒⃞ , fusion in the presence of 36 μM stearoyl LPC; ▪, unbound LPC was removed before low pH application. Cells were incubated with LPC (same concentration as above) for 10 min and then washed out with LPC-free PBS; ▨ , LPC was extracted from membranes by BSA before low pH application. Cells were incubated with LPC as above and then washed out with LPC-free PBS containing BSA. Each point is mean ± SEM, n = 3. ( b ) LPC inhibits fusion when present either in HA-expressing membrane or in RBC membrane. □, fusion of HAb2 cells with RBCs, no lipids added, ▒⃞ , only HAb2 cells were treated by stearoyl LPC (42 μM); ▪, only RBCs were treated by stearoyl LPC (1 μM/5 × 10 6 RBCs); ▨ , both RBCs and HAb2 cells were separately treated with stearoyl LPC (same concentrations as above).

    Techniques Used: Fluorescence, Microscopy, Inhibition, Incubation, Concentration Assay, Expressing

    36) Product Images from "Impairment of Fatty Acid Oxidation in Alveolar Epithelial Cells Mediates Acute Lung Injury"

    Article Title: Impairment of Fatty Acid Oxidation in Alveolar Epithelial Cells Mediates Acute Lung Injury

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2018-0152OC

    AECs use both glucose and fatty acids, and FAO is impaired in AECs of mice with LPS-induced ALI. ( A ) Primary AECs were seeded in Seahorse XF-24 microplates and incubated with substrate-limited media for 3 hours. Basal oxygen consumption rate (OCR) was then recorded before BSA or palmitate-BSA (final concentration: 150 μM) was injected into the wells. After injection, OCR was continuously monitored until it reached the maximum value. n = 4 per group; mean ± SEM. ( B ) Primary AECs were seeded in Seahorse XF-24 microplates and incubated with extracellular acidification rate (ECAR) media for 1 hour. Basal OCR and ECAR were then recorded before glucose (final concentration: 10 mM) was injected into the wells. After injection, OCR and ECAR were continuously monitored until they reached the maximum values. n = 3 per group; mean ± SEM. ( C ) Primary AECs purified from saline or intratracheal LPS–treated mice were seeded in Seahorse XF-24 microplates and incubated with substrate-limited media for 3 hours. Basal OCR was then recorded before palmitate-BSA was injected into the wells. After injection, OCR was continuously monitored until it reached the maximum value. OCR values were normalized to total protein content of the cellular extract from each well. n = 4 mice per group; mean ± SEM.
    Figure Legend Snippet: AECs use both glucose and fatty acids, and FAO is impaired in AECs of mice with LPS-induced ALI. ( A ) Primary AECs were seeded in Seahorse XF-24 microplates and incubated with substrate-limited media for 3 hours. Basal oxygen consumption rate (OCR) was then recorded before BSA or palmitate-BSA (final concentration: 150 μM) was injected into the wells. After injection, OCR was continuously monitored until it reached the maximum value. n = 4 per group; mean ± SEM. ( B ) Primary AECs were seeded in Seahorse XF-24 microplates and incubated with extracellular acidification rate (ECAR) media for 1 hour. Basal OCR and ECAR were then recorded before glucose (final concentration: 10 mM) was injected into the wells. After injection, OCR and ECAR were continuously monitored until they reached the maximum values. n = 3 per group; mean ± SEM. ( C ) Primary AECs purified from saline or intratracheal LPS–treated mice were seeded in Seahorse XF-24 microplates and incubated with substrate-limited media for 3 hours. Basal OCR was then recorded before palmitate-BSA was injected into the wells. After injection, OCR was continuously monitored until it reached the maximum value. OCR values were normalized to total protein content of the cellular extract from each well. n = 4 mice per group; mean ± SEM.

    Techniques Used: Mouse Assay, Incubation, Concentration Assay, Injection, Purification

    Alveolar epithelial line MLE-12 cells use fatty acids, and FAO is impaired in MLE-12 treated with BAL fluid (BALF) from ALI mice. ( A ) Alveolar epithelial line MLE-12 cells were seeded in Seahorse XF-24 microplates and incubated with substrate-limited media for 12 hours. Basal OCR was then recorded before BSA or palmitate-BSA was injected into the wells. After injection, OCR was continuously monitored until it reached the maximum value. n = 5 or 6 for the palmitate-BSA or BSA group; mean ± SEM. ( B ) MLE-12 cells were seeded in Seahorse XF-24 microplates and treated with saline or 100 μM etomoxir (ETO) for 1 hour. The media were then replaced with FAO assay media supplemented with 150 μM palmitate-BSA and cultured for 1 hour, followed by sequential treatments with 3 μg/ml oligomycin (Oligo), 6 μM FCCP, and 1 μM rotenone (Rot) and 0.5 μM antimycin A (Ant). Real-time OCR was recorded. n = 6 or 5 for the saline or ETO group; mean ± SEM. ( C ) MLE-12 cells were seeded in Seahorse XF-24 microplates and incubated with ECAR media for 1 hour. Basal OCR and ECAR were then recorded before glucose (final concentration: 10 mM) was injected into the wells. After injection, OCR and ECAR were continuously monitored until they reached the maximum values. n = 7; mean ± SEM. ( D ) MLE-12 cells were seeded in Seahorse XF-24 microplates and treated for 16 hours with BALF pooled from five mice instilled intratracheally with saline or LPS. The media were then replaced with FAO assay media supplemented with 150 μM palmitate-BSA and cultured for 1 hour, followed by sequential treatments with 3 μg/ml oligomycin, 6 μM FCCP, and 1 μM Rot and 0.5 μM Ant. Real-time OCR was recorded. n = 6 per group; mean ± SEM. ( E – F ) MLE-12 cells were incubated for 16 hours with BALFs from mice instilled intratracheally with saline or LPS for 24 hours. The mRNA ( E ) and/or protein ( F ) levels of indicated genes were determined by real-time PCR and Western blotting. ( E ) n = 3; mean ± SD; * P
    Figure Legend Snippet: Alveolar epithelial line MLE-12 cells use fatty acids, and FAO is impaired in MLE-12 treated with BAL fluid (BALF) from ALI mice. ( A ) Alveolar epithelial line MLE-12 cells were seeded in Seahorse XF-24 microplates and incubated with substrate-limited media for 12 hours. Basal OCR was then recorded before BSA or palmitate-BSA was injected into the wells. After injection, OCR was continuously monitored until it reached the maximum value. n = 5 or 6 for the palmitate-BSA or BSA group; mean ± SEM. ( B ) MLE-12 cells were seeded in Seahorse XF-24 microplates and treated with saline or 100 μM etomoxir (ETO) for 1 hour. The media were then replaced with FAO assay media supplemented with 150 μM palmitate-BSA and cultured for 1 hour, followed by sequential treatments with 3 μg/ml oligomycin (Oligo), 6 μM FCCP, and 1 μM rotenone (Rot) and 0.5 μM antimycin A (Ant). Real-time OCR was recorded. n = 6 or 5 for the saline or ETO group; mean ± SEM. ( C ) MLE-12 cells were seeded in Seahorse XF-24 microplates and incubated with ECAR media for 1 hour. Basal OCR and ECAR were then recorded before glucose (final concentration: 10 mM) was injected into the wells. After injection, OCR and ECAR were continuously monitored until they reached the maximum values. n = 7; mean ± SEM. ( D ) MLE-12 cells were seeded in Seahorse XF-24 microplates and treated for 16 hours with BALF pooled from five mice instilled intratracheally with saline or LPS. The media were then replaced with FAO assay media supplemented with 150 μM palmitate-BSA and cultured for 1 hour, followed by sequential treatments with 3 μg/ml oligomycin, 6 μM FCCP, and 1 μM Rot and 0.5 μM Ant. Real-time OCR was recorded. n = 6 per group; mean ± SEM. ( E – F ) MLE-12 cells were incubated for 16 hours with BALFs from mice instilled intratracheally with saline or LPS for 24 hours. The mRNA ( E ) and/or protein ( F ) levels of indicated genes were determined by real-time PCR and Western blotting. ( E ) n = 3; mean ± SD; * P

    Techniques Used: Mouse Assay, Incubation, Injection, Cell Culture, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot

    37) Product Images from "The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity [S]"

    Article Title: The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M900146-JLR200

    Protein markers of ER stress. WT ( Ncb5or +/+ ) or KO ( Ncb5or −/− ) primary hepatocytes were treated for 12 h with palmitic acid (0.5 mM) or fatty acid free BSA only. In addition WT hepatocytes were treated for 12 h with tunicamycin 1 μg/ml.
    Figure Legend Snippet: Protein markers of ER stress. WT ( Ncb5or +/+ ) or KO ( Ncb5or −/− ) primary hepatocytes were treated for 12 h with palmitic acid (0.5 mM) or fatty acid free BSA only. In addition WT hepatocytes were treated for 12 h with tunicamycin 1 μg/ml.

    Techniques Used:

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    Article Snippet: .. Cells were treated for 20 h with 5 mmol/L of AGE-modified BSA (AGE-BSA) (Sigma) 4 h after transfection. ..

    Mouse Assay:

    Article Title: Diagnosis of hepatocellular carcinoma using a novel anti-glycocholic acid monoclonal antibody-based method
    Article Snippet: .. As previously described , mice were subcutaneously immunized with 100 µg GCA-BSA emulsified with Freund's complete adjuvant (Sigma-Aldrich; Merck KGaA) with a volume ratio of 1:1. ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: An Essential Role for RGS Protein/Gαi2 Interactions in B Lymphocyte Directed Cell Migration and Trafficking
    Article Snippet: .. Briefly, 96 well ELISA plates (Nunc) were coated with TNP-BSA (Biosearch Technology) overnight at 4°C, washed and blocked with 1% BSA fraction V (Sigma-Aldrich), serum titers were then added to the plates and incubated 4h at 4°C. .. After washing alkaline phosphatase-labeled goat anti-mouse Ig isotype specific antibodies were added for 2h at RT (SouthernBiotech).

    Incubation:

    Article Title: Impaired Fc?RI stability, signaling, and effector functions in murine mast cells lacking glycosylphosphatidylinositol-anchored proteins
    Article Snippet: .. After stimulation with IgE anti-DNP (IGEL-A2) and DNP-BSA, BMMC were lysed by incubation for 30 minutes on ice in lysis buffer (20mM Tris-HCl, at pH 7.4 containing 0.5% Triton X-100, 150mM NaCl, 2mM EDTA, 50mM NaF, 1mM Na3 VO4 , 1mM PMSF, 10 μg aprotinin/mL, and 10 μg leupeptin/mL). ..

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    other:

    Article Title: Low-Temperature Storage Improves the Over-Time Stability of Implantable Glucose and Lactate Biosensors
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    Lysis:

    Article Title: Impaired Fc?RI stability, signaling, and effector functions in murine mast cells lacking glycosylphosphatidylinositol-anchored proteins
    Article Snippet: .. After stimulation with IgE anti-DNP (IGEL-A2) and DNP-BSA, BMMC were lysed by incubation for 30 minutes on ice in lysis buffer (20mM Tris-HCl, at pH 7.4 containing 0.5% Triton X-100, 150mM NaCl, 2mM EDTA, 50mM NaF, 1mM Na3 VO4 , 1mM PMSF, 10 μg aprotinin/mL, and 10 μg leupeptin/mL). ..

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    Millipore fatty acid free bsa
    PA-induced lipotoxicity and DHA neuroprotective action on primary rat cortical cells <t>(RCC).</t> A : Cellular morphology of control and treated <t>(PA/BSA,</t> 2:1, 48 hr) RCC. B : Nuclear morphology of control and treated RCC. Arrows indicate fragmented nuclei. C : Viability of RCC treated for 48 hr with PA/BSA (2:1 ratio; PA) or in cotreatment with DHA (PA + DHA). Viability was determined using the WST-1 assay. Data represent mean ± SEM of three independent experiments. ★ P
    Fatty Acid Free Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fatty acid free bsa/product/Millipore
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    Millipore elisa grade bsa
    Anti-glycan IgG in the sera of lambs vaccinated with Haemonchus contortus excretory/secretory (ES) products analyzed by <t>ELISA.</t> The amount of IgG against Galα1-3GalNAc-polyacrylamide (PAA), <t>GalNAcβ1-4(Fucα1-3)GlcNAc-BSA</t> (LDNF) and
    Elisa Grade Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of OF and CM on the mRNA expressions of lipogenesis-related genes. Real-time RT-PCR analysis of sterol regulatory element-binding protein-1 ( SREBP-1c ) (a) and glycerol-3-phosphate acyltransferase ( GPAT ) (b) mRNA levels in 1 mM <t>FFA/BSA-treated</t> HepG2 cells. All data were normalized to GAPDH mRNA, and the fold changes in expression were calculated relative to control cells (treated with 1% BSA). Each experiment was independently performed three times. Data were presented as the mean ± SD. Values not sharing common superscripts are significantly different ( p
    Ffa Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PA-induced lipotoxicity and DHA neuroprotective action on primary rat cortical cells (RCC). A : Cellular morphology of control and treated (PA/BSA, 2:1, 48 hr) RCC. B : Nuclear morphology of control and treated RCC. Arrows indicate fragmented nuclei. C : Viability of RCC treated for 48 hr with PA/BSA (2:1 ratio; PA) or in cotreatment with DHA (PA + DHA). Viability was determined using the WST-1 assay. Data represent mean ± SEM of three independent experiments. ★ P

    Journal: Journal of neuroscience research

    Article Title: Activation and Reversal of Lipotoxicity in PC12 and Rat Cortical Cells Following Exposure to Palmitic Acid

    doi: 10.1002/jnr.21918

    Figure Lengend Snippet: PA-induced lipotoxicity and DHA neuroprotective action on primary rat cortical cells (RCC). A : Cellular morphology of control and treated (PA/BSA, 2:1, 48 hr) RCC. B : Nuclear morphology of control and treated RCC. Arrows indicate fragmented nuclei. C : Viability of RCC treated for 48 hr with PA/BSA (2:1 ratio; PA) or in cotreatment with DHA (PA + DHA). Viability was determined using the WST-1 assay. Data represent mean ± SEM of three independent experiments. ★ P

    Article Snippet: PA and/or DHA (Sigma, St. Louis, MO) was first dissolved/diluted in 100% ethanol and further diluted in warm low-serum medium for NGFDPC12 cells, or serum-free medium for RCC, with fatty acid-free BSA (EMD Biosciences, La Jolla, CA).

    Techniques: WST-1 Assay

    Palmitic acid-induced lipotoxicity in NGFDPC12 cells. A : NGFDPC12 cells were exposed to PA/BSA (2:1 ratio), and viability was determined using WST-1 assay at the indicated times. B : NGFDPC12 cells were exposed to different PA/BSA ratios (0.25:1, 0.5:1, 1:1, and 2:1 with BSA at a concentration of 0.150 mM) for 24 hr. Cell viability was measured by the WST-1 assay. C : NGFDPC12 cells were exposed to 0.150 mM BSA without PA (control) or PA/BSA (2:1; PA) for 12 hr. Cells were stained with Hoechst (10 ng/liter), and nuclei were visualized under fluorescent microscopy. Arrows indicate nuclei showing chromatin condensation and fragmentation. D : Regulation of apoptosis-asociated genes in NGFDPC12 cultures exposed to PA/BSA (2:1). Quantitative RT-PCR experiments show the time-dependent mRNA expression of Bim, Bad, AIF, BNIP-3, 14.3.3, and FAS-R. GAPDH mRNA expression was used as the internal control. Data represent mean 6 SEM of three independent experiments. ★ P

    Journal: Journal of neuroscience research

    Article Title: Activation and Reversal of Lipotoxicity in PC12 and Rat Cortical Cells Following Exposure to Palmitic Acid

    doi: 10.1002/jnr.21918

    Figure Lengend Snippet: Palmitic acid-induced lipotoxicity in NGFDPC12 cells. A : NGFDPC12 cells were exposed to PA/BSA (2:1 ratio), and viability was determined using WST-1 assay at the indicated times. B : NGFDPC12 cells were exposed to different PA/BSA ratios (0.25:1, 0.5:1, 1:1, and 2:1 with BSA at a concentration of 0.150 mM) for 24 hr. Cell viability was measured by the WST-1 assay. C : NGFDPC12 cells were exposed to 0.150 mM BSA without PA (control) or PA/BSA (2:1; PA) for 12 hr. Cells were stained with Hoechst (10 ng/liter), and nuclei were visualized under fluorescent microscopy. Arrows indicate nuclei showing chromatin condensation and fragmentation. D : Regulation of apoptosis-asociated genes in NGFDPC12 cultures exposed to PA/BSA (2:1). Quantitative RT-PCR experiments show the time-dependent mRNA expression of Bim, Bad, AIF, BNIP-3, 14.3.3, and FAS-R. GAPDH mRNA expression was used as the internal control. Data represent mean 6 SEM of three independent experiments. ★ P

    Article Snippet: PA and/or DHA (Sigma, St. Louis, MO) was first dissolved/diluted in 100% ethanol and further diluted in warm low-serum medium for NGFDPC12 cells, or serum-free medium for RCC, with fatty acid-free BSA (EMD Biosciences, La Jolla, CA).

    Techniques: WST-1 Assay, Concentration Assay, Staining, Microscopy, Quantitative RT-PCR, Expressing

    DNA fragmentation and caspase-3 activity of NGFDPC12 cells exposed to PA. A : Representative experimental results of BRDU-FITC plots (apoptotic cells) vs. PI (total DNA marker) for controls and cells exposed to PA/BSA (2:1) for 12 hr. The R2 quadrant shows cells exhibiting increased BrdU-FITC fluorescence (green). Nonapoptotic cells are indicated in red. B : Representative distribution diagram of positive apoptotic NGFDPC12 cells. Cells were treated with BSA for negative control (green), PA/BSA (2:1) for 12 hr (red) or 24 hr (blue), or staurosporine for positive control (orange). C : Quantitative analysis of positive apoptotic NGFDPC12 cells as shown in B. The data represent the mean ± SEM of three independent experiments done in triplicate. D : Caspase-3 activity in cell extracts was determined in control or NGFDPC12 cells treated with PA/BSA for 12 hr. E : Early posttreatment with BSA protects NGFDPC12 cells from PA-induced lipotoxicity. Additional BSA (0.6 mM final concentration) was added to NGFDPC-12 cells at 2 or 6 hr after initial PA/BSA (2:1 ratio) treatment. Percentage of viable cells was measured by the WST-1 assay at the end of a 24-hr incubation period. Nuclei in bottom panel were visualized with Hoechst staining. Data represent mean ± SEM of three independent experiments. ★ P

    Journal: Journal of neuroscience research

    Article Title: Activation and Reversal of Lipotoxicity in PC12 and Rat Cortical Cells Following Exposure to Palmitic Acid

    doi: 10.1002/jnr.21918

    Figure Lengend Snippet: DNA fragmentation and caspase-3 activity of NGFDPC12 cells exposed to PA. A : Representative experimental results of BRDU-FITC plots (apoptotic cells) vs. PI (total DNA marker) for controls and cells exposed to PA/BSA (2:1) for 12 hr. The R2 quadrant shows cells exhibiting increased BrdU-FITC fluorescence (green). Nonapoptotic cells are indicated in red. B : Representative distribution diagram of positive apoptotic NGFDPC12 cells. Cells were treated with BSA for negative control (green), PA/BSA (2:1) for 12 hr (red) or 24 hr (blue), or staurosporine for positive control (orange). C : Quantitative analysis of positive apoptotic NGFDPC12 cells as shown in B. The data represent the mean ± SEM of three independent experiments done in triplicate. D : Caspase-3 activity in cell extracts was determined in control or NGFDPC12 cells treated with PA/BSA for 12 hr. E : Early posttreatment with BSA protects NGFDPC12 cells from PA-induced lipotoxicity. Additional BSA (0.6 mM final concentration) was added to NGFDPC-12 cells at 2 or 6 hr after initial PA/BSA (2:1 ratio) treatment. Percentage of viable cells was measured by the WST-1 assay at the end of a 24-hr incubation period. Nuclei in bottom panel were visualized with Hoechst staining. Data represent mean ± SEM of three independent experiments. ★ P

    Article Snippet: PA and/or DHA (Sigma, St. Louis, MO) was first dissolved/diluted in 100% ethanol and further diluted in warm low-serum medium for NGFDPC12 cells, or serum-free medium for RCC, with fatty acid-free BSA (EMD Biosciences, La Jolla, CA).

    Techniques: Activity Assay, Marker, Fluorescence, Negative Control, Positive Control, Concentration Assay, WST-1 Assay, Incubation, Staining

    ROS generation and changes in mitochondrial membrane permeability in NGFDPC12 cells undergoing PA-induced lipotoxicity. A : ROS generation in NGFDPC12 cells was detected by the 2′,7′ -dichlorofluorescein diacetate (DCF) method at 12 hr after the initial exposure to PA using flow cytometry. NGFDPC12 cells were treated with no DCF (light green), DCF (dark green), PA + DCF (blue), or hydrogen peroxide + DCF (red). B : Quantification of ROS production in NGFDPC12 cells at 3, 6, 9, and 12 hr after PA/BSA treatment. C : ROS modulation after addition of DHA, MCI-186, or BSA to NCGDPC12 cells treated with PA for 6 hr. D: Mitochondrial depolarization in NGFDPC12 cells, control or PA/BSA treated, detected by JC-1 flow cytometric analysis. JC-1 was detected in the FL1 or FL2 channel depending on its aggregation status (see Mqaterials and Methods). The R2 quadrant defines cells with leaky mitochondria showing reduced FL2 readings (green). Cells with intact mitochondria are indicated in R1 (red). Data represent mean ± SEM sent of at least three independent experiments. ★ P

    Journal: Journal of neuroscience research

    Article Title: Activation and Reversal of Lipotoxicity in PC12 and Rat Cortical Cells Following Exposure to Palmitic Acid

    doi: 10.1002/jnr.21918

    Figure Lengend Snippet: ROS generation and changes in mitochondrial membrane permeability in NGFDPC12 cells undergoing PA-induced lipotoxicity. A : ROS generation in NGFDPC12 cells was detected by the 2′,7′ -dichlorofluorescein diacetate (DCF) method at 12 hr after the initial exposure to PA using flow cytometry. NGFDPC12 cells were treated with no DCF (light green), DCF (dark green), PA + DCF (blue), or hydrogen peroxide + DCF (red). B : Quantification of ROS production in NGFDPC12 cells at 3, 6, 9, and 12 hr after PA/BSA treatment. C : ROS modulation after addition of DHA, MCI-186, or BSA to NCGDPC12 cells treated with PA for 6 hr. D: Mitochondrial depolarization in NGFDPC12 cells, control or PA/BSA treated, detected by JC-1 flow cytometric analysis. JC-1 was detected in the FL1 or FL2 channel depending on its aggregation status (see Mqaterials and Methods). The R2 quadrant defines cells with leaky mitochondria showing reduced FL2 readings (green). Cells with intact mitochondria are indicated in R1 (red). Data represent mean ± SEM sent of at least three independent experiments. ★ P

    Article Snippet: PA and/or DHA (Sigma, St. Louis, MO) was first dissolved/diluted in 100% ethanol and further diluted in warm low-serum medium for NGFDPC12 cells, or serum-free medium for RCC, with fatty acid-free BSA (EMD Biosciences, La Jolla, CA).

    Techniques: Permeability, Flow Cytometry, Cytometry

    Inhibition of fatty acid-induced lipotoxicity by DHA. A : DHA inhibits induction of cell death by PA in NGFDPC12 cells. Cell viability was determined after treatment for 24 hr as follows: 0.150 mM BSA alone (control), PA/BSA 2:1 ratio (PA), PA/BSA 2:1 cotreated with DHA followed by an increase in BSA final concentration to 0.6 mM at 6 hr (PA + DHA + BSA at 6 hr), and DHA pretreatment for 12 hr followed by treatment with PA/BSA 2:1 (DHA 12 hr pre + PA). B : DHA treatment inhibits apoptotic features of NGFDPC12 cells treated with PA/BSA. Morphology of NGFDPC12 cells treated with BSA alone (control), PA/BSA 2:1 cotreated with DHA, or PA/BSA 2:1 for 24 hr. C : Nuclear morphology of NGFDPC12 cells treated with BSA alone (control), PA/BSA 2:1 cotreated with DHA or PA/BSA 2:1 for 24 hr. Arrows indicate nuclei exhibiting fragmentation. D : DHA cotreatment produced a significant inhibitory effect of caspase-3 activity in cellular extracts of NGFDPC12 cells exposed to PA/BSA (2:1). E : Cleavage of PARP and lamin B in NGFDPC-12 cells exposed to PA/BSA (2:1), as assessed by Western blotting. PA treatment induced the appearance of the signature apoptotic fragments of PARP (85 kDa) or lamin B (46 kDa). DHA inhibited PARP and lamin B cleavage. Data in A and D represent mean ± SEM of three independent experiments. ★ P

    Journal: Journal of neuroscience research

    Article Title: Activation and Reversal of Lipotoxicity in PC12 and Rat Cortical Cells Following Exposure to Palmitic Acid

    doi: 10.1002/jnr.21918

    Figure Lengend Snippet: Inhibition of fatty acid-induced lipotoxicity by DHA. A : DHA inhibits induction of cell death by PA in NGFDPC12 cells. Cell viability was determined after treatment for 24 hr as follows: 0.150 mM BSA alone (control), PA/BSA 2:1 ratio (PA), PA/BSA 2:1 cotreated with DHA followed by an increase in BSA final concentration to 0.6 mM at 6 hr (PA + DHA + BSA at 6 hr), and DHA pretreatment for 12 hr followed by treatment with PA/BSA 2:1 (DHA 12 hr pre + PA). B : DHA treatment inhibits apoptotic features of NGFDPC12 cells treated with PA/BSA. Morphology of NGFDPC12 cells treated with BSA alone (control), PA/BSA 2:1 cotreated with DHA, or PA/BSA 2:1 for 24 hr. C : Nuclear morphology of NGFDPC12 cells treated with BSA alone (control), PA/BSA 2:1 cotreated with DHA or PA/BSA 2:1 for 24 hr. Arrows indicate nuclei exhibiting fragmentation. D : DHA cotreatment produced a significant inhibitory effect of caspase-3 activity in cellular extracts of NGFDPC12 cells exposed to PA/BSA (2:1). E : Cleavage of PARP and lamin B in NGFDPC-12 cells exposed to PA/BSA (2:1), as assessed by Western blotting. PA treatment induced the appearance of the signature apoptotic fragments of PARP (85 kDa) or lamin B (46 kDa). DHA inhibited PARP and lamin B cleavage. Data in A and D represent mean ± SEM of three independent experiments. ★ P

    Article Snippet: PA and/or DHA (Sigma, St. Louis, MO) was first dissolved/diluted in 100% ethanol and further diluted in warm low-serum medium for NGFDPC12 cells, or serum-free medium for RCC, with fatty acid-free BSA (EMD Biosciences, La Jolla, CA).

    Techniques: Inhibition, Concentration Assay, Produced, Activity Assay, Western Blot

    DHA reduces mitochondrial depolarization of NGFDPC12 cells undergoing PA-induced lipotoxicity. A : NGFDPC12 cells were treated with PA/BSA alone (PA) or in the presence DHA (PA + DHA) for 9 or 12 hr, and mitochondrial depolarization was determined by JC-1 flow cytometry assays. Representative flow cytometric plots are shown. B : Quantification of mitochondria depolarization (R2 in JC-1 flow cytometry) in NGFDPC12 cells undergoing PA-induced lipotoxicity. Data represent mean ± SEM of three independent experiments. ★ P

    Journal: Journal of neuroscience research

    Article Title: Activation and Reversal of Lipotoxicity in PC12 and Rat Cortical Cells Following Exposure to Palmitic Acid

    doi: 10.1002/jnr.21918

    Figure Lengend Snippet: DHA reduces mitochondrial depolarization of NGFDPC12 cells undergoing PA-induced lipotoxicity. A : NGFDPC12 cells were treated with PA/BSA alone (PA) or in the presence DHA (PA + DHA) for 9 or 12 hr, and mitochondrial depolarization was determined by JC-1 flow cytometry assays. Representative flow cytometric plots are shown. B : Quantification of mitochondria depolarization (R2 in JC-1 flow cytometry) in NGFDPC12 cells undergoing PA-induced lipotoxicity. Data represent mean ± SEM of three independent experiments. ★ P

    Article Snippet: PA and/or DHA (Sigma, St. Louis, MO) was first dissolved/diluted in 100% ethanol and further diluted in warm low-serum medium for NGFDPC12 cells, or serum-free medium for RCC, with fatty acid-free BSA (EMD Biosciences, La Jolla, CA).

    Techniques: Flow Cytometry, Cytometry

    Analysis of de novo sphingolipid biosynthesis in RAW264.7 cells by [ 13 C]palmitate labeling. RAW264.7 cells were incubated with 0.1 m m [U- 13 C]palmitic acid (as the BSA complex) with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μ m ), or KLA+ ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA for the times shown. The appearance of 13 C in newly synthesized sphingolipids was quantified by mass spectrometry as described under supplemental “Materials and Methods” . A , summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer subspecies. Data represent the means ± S.E. ( n = 3). B , summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species in cells treated with control ( left ) or KLA ( right ), Data represent the means ( n = 3). Because of the difficulties in visualization, the quantities of ceramide subspecies in control treated cells at the 24-h time point are as follows: C16 (7.6 pmol/μg DNA); C18 (0.7 pmol/μg DNA); C20 (0.3 pmol/μg DNA); C22 (3.2 pmol/μg DNA); C24:1 (6 pmol/μg DNA); C24 (6.4 pmol/μg DNA); C26:1 (0.2 pmol/μg DNA); C26 (0.1 pmol/μg DNA). C , summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species ( left ) and 12 C (unlabeled) Cer species ( right ) in cells treated with KLA + ISP1. Data represent the means ( n = 3).

    Journal: The Journal of Biological Chemistry

    Article Title: Kdo2-Lipid A, a TLR4-specific Agonist, Induces de Novo Sphingolipid Biosynthesis in RAW264.7 Macrophages, Which Is Essential for Induction of Autophagy *

    doi: 10.1074/jbc.M110.170621

    Figure Lengend Snippet: Analysis of de novo sphingolipid biosynthesis in RAW264.7 cells by [ 13 C]palmitate labeling. RAW264.7 cells were incubated with 0.1 m m [U- 13 C]palmitic acid (as the BSA complex) with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μ m ), or KLA+ ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA for the times shown. The appearance of 13 C in newly synthesized sphingolipids was quantified by mass spectrometry as described under supplemental “Materials and Methods” . A , summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer subspecies. Data represent the means ± S.E. ( n = 3). B , summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species in cells treated with control ( left ) or KLA ( right ), Data represent the means ( n = 3). Because of the difficulties in visualization, the quantities of ceramide subspecies in control treated cells at the 24-h time point are as follows: C16 (7.6 pmol/μg DNA); C18 (0.7 pmol/μg DNA); C20 (0.3 pmol/μg DNA); C22 (3.2 pmol/μg DNA); C24:1 (6 pmol/μg DNA); C24 (6.4 pmol/μg DNA); C26:1 (0.2 pmol/μg DNA); C26 (0.1 pmol/μg DNA). C , summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species ( left ) and 12 C (unlabeled) Cer species ( right ) in cells treated with KLA + ISP1. Data represent the means ( n = 3).

    Article Snippet: Materials The suppliers for the reagents were as follows: Kdo2 -lipid A and the internal standard mixture for SL analysis by LC ESI-MS/MS (ceramide/sphingoid internal standard mixture II, LM-6005) (Avanti Polar Lipids, Alabaster, AL); [U-13 C]palmitic acid (98%) (Cambridge Isotopes, Andover, MA); fatty acid-free BSA (Calbiochem) ; Hoechst 33342 (Invitrogen); ISP1 (Biomol, Plymouth Meeting, PA); mouse monoclonal antibody to SPT1 (anti-LCB1), anti-BiP/GRP78, and anti-GM130 (BD Biosciences); rabbit monoclonal anti-HA tag and rabbit polyclonal LC3B antibody (Cell Signaling Technology, Boston); mouse monoclonal anti-Cer antibody clone 15B4 (Alexis Biochemicals, San Diego) This antibody clone has been successfully used in immunocytochemistry applications with no indication of nonspecific reactions with other lipids ( ) The secondary Alexa Fluor-conjugated F(ab)2 fragment goat anti-mouse antibody and goat anti-rabbit antibody were from Molecular Probes, Inc. (Eugene, OR.).

    Techniques: Labeling, Incubation, Synthesized, Mass Spectrometry

    Anti-glycan IgG in the sera of lambs vaccinated with Haemonchus contortus excretory/secretory (ES) products analyzed by ELISA. The amount of IgG against Galα1-3GalNAc-polyacrylamide (PAA), GalNAcβ1-4(Fucα1-3)GlcNAc-BSA (LDNF) and

    Journal: International journal for parasitology

    Article Title: Vaccination-induced IgG response to Gal?1-3GalNAc glycan epitopes in lambs protected against Haemonchus contortus challenge infection

    doi: 10.1016/j.ijpara.2009.07.009

    Figure Lengend Snippet: Anti-glycan IgG in the sera of lambs vaccinated with Haemonchus contortus excretory/secretory (ES) products analyzed by ELISA. The amount of IgG against Galα1-3GalNAc-polyacrylamide (PAA), GalNAcβ1-4(Fucα1-3)GlcNAc-BSA (LDNF) and

    Article Snippet: After blocking (60 min 37°C) with 1% ELISA-grade BSA (Fraction V, fatty acid free; Calbiochem, San Diego, USA) in TSM (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM CaCl2 , 2 mM MgCl2 ) and washing with TSM containing 0.1% Tween-20, glycan-specific antibodies or biotinylated GSI-B4 were added for 60 min at 37°C.

    Techniques: Enzyme-linked Immunosorbent Assay

    Effects of OF and CM on the mRNA expressions of lipogenesis-related genes. Real-time RT-PCR analysis of sterol regulatory element-binding protein-1 ( SREBP-1c ) (a) and glycerol-3-phosphate acyltransferase ( GPAT ) (b) mRNA levels in 1 mM FFA/BSA-treated HepG2 cells. All data were normalized to GAPDH mRNA, and the fold changes in expression were calculated relative to control cells (treated with 1% BSA). Each experiment was independently performed three times. Data were presented as the mean ± SD. Values not sharing common superscripts are significantly different ( p

    Journal: Mediators of Inflammation

    Article Title: Antilipotoxicity Activity of Osmanthus fragrans and Chrysanthemum morifolium Flower Extracts in Hepatocytes and Renal Glomerular Mesangial Cells

    doi: 10.1155/2017/4856095

    Figure Lengend Snippet: Effects of OF and CM on the mRNA expressions of lipogenesis-related genes. Real-time RT-PCR analysis of sterol regulatory element-binding protein-1 ( SREBP-1c ) (a) and glycerol-3-phosphate acyltransferase ( GPAT ) (b) mRNA levels in 1 mM FFA/BSA-treated HepG2 cells. All data were normalized to GAPDH mRNA, and the fold changes in expression were calculated relative to control cells (treated with 1% BSA). Each experiment was independently performed three times. Data were presented as the mean ± SD. Values not sharing common superscripts are significantly different ( p

    Article Snippet: The FFA/BSA or oleic acid (OA)/BSA complex solution was sterile-filtered through 0.22 μ m sterile filters (Millipore S.A.S., Molsheim, France) and then stored at −20°C until use.

    Techniques: Quantitative RT-PCR, Binding Assay, Expressing

    Effect of OF and CM on FFA-induced ROS production. HepG2 cells were incubated with 1 mM FFAs/BSA for 24 h in the presence of OF or CM. Vehicle cells were incubated with 0.1% DMSO in the presence of FFAs/BSA. Control cells were incubated with 1% BSA. Intracellular ROS production was quantified using the fluorescent probe DCFDA. Data were presented as mean ± SD of three independent experiments. Values not sharing common superscripts are significantly different ( p

    Journal: Mediators of Inflammation

    Article Title: Antilipotoxicity Activity of Osmanthus fragrans and Chrysanthemum morifolium Flower Extracts in Hepatocytes and Renal Glomerular Mesangial Cells

    doi: 10.1155/2017/4856095

    Figure Lengend Snippet: Effect of OF and CM on FFA-induced ROS production. HepG2 cells were incubated with 1 mM FFAs/BSA for 24 h in the presence of OF or CM. Vehicle cells were incubated with 0.1% DMSO in the presence of FFAs/BSA. Control cells were incubated with 1% BSA. Intracellular ROS production was quantified using the fluorescent probe DCFDA. Data were presented as mean ± SD of three independent experiments. Values not sharing common superscripts are significantly different ( p

    Article Snippet: The FFA/BSA or oleic acid (OA)/BSA complex solution was sterile-filtered through 0.22 μ m sterile filters (Millipore S.A.S., Molsheim, France) and then stored at −20°C until use.

    Techniques: Incubation

    Effects of OF and CM on lipid accumulation in free fatty acid-overloaded HepG2 cells. HepG2 cells were incubated with 1 mM FFAs/BSA and cotreated with various concentrations of OF and CM for 24 h. Vehicle cells were incubated with 0.1% DMSO in the presence of FFAs/BSA. Control cells were incubated with 1% BSA. Cell viability was measured by the MTT assay (a). Quantitative analysis of lipid deposition in cells by the OD 500 nm values using Oil Red O staining (b). Intracellular triglyceride (c) and cholesterol (d) contents were determined in cell lysates by an enzymatic colorimetric method using a commercially available kit. Total cholesterol and TG levels of the control cells were 25.3 ± 7.1 and 28.7 ± 2.7 μ g/mg of cellular protein, respectively. Data were presented as mean ± SD of three independent experiments. Values not sharing common superscripts are significantly different ( p

    Journal: Mediators of Inflammation

    Article Title: Antilipotoxicity Activity of Osmanthus fragrans and Chrysanthemum morifolium Flower Extracts in Hepatocytes and Renal Glomerular Mesangial Cells

    doi: 10.1155/2017/4856095

    Figure Lengend Snippet: Effects of OF and CM on lipid accumulation in free fatty acid-overloaded HepG2 cells. HepG2 cells were incubated with 1 mM FFAs/BSA and cotreated with various concentrations of OF and CM for 24 h. Vehicle cells were incubated with 0.1% DMSO in the presence of FFAs/BSA. Control cells were incubated with 1% BSA. Cell viability was measured by the MTT assay (a). Quantitative analysis of lipid deposition in cells by the OD 500 nm values using Oil Red O staining (b). Intracellular triglyceride (c) and cholesterol (d) contents were determined in cell lysates by an enzymatic colorimetric method using a commercially available kit. Total cholesterol and TG levels of the control cells were 25.3 ± 7.1 and 28.7 ± 2.7 μ g/mg of cellular protein, respectively. Data were presented as mean ± SD of three independent experiments. Values not sharing common superscripts are significantly different ( p

    Article Snippet: The FFA/BSA or oleic acid (OA)/BSA complex solution was sterile-filtered through 0.22 μ m sterile filters (Millipore S.A.S., Molsheim, France) and then stored at −20°C until use.

    Techniques: Incubation, MTT Assay, Staining