Journal: International Journal of Molecular Sciences

Article Title: Acute Increases in Intracellular Zinc Lead to an Increased Lysosomal and Mitochondrial Autophagy and Subsequent Cell Demise in Malignant Melanoma

doi: 10.3390/ijms22020667

Figure Lengend Snippet: Mitochondrial membrane potential (Δψm) changes, ATP production and mitophagy in explant human melanoma cultures with lower free zinc stores (M5), average free zinc stores (M10) and higher free zinc stores (M9) exposed to 0.5 μM zinc pyrithione during 72 h. Cells were exposed to external zinc pyrithione and ( A ) loss of Δψm measured by decreased red fluorescence of JC-1 was determined in at least 1000 cells visualized by fluorescence microscopy. Results represent means ± SD of at least three independent experiments. # p < 0.05 significantly lower compared to the beginning of treatment with one-way ANOVA test and Dunnett’s post-test for multiple comparisons. ( B ) ATP production was measured in cell lysates by ATP bioluminescent assay kit ( C ). Mitophagy-specific fluorescence (Mitophagy Detection Kit) in cells exposed to zinc pyrithione alone or together with autophagy inhibitor chloroquine was determined fluorimetrically. Results represent means ± SD of at least three independent experiments. * p < 0.05 significantly higher compared to the beginning of treatment, # p < 0.05 significantly lower compared to the beginning of treatment with one-way ANOVA test and Dunnett’s post-test for multiple comparisons.

Article Snippet: Control and zinc pyrithione-treated cells of explant human melanoma grown in 96-well plates with black bottom were washed with PBS and incubated in 100 nM Mitophagy Dye Solution (Mitophagy Detection Kit (Dojindo Laboratories, Kumamoto, Japan)) for 35 min. After removal of the culture medium and washing with PBS, mitophagy-specific fluorescence was evaluated using a cell scoring module of MetaXpress ® Image Acquisition and Analysis Software.

Techniques: Membrane, Fluorescence, Microscopy, ATP Bioluminescent Assay

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: ATDC5 cells adhere more extensively to fibronectin, collagen I, and collagen IV. ATDC5 cells were screened with extracellular matrix array printed with collagen I (COL I), collagen III (COL III), collagen IV (COL IV), collagen V (COL V), collagen VI (COL VI), fibronectin (FN), vitronectin (VTN), laminin (LMN), tropoelastin (TE), and BSA as a negative control. (A) Representative bright-field images of ATDC5 cells incubated for 30 h indicated differential binding of a number of extracellular proteins. Scale bar: 40 μm. (B) Attached cell counts determined for each of the nine replicates, as well as mean and standard deviation are shown ( n = 9).

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Negative Control, Incubation, Binding Assay, Standard Deviation

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Fibronectin interaction with graphene is stabilized by arginine residues. (A) Graphical rendering of the stabilized fibronectin atop the three graphene sheets with the four best arginine binders highlighted (Arg1166, Arg1369, Arg1374, Arg1403). The time evolution of the binding energy of these arginine residues with graphene is shown in the lower panel, color-coded for the amino acid residues. (B) Analogous to A but showing the data for the second studied configuration. This configuration features five arginine residue binders (Arg1166, Arg1351, Arg1379, Arg1445, Arg1493). (C) Binding energy with graphene computed for every amino acid with average binding energy above 1 kcal/mol, averaged over the 400 ns simulation. (D) Analogous to C, for the second studied configuration. The residue numbers are indicated, while the corresponding amino acid types are color-coded for both panels (C and D). (E and F) Time evolution of the fibronectin and arginine interaction energy with graphene for the two configurations. The lower plots in both panels show the fraction of arginine residue binding energy with respect to the total fibronectin-binding energy as a function of simulation time.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Binding Assay, Residue

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Mechanical properties. The measured quasi-static (A and B) and dynamic (C – E) properties of GF (hatched bars), GF coated in fibronectin (dark blue bars), and GF coated in fibronectin and cultured with ATDC5 cells (light blue bars) for 28 days. Fibronectin changed the elasticity of the composite (i.e., modulus values), but did not increase the viscoelastic properties (stress relaxation and phase shift).

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Cell Culture

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Actin cytoskeleton of cells on GF and fibronectin-coated GF. Fluorescence of ATDC5 cells grown on glass-bottom tissue culture wells compared to GF, with or without fibronectin. Cell nuclei are stained blue (DAPI); Green, F-actin (Alexa Fluor 488 phalloidin); (A–D) ATDC5 cells were grown on glass-bottom tissue culture wells without (A and E) and with fibronectin (B and F); ATDC5 cells were grown on GF without (C and G) and with fibronectin (D and H). Note the prevalence of stress fibers and the absence of puncta in F and H compared to E and G, respectively. Additionally, note the relative abundance of puncta of actin which are more prevalent in the absence of fibronectin on glass-bottomed tissue culture wells as well as on GF. (A–D) Scale-bar: 50 μm. (E–H) Scale-bar: 10 μm.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Fluorescence, Staining

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: ActB and Hsp90ab1 housekeeping genes. ActB and Hsp90ab1 are stably expressed by ATDC5 cells under all experimental conditions used in this study (i.e., on glass-bottom tissue culture wells, GF, and fibronectin-GF). (A) ActB and Hsp90ab1 cycle threshold levels were most consistent among all samples analyzed by qRT-PCR for candidate HKGs considered, based on pairwise analysis of variance for differences between threshold values, variance equal to 0.12. (B) Correlation analysis of cycle threshold values for Hsp90ab1 and ActB indicate a slope and an R 2 value close to 1. ( n = 15).

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Stable Transfection, Quantitative RT-PCR

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: GF supports or enhances gene expression levels. The effect of fibronectin, GF, and fibronectin in combination with GF on ATDC5 cell gene expression was investigated. Correlation analysis of relative expression levels was carried out to detect differential gene expression as a function of the cell culture substrate. The mRNA levels were compared for cells seeded on four distinct surfaces. Data points above the diagonal line indicate genes that are upregulated and data points below the diagonal line indicate genes that are downregulated. Data points falling on the diagonal line are not differentially expressed in experimental compared to control conditions. The effect of GF on gene expression is demonstrated in panels A and B. The effect of fibronectin on gene expression is demonstrated in panels C and D. (A) Relative gene expression levels in 2D cell culture conditions compared to cells grown in 3D on GF in the absence of fibronectin. (B) Relative gene expression levels in 2D cell culture conditions compared to cells grown in 3D on GF in the presence of fibronectin. (C) Relative gene expression levels in 2D cell culture conditions comparing the presence and absence of fibronectin. (D) Relative gene expression levels by cells grown in 3D on GF comparing the presence and absence of fibronectin. Genes for which expression levels met or exceeded the control are indicated in magenta, while those genes that were supported by substrate conditions are indicated by turquoise. Col2a1, a marker for chondrocyte differentiation, is shown as a diamond shape and bolded in each frame. Col2a1 is found above the diagonal line in A and B indicating upregulation as a function of 3D GF culture, and below the line in C and D, indicating downregulation as a function of fibronectin in either 2D or 3D culture. Genes included in this analysis are listed in Tables – .

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Gene Expression, Expressing, Cell Culture, Control, Marker

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Expression of genes encoding mediators of cell attachment by ATDC5 cells on glass-bottom tissue culture wells, GF, and fibronectin-GF. (A) Time course of gene expression during chondrogenic differentiation for Ctnnal (triangle) and Ctnnb1 (circle). (B) Relative gene expression levels of Ctnnal (gray) and Ctnnb1 (black) at day 17 of chondrogenic differentiation in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (C) Time course of gene expression during chondrogenic differentiation for Cd44 (triangle), Ncam1 (circle), and Sgce (square). (D) Relative gene expression levels of Cd44 (gray), Ncam1 (black), and Sgce (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (E) Time course of gene expression during chondrogenic differentiation for Itga3 (triangle), Itga5 (circle), and Itgav (square). (F) Relative gene expression levels of Itga3 (gray), Itga5 (black ) , and Itgav (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (G) Time course of gene expression during chondrogenic differentiation for Itgb1 . (H) Relative gene expression levels of Itgb1 at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. Error bars = Mean ± SD. These genes are listed in Table with references from current literature indicating an association with chondrocyte differentiation.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Expressing, Cell Attachment Assay, Gene Expression, Control

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Expression of genes encoding extracellular matrix proteins by ATDC5 cells on glass-bottom tissue culture wells, GF, and fibronectin-GF. (A) Time course of gene expression during chondrogenic differentiation for Col1a1 (circle) and Col3a1 (triangle). (B) Relative gene expression levels of Col1a1 (gray) and Col3a1 (black) at day 17 of chondrogenic differentiation in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (C) Time course of gene expression during chondrogenic differentiation for Col2a1 (circle), Col5a1 (triangle), and Col6a1 (square). (D) Relative gene expression levels of Col2a1 (gray), Col5a1 (black), and Col6a1 (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (E) Time course of gene expression during chondrogenic differentiation for Ecm1 (circle), Emilin1 (triangle), and Tnc (square). (F) Relative gene expression levels of Ecm1 (gray), Emilin1 (black), and Tnc (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (G) Time course of gene expression during chondrogenic differentiation for Fn (circle), Sparc (triangle), and Spp1 (square). (H) Relative gene expression levels of Fn (gray), Sparc (black), and Spp1 (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (I) Time course of gene expression during chondrogenic differentiation for Thbs1 (circle), Thbs2 (triangle), and Postn (square). (J) Relative gene expression levels of Thbs1 (black), Thbs2 (white), and Postn (gray) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (K) Time course of gene expression during chondrogenic differentiation for Hapln1 (circle) and Lamb3 (triangle). (L) Relative gene expression levels of Hapln1 (gray) and Lamb3 (black) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. Error bars = Mean ± SD Table lists extracellular matrix genes with description, function, and literature citations that corroborate an upregulation during early chondrogenic differentiation.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Expressing, Gene Expression, Control

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Expression of genes encoding matrix remodeling proteins and their endogenous inhibitors by ATDC5 cells on glass-bottom tissue culture wells, GF, and fibronectin-GF. (A) Time course of gene expression during chondrogenic differentiation for Adamts1 (circle) and Adamts2 (triangle). (B) Relative gene expression levels of Adamts1 (gray) and Adamts2 (black) at day 17 of chondrogenic differentiation in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (C) Time course of gene expression during chondrogenic differentiation for Mmp2 (triangle) and Mmp14 (circle). (D) Relative gene expression levels of Mmp2 (black) and Mmp14 (gray) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (E) Time course of gene expression during chondrogenic differentiation for Timp1 (circle), Timp2 (triangle), and Timp3 (square). (F) Relative gene expression levels of Timp1 (gray), Timp2 (black), and Timp3 (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (G) Time course of gene expression during chondrogenic differentiation for Ctgf (circle) and Tgfbi (triangle). (H) Relative gene expression levels of Ctgf (gray) and Tgfbi (black) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. Error bars = Mean ± SD Table lists matrix remodeling genes analyzed in this study with descriptions and literature citations that have demonstrated a link between increases in gene expression and chondrogenic differentiation.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Expressing, Gene Expression, Control

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: ECM Genes Expressed during Chondroprogenitor Cell Differentiation on GF

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Cell Differentiation, Binding Assay, Activity Assay, Membrane

Journal: Journal of Biological Chemistry

Article Title: Topogenesis of Peroxisomal Membrane Protein Requires a Short, Positively Charged Intervening-loop Sequence and Flanking Hydrophobic Segments

doi: 10.1074/jbc.m003304200

Figure Lengend Snippet: FIG. 2. Functional and topogenic regions of PMP34. C-terminally HA- tagged or GFP-fused PMP34 and its vari- ants were verified for intracellular local- ization in CHO-K1. A, constructs of deletion mutants of PMP34. DN30HA, PMP34-HA with deletion of N-terminal residues from 1 to 30; 204HA, HA-tagged PMP34 with residues 1–204; 204GFP, PMP34 comprising residues 1–204 fused with GFP. Others likewise representing respective constructs were indicated. Numbers in box represent the positions of transmembrane segments; L1–L5 desig- nate the intervening-loop region between two flanking TMs. Peroxisomal targeting activity of each variant verified (see be- low) was shown: 1, active; 1/2, partially active; 2, inactive. B, PMP34 variants represented in A were expressed in CHO- K1. a and b, DN30HA; c and d, DN125HA; e and f, DN186HA; g and h, DN125GFP; i and j, DN186GFP; k, DN204HA; l, 186HA; m and n, 204HA; o and p, 204GFP. C- terminally HA-tagged PMP34 variants were verified for peroxisomal localization by immunostaining using mouse (a, c, e, and m) and rabbit (k and l) anti-HA anti- body and FITC-labeled second antibody, where peroxisomes were assessed by anti- Pex14p antibody and Texas Red-labeled second antibody (b, d, f, h, j, n, and p). PMP34 truncation mutants fused with GFP were verified by GFP fluorescence (g, i, and o). Arrowheads indicate PMP34- positive particles, positive in expressed PMP34-variants, that were absent from Pex14p. Original magnification, 3630; bar, 20 mm. C, transmembrane topology of GFP fusion proteins, DN125GFP and 204GFP, was determined. CHO-K1 cells expressing DN125GFP (a and b) and 204GFP (c and d) were fixed, then treated with 25 mg/ml digitonin. Localization and membrane orientation were verified by GFP fluorescence (a and c) and immuno- fluorescence staining of GFP with anti- GFP antibody and Texas Red-labeled sec- ond antibody (b and d). Bar, 20 mm.

Article Snippet: Antigen-antibody complexes were detected under a Carl Zeiss Axioskop FL microscope, using fluorescein isothiocyanate (FITC)-labeled sheep anti-mouse antibody (Amersham Pharmacia Biotech, Tokyo, Japan), FITC-labeled sheep anti-rabbit immunoglobulin (Ig) G antibody (Cappel), or Texas Red-labeled goat antibodies to guinea pig IgG (Vector Laboratories) and rabbit IgG (Leinco Technologies).

Techniques: Functional Assay, Construct, Activity Assay, Variant Assay, Immunostaining, Labeling, Fluorescence, Expressing, Membrane, Staining

Journal: Journal of Biological Chemistry

Article Title: Topogenesis of Peroxisomal Membrane Protein Requires a Short, Positively Charged Intervening-loop Sequence and Flanking Hydrophobic Segments

doi: 10.1074/jbc.m003304200

Figure Lengend Snippet: FIG. 5. Coordinated function of the membrane targeting se- quence and transmembrane segments. A, constructs of the loop region and transmembrane segments (loop plus TM) fused with GFP. B, intracellular localization of the (loop plus TM)-GFP fusion protein. a and b, 86/204GFP; c and d, 30/204GFP; e, 125/273GFP; f, 86/273GFP. Each construct was expressed in CHO-K1 cells and detected by GFP fluorescence (a, c, e, and f). Cells expressing 86/204GFP were also stained using anti-malate dehydrogenase antibody and Texas Red- labeled second antibody (b); peroxisomes in 30/204GFP-expressing cells were assessed by anti-Pex14p antibody (d). Bar, 20 mm.

Article Snippet: Antigen-antibody complexes were detected under a Carl Zeiss Axioskop FL microscope, using fluorescein isothiocyanate (FITC)-labeled sheep anti-mouse antibody (Amersham Pharmacia Biotech, Tokyo, Japan), FITC-labeled sheep anti-rabbit immunoglobulin (Ig) G antibody (Cappel), or Texas Red-labeled goat antibodies to guinea pig IgG (Vector Laboratories) and rabbit IgG (Leinco Technologies).

Techniques: Membrane, Construct, Fluorescence, Expressing, Staining, Labeling

Journal: Endocrinology

Article Title: Prolonged Exposure to Insulin Induces Mitochondrion-Derived Oxidative Stress through Increasing Mitochondrial Cholesterol Content in Hepatocytes

doi: 10.1210/en.2011-2119

Figure Lengend Snippet: Prolonged exposure to insulin increases mitochondrial content of cholesterol in hepatocytes. Hepa1c1c7 cells were incubated with insulin for 16 h (n = 3). Mitochondria were then isolated and mitochondrial content of free cholesterol was quantified (A). Levels of phosphorylated (P-Akt), total Akt (T-Akt), and β-actin in these same cells were evaluated by immunoblotting with specific antibodies (B). Results represent mean ± se of three independent experiments. **, P < 0.01 vs. no insulin.

Article Snippet: The ample red cholesterol assay kit was obtained from Invitrogen (Paisley, UK).

Techniques: Incubation, Isolation, Western Blot

Journal: Endocrinology

Article Title: Prolonged Exposure to Insulin Induces Mitochondrion-Derived Oxidative Stress through Increasing Mitochondrial Cholesterol Content in Hepatocytes

doi: 10.1210/en.2011-2119

Figure Lengend Snippet: Prolonged exposure to insulin increases mitochondrial content of cholesterol in liver of mice. B6 mice were treated with the long- and slow-acting insulin (glargine) for 8 wk as detailed in Materials and Methods. Liver mitochondria were immediately isolated after animals were killed. Mitochondrial content of cholesterol was quantified. Results represent mean ± sd of six mice per group. **:, P < 0.01.

Article Snippet: The ample red cholesterol assay kit was obtained from Invitrogen (Paisley, UK).

Techniques: Isolation

Journal: Endocrinology

Article Title: Prolonged Exposure to Insulin Induces Mitochondrion-Derived Oxidative Stress through Increasing Mitochondrial Cholesterol Content in Hepatocytes

doi: 10.1210/en.2011-2119

Figure Lengend Snippet: Exposure of isolated mitochondria to cholesterol increases ROS production. Mitochondria isolated from primary hepatocytes were incubated with cholesterol bound to BSA at noted concentration for 5 min and then with carboxy-H2-DCF-DA for 30 min. ROS levels were subsequently quantified as detailed in Materials and Methods. Results represent mean ± se of three independent experiments. **, P < 0.01 vs. no cholesterol.

Article Snippet: The ample red cholesterol assay kit was obtained from Invitrogen (Paisley, UK).

Techniques: Isolation, Incubation, Concentration Assay

Journal: Endocrinology

Article Title: Prolonged Exposure to Insulin Induces Mitochondrion-Derived Oxidative Stress through Increasing Mitochondrial Cholesterol Content in Hepatocytes

doi: 10.1210/en.2011-2119

Figure Lengend Snippet: ROS production induced by the prolonged exposure to insulin is cholesterol synthesis dependent in hepatocytes. Hepa1c1c7 cells (A) or primary mouse hepatocytes (B) were treated with either the vehicle solution or insulin (5 nm) in the presence or absence of simvastatin (10 nm) for 16 h as indicated (n = 3). Cells were incubated with carboxy-H2-DCF-DA for 30 min, followed by quantification of ROS level. C, Levels of HMG-CoA-R activity in the cells described in A were quantified by using an HMG-CoA-R assay kit as detailed in Materials and Methods. Results represent mean ± se of three independent experiments. **, P < 0.01 vs. no insulin; #, P < 0.05 and ##, P < 0.01 vs. insulin alone.

Article Snippet: The ample red cholesterol assay kit was obtained from Invitrogen (Paisley, UK).

Techniques: Incubation, Activity Assay

Journal: Endocrinology

Article Title: Prolonged Exposure to Insulin Induces Mitochondrion-Derived Oxidative Stress through Increasing Mitochondrial Cholesterol Content in Hepatocytes

doi: 10.1210/en.2011-2119

Figure Lengend Snippet: Prolonged exposure of hepatocytes to insulin decreases mitochondrial membrane potential in a cholesterol synthesis-dependent manner. Hepa1c1c7cells (A and B) or primary mouse hepatocytes (C) were treated with either the vehicle solution or insulin (5 nm) in the presence of simvastatin (10 nm) for 16 h as indicated (n = 3). Mitochondrial membrane potential was then either visualized by using fluorescent microscopy (A) or quantification of fluorescence density (B and C) as detailed in Materials and Methods. Results represent mean ± se of three independent experiments. **, P < 0.01 vs. no insulin control. #, P < 0.05 and ##, P < 0.01 vs. insulin alone.

Article Snippet: The ample red cholesterol assay kit was obtained from Invitrogen (Paisley, UK).

Techniques: Membrane, Microscopy, Fluorescence, Control

Journal: Endocrinology

Article Title: Prolonged Exposure to Insulin Induces Mitochondrion-Derived Oxidative Stress through Increasing Mitochondrial Cholesterol Content in Hepatocytes

doi: 10.1210/en.2011-2119

Figure Lengend Snippet: ROS production induced by cholesterol was prevented by the inhibition of mitochondrial respiration complex I. A, Mitochondria isolated from Hepa1c1c7 cells were incubated with cholesterol (10 μm) bound to BSA in the presence or absence of rotenone (10 nm) for 5 min as noted. Carboxy-H2-DCF-DA was then added to the mitochondria for 30 min, followed by measurements of ROS as detailed in Materials and Methods. Results represent mean ± se of three independent experiments. **, P < 0.01 vs. no cholesterol control; ##, P < 0.01 vs. cholesterol alone. B, Mitochondria isolated from Hepa1c1c7 cells were incubated with cholesterol (10 μm) bound to BSA in the presence of CoQ10 at noted concentration for 5 min (n = 3) and were then incubated with carboxy-H2-DCF-DA for 30 min, followed by quantifications of ROS as detailed in Materials and Methods. Results represent mean ± se of three independent experiments. **, P < 0.01 vs. no cholesterol control; ##, P < 0.01 vs. cholesterol alone.

Article Snippet: The ample red cholesterol assay kit was obtained from Invitrogen (Paisley, UK).

Techniques: Inhibition, Isolation, Incubation, Control, Concentration Assay

Journal: Endocrinology

Article Title: Prolonged Exposure to Insulin Induces Mitochondrion-Derived Oxidative Stress through Increasing Mitochondrial Cholesterol Content in Hepatocytes

doi: 10.1210/en.2011-2119

Figure Lengend Snippet: Prolonged exposure to insulin decreases mitochondrial membrane fluidity in a cholesterol synthesis-dependent manner. A and B, Hepa1c1c7cells were treated with either the vehicle solution or insulin (5 nm) in the presence or absence of simvastatin (10 nm) for 16 h as indicated (n = 3). Mitochondria were then isolated from these cells and labeled with either TMA-DPH or DPH. Fluorescence density was quantified at 366 nm (emission = 440 nm) using polarizing filters in excitation and emission planes and normalized to mitochondrial protein level. C and D, Mitochondria (50 mg proteins) isolated from Hepa1c1c7 cells were incubated cholesterol-BSA complex for 5 min at 4 C (n = 3), washed three times to eliminate the free cholesterol, and labeled with TMA-DPH or DPH. Fluorescence density was quantified at 366 nm (emission = 440 nm) using polarizing filters in excitation and emission planes and normalized to mitochondrial protein level. Results represent mean ± se of three independent experiments. **, P < 0.01 vs. control; ##, P < 0.01 vs. insulin alone.

Article Snippet: The ample red cholesterol assay kit was obtained from Invitrogen (Paisley, UK).

Techniques: Membrane, Isolation, Labeling, Fluorescence, Incubation, Control

Journal: PLoS Genetics

Article Title: A single class of ARF GTPase activated by several pathway-specific ARF-GEFs regulates essential membrane traffic in Arabidopsis

doi: 10.1371/journal.pgen.1007795

Figure Lengend Snippet: ( A-F ) Co-immunoprecipitation (Co-IP) studies. ( A ) Co-IP of GNOM-Myc with ARF1-YFP, using GFP-Trap-agarose beads (IP: α-GFP) followed by immunoblot analysis (IB) with α-Myc antibody. Seedlings expressing only GNOM-Myc (GN-Myc) were used as control. ( B ) Co-IP of endogenous and YFP-tagged ARF1 with GNOM-Myc, using α-Myc-agarose beads (IP) followed by IB with α-GFP antibody and α-ARF1 antibody to detect YFP-tagged and endogenous ARF1. ( C ) Co-IP of endogenous and YFP-tagged ARF1 with GNL1-Myc, using α-Myc-agarose beads (IP) followed by IB analysis with α-GFP antibody and α-ARF1 antibody. ( D ) Co-IP of endogenous ARF1 with BIG3-YFP, using GFP-Trap-agarose beads (IP: α-GFP) followed by IB with α-ARF1 antibody. Col, wild-type control. ( E ) Co-IP of ARFA (ARFB1c)-RFP and endogenous ARF1 with BIG5-YFP, using GFP-Trap-agarose beads (IP: α-GFP) followed by IB analysis with α-RFP antibody and α-ARF1 antibody. ( F ) Co-IP of ARFA (ARFB1c)-RFP and endogenous ARF1 with BIG4-YFP, using GFP-Trap-agarose beads (IP: α-GFP) followed by IB analysis using α-RFP antibody and α-ARF1 antibody. IN, input; IP; immunoprecipitate; IB, immunoblot; kDa, kilodalton. ( G-J ) ARFA-YFP localization in wild-type (WT) (G-H) and big5 mutant (I-J). Note the highly cytosolic signal of ARFA-YFP in big5 . Scale bars, 10μm. ( K ) In-vitro GDP-GTP exchange activity of the catalytic SEC7 domain of BIG3 (blue) and BIG5 (red) on ARFA. Negative control, ARFA alone (black). See also .

Article Snippet: Proteins were separated on SDS-PAGE, transferred to the PVDF membrane (Millipore) and detected using one of the following antibodies: α-GFP (mouse, 1:1,000, Roche), α-RFP (rat, 1:1,000, Chromotek), α-Myc (1:1,000, Sigma), α-ARF1 (rabbit, 1:2,000, Agrisera), α-GN-SEC7 (rabbit, 1:2,500) [ ], and POD-conjugated secondary antibodies.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Expressing, Control, Mutagenesis, In Vitro, Activity Assay, Negative Control

Journal: The Journal of investigative dermatology

Article Title: Inhibition of Akt signaling by exclusion from lipid rafts in normal and transformed epidermal keratinocytes.

doi: 10.1038/jid.2009.415

Figure Lengend Snippet: Figure 1. Cholesterol depletion induces dephosphorylation of Akt, mammalian target of rapamycin (mTOR), and translocation of FoxO3a to the nucleus. (a) Serum-starved HaCaT cells were treated with methyl-b-cyclodextrin (MBCD) for 15 minutes at indicated concentrations followed by 5 mM cholesterol for a total of 1 hour (solid bars) or serum-free DMEM (open bars). Cholesterol concentration in plasma membrane was determined by cholesterol oxidase Amplex red assay as described in Materials and Methods. Data are mean values (n ¼ 6 experiments) with SD and represent percentage of the negative control (cells treated only with DMEM). (b) Serum-starved HaCaT cells were pretreated with a vehicle (control) or with phosphatidylinositol-3 kinase (PI3K) inhibitors LY294002 (20 mM) or wortmannin (0.5 mM) for 30 minutes at 37 1C before incubation with 1% MBCD in serum-free DMEM for the indicated times at 37 1C. Whole-cell lysates were resolved by SDS–PAGE and immunoblotted with antibodies directed against P-Ser473 Akt, P-Thr308 Akt, P-mTOR, P-Thr389 p70S6K, P-Thr421/Ser424 p70S6K, P-FoxO3a, total Akt, and actin (loading control). (c) HaCaT cells were cholesterol depleted with 1% MBCD for 15 minutes and then repleted with 5 mM cholesterol (or left in DMEM for control) for an additional 45 minutes, as in a. Phosphorylation of Akt, mTOR, and FoxO3a was determined as in b. (d and e) Translocation of FoxO3a to the nucleus in MBCD-treated cells. HaCaT or A431 cells were incubated with LY294002 (20 mM), wortmannin (0.5 mM), Akt inhibitor X (5 mM) or 1% MBCD or left untreated for 30 minutes at 37 1C. Cells were fixed in acetone at 4 1C, rehydrated in phosphate-buffered saline/BSA (0.5%, 15 minutes) and stained with rabbit anti-FoxO3a followed by labeling with secondary Alexa Fluor 488 antibody (both at 4 1C for 30 minutes). Nuclei were counterstained with propidium iodide, outlined, and the FoxO3a-specific fluorescence was determined in the nuclear (black bars) and cytoplasmic (white bars) areas by image analysis of confocal images (d). *Po0.05, t-test against control. (e) Nuclear and cytoplasmic fractions from HaCaT cells were prepared as in Materials and Methods and immunoblotted with antibodies against total FoxO3 and actin. (f and g) Influence of MBCD on Akt/mTOR pathway in A431 cells (f) and normal human keratinocytes (g). Western blots were performed as in b. (h) HaCaT cells were incubated with simvastatin (20 mM, overnight), filipin III (2 mM, 1 hour), cholesterol oxidase (Chol. ox., 1 U ml1, 1 h), 5-cholesten-5-b-ol (5-chol, 5 mM, 2 hours), or with DMSO (vehicle) at 37 1C. Cell lysates were subjected to SDS–PAGE and immunoblotted with the antibodies indicated above.

Article Snippet: The human epidermoid carcinoma A431 cell line was from the ATCC (Manassas, VA).

Techniques: De-Phosphorylation Assay, Translocation Assay, Concentration Assay, Clinical Proteomics, Membrane, Amplex Red Assay, Negative Control, Control, Incubation, SDS Page, Phospho-proteomics, Saline, Staining, Labeling, Fluorescence, Western Blot

Journal: The Journal of investigative dermatology

Article Title: Inhibition of Akt signaling by exclusion from lipid rafts in normal and transformed epidermal keratinocytes.

doi: 10.1038/jid.2009.415

Figure Lengend Snippet: Figure 6. Cholesterol depletion sensitizes HaCaT and A431 cells to the cytotoxic effect of death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), etoposid, and doxorubicin. (a–f) HaCaT cells (a, c, e) or A431 cells (b, d, f) were cholesterol-depleted for 15 minutes with 1% methyl-b- cyclodextrin (MBCD) and cultured in the presence of different concentrations of TRAIL, etoposid, or doxorubicin for 24 hours, alone or in combination with Akt inhibitors: LY294002 (20 mM) or Akt inhibitor X (5 mM). To eliminate MBCD-induced toxicity, we removed this compound after total 3 hours incubation. The number of cells was determined by the methylene blue assay. Shown are means with SD. *Po0.05, t-test. (g) HaCaT cells were treated as above and apoptosis was determined after 24 hours using the caspase 3/7 assay as described in Materials and Methods. The following concentrations of apoptosis inducers were used: 5 ng ml1 TRAIL, 20 mM etoposid, and 1 mg ml1 doxorubicin.

Article Snippet: The human epidermoid carcinoma A431 cell line was from the ATCC (Manassas, VA).

Techniques: Cell Culture, Incubation

Journal: PLoS ONE

Article Title: DPAGT1-CDG: Functional analysis of disease-causing pathogenic mutations and role of endoplasmic reticulum stress

doi: 10.1371/journal.pone.0179456

Figure Lengend Snippet: Double labelled immunofluorescence analysis of control (C) and patient cell lines (patients 1, 2 and 3). Cells were double-labelled with GPT (red fluorescence) and Calnexin (green fluorescence) antibodies (ER marker).

Article Snippet: The fixed cells were blocked with blocking solution (PBS 1x, 0.1% triton, 5% foetal bovine serum) for 30 min and the cells then incubated overnight at 4°C with anti-GPT antibody (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:50 in blocking solution plus anti-calnexin antibody (StressMarq, Victoria, British Columbia) diluted 1:200 in blocking solution.

Techniques: Immunofluorescence, Fluorescence, Marker

Journal: PLoS ONE

Article Title: DPAGT1-CDG: Functional analysis of disease-causing pathogenic mutations and role of endoplasmic reticulum stress

doi: 10.1371/journal.pone.0179456

Figure Lengend Snippet: A) COS-7 cells were cotransfected with the wild type GPT-GFP fused protein (green fluorescence) or the protein bearing the mutations (p.Phe110Ser, p.Leu120Met, p.Val264Gly, p.Arg301Cys, p.Arg301His and p.Leu385Arg) and with the Calreticulin-DsRed fused protein (red fluorescence) as an ER marker. B) Quantification of GPT-GFP (colocalised with Calnexin-DsRed protein) fluorescence intensity. Data were collected from two different experiments; at least 80 images were analysed. Data represent mean ± SD. ***p<0.001.

Article Snippet: The fixed cells were blocked with blocking solution (PBS 1x, 0.1% triton, 5% foetal bovine serum) for 30 min and the cells then incubated overnight at 4°C with anti-GPT antibody (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:50 in blocking solution plus anti-calnexin antibody (StressMarq, Victoria, British Columbia) diluted 1:200 in blocking solution.

Techniques: Fluorescence, Marker

Journal: Genetics and Molecular Research

Article Title: Apoptosis induced by lipid-associated membrane proteins from Mycoplasma hyopneumoniae in a porcine lung epithelial cell line with the involvement of caspase 3 and the MAPK pathway

doi: 10.4238/2015.september.25.10

Figure Lengend Snippet: Figure 2. Apoptotic features induced by lipid-associated membrane proteins (LAMPs) in St. Jude porcine lung epithelial cell line (SJPL) cells. Stimulated groups were treated with 0.2 mg/mL LAMPs for 24 h while untreated cells served as controls. (A) Representative images show nuclear morphology as detected by diamidino- phenylindole (DAPI) staining (blue) under fluorescence microscopy. (B) Representative images of terminal dexynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining showing that LAMPs induced more apoptotic cells. (C) Representative images of acridine orange (AO)-stained cells (red channel) and ethidium bromide (EB)-stained cells (green channel) were merged to distinguish living and apoptotic cells.

Article Snippet: Diamidino-phenylindole (DAPI), acridine orange/ethidium bromide (AO/EB), and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assays DAPI staining solution (Beyotime, China) was used to detect the changes in nuclear morphology.

Techniques: Membrane, Staining, Fluorescence, Microscopy, End Labeling, TUNEL Assay