Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) Immunoblot of Yap1 to verify knockout in Yap1 KO cells. β-actin was used as a loading control. ( B ) Representative brightfield microscopy images of WT and Yap1 KO ES cells in ±LIF. Scale bar, 200 μm. ( C ) Immunoblot of Yap1 to verify knockout of Yap1 in three different ESC lines (J1, E14, and CJ7). β-actin was used as a loading control. J1 clone #5 was used as a positive control for knockout. ( D ) RT-qPCR measuring the expression of Yap1 after lentiviral shRNA-mediated Yap1 KD in differentiating WT ESCs (-LIF 72 hr). ( E ) LDH assay measuring cell death of Yap1 KD vs. control KD cells during differentiation (-LIF 72 hr). ( F ) Immunoblot of Yap1 to verify stable overexpression (OE) of FLAG-Bio-Yap1 in three different clones compared to WT ESCs. β-actin was used as a loading control. ( G ) Immunoblot of cleaved Casp3 and cleaved Parp1 in WT and Yap1 KO cells that had been treated with 1 μM STS for the indicated number of hours during differentiation (treatment started 43–48 hr after withdrawal of LIF depending on the length of STS treatment). ( H ) RT-qPCR measuring the expression of Casp9 upon shRNA-mediated lentiviral KD in WT and Yap1 KO cells during differentiation (72 hr) relative to empty vector KD. ( I ) RT-qPCR measuring the expression of Casp2, Casp3, Casp6, Casp7, Casp8, and Casp9 in Yap1 KO cells compared to WT cells in ±LIF. All data are expressed as mean ±standard deviation (n = 3 independent samples). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Article Snippet: Cells were then resuspended in 200 μL 1X Annexin V binding buffer plus 5 μL of 0.2 mM NucView 488 caspase-3 substrate solution and 5 μL CF 594 annexin V solution (Biotium).

Techniques: Western Blot, Knock-Out, Microscopy, Positive Control, Quantitative RT-PCR, Expressing, shRNA, Lactate Dehydrogenase Assay, Over Expression, Clone Assay, Plasmid Preparation, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) Lactate dehydrogenase (LDH) assay of WT and Yap1 KO ESCs in ±LIF. Cells were treated with either Z-VAD-FMK (Z-VAD), necrostatin-1, DMSO, or no treatment. Values were normalized to wells that had been lysed completely. ( B ) LDH assay measuring cell death after Yap1 KO in three different ESC lines during differentiation (72 hr) or self-renewal. ( C ) LDH assay measuring cell death in Yap1 KO, WT, and three different stable FLAG-Bio (FB) Yap1 overexpression cell lines during differentiation (72 hr). ( D ) Representative brightfield and fluorescence microscopy images of WT and Yap1 KO ESCs incubated with NucView 488 Casp3 substrate at the indicated times after LIF withdrawal. ( E ) Representative flow cytometry density plots of WT and Yap1 KO ESCs detecting fluorescent signal from annexin-V (conjugated to CF594) and NucView 488 reagent during differentiation (60 hr). ( F ) Fold enrichment of annexin-V and active Casp3-positive Yap1 KO vs. WT ESCs according to flow cytometry. ( G ) Immunoblot of Casp9, Casp8, Casp3, cleaved Casp3, and cleaved Parp1 in WT and Yap1 KO cells during differentiation. β-actin was used as a loading control. ( H ) Luminescent assay of caspase activity in Yap1 KO vs. WT ESCs in ±LIF media. ( I ) LDH assay of WT and Yap1 KO cells ± KD of Casp9 during differentiation (72 hr). All data are expressed as mean ±standard deviation (n = 4 independent samples for LDH assays and n = 3 for other experiments). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Article Snippet: Cells were then resuspended in 200 μL 1X Annexin V binding buffer plus 5 μL of 0.2 mM NucView 488 caspase-3 substrate solution and 5 μL CF 594 annexin V solution (Biotium).

Techniques: Lactate Dehydrogenase Assay, Over Expression, Fluorescence, Microscopy, Incubation, Flow Cytometry, Western Blot, Luminescence Assay, Activity Assay, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) JC-10 mitochondrial membrane potential assay in WT and Yap1 KO cells during various forms of differentiation (72 hr for Pan and EpiLC, 48 hr for Neural and Endo) and self-renewal (maintained for an equal amount of time). Values (525/570 nm ratio, n = 6) corresponding to loss in ∆ψ (mitochondrial membrane potential) in Yap1 KO cells were normalized to WT cells. ( A ) JC-10 assay in WT and Yap1 KO cells in ±LIF after 12 hr of treatment with BH3 mimetics ABT-737, Venetoclax, A-1210477, and A1155463 (total differentiation time: 36 hr). Values (525/570 nm ratio) corresponding to loss in ∆ψ were normalized to DMSO as a control. ( C ) LDH assays of BH3 mimetic dose response curves after 24 hr of treatment in WT and Yap1 KO cells in ±LIF (48 hr differentiation). ( D ) LDH assay of WT and Yap1 KO cells after KD of Bmf or Puma in -LIF conditions (72 hr). ( E ) LDH assay of inducible Bmf and Puma OE (±Dox, 48 hr, 500 ng/mL) in WT and Yap1 KO cells in ±LIF (48 hr differentiation). ( F ) Immunoblot of cleaved Casp3, cleaved Parp1, and Mcl-1 in WT and Yap1 KO dESCs (28 hr) after 4 hr of treatment with BH3 mimetics A-1210477 (Mcl-1 inhibitor) and ABT-737 (inhibitor of Bcl-2, Bcl-xL, and Bcl-w). β-actin was used as a loading control. All data are expressed as mean ±standard deviation (n = 4 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Article Snippet: Cells were then resuspended in 200 μL 1X Annexin V binding buffer plus 5 μL of 0.2 mM NucView 488 caspase-3 substrate solution and 5 μL CF 594 annexin V solution (Biotium).

Techniques: Membrane, Lactate Dehydrogenase Assay, Western Blot, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) LDH assay of WT, Yap1 KO, and Yap1 KO constitutively overexpressing Bcl-xL or Yap1 in -LIF (72 hr). ( B ) LDH assay of inducible Bcl-2 (±Dox, 48 hr, 500 ng/mL) in WT and Yap1 KO cells -LIF (72 hr). ( C ) LDH assay of inducible Taz (±Dox, 48 hr, 500 ng/mL) in WT and Yap1 KO cells ± LIF (72 hr differentiation). ( D ) Immunoblot of cleaved Parp1, cleaved Casp3, Bcl-xL, and Mcl-1 in Yap1 KO cells inducibly overexpressing Taz (±Dox, 48 hr, 500 ng/mL) in -LIF (72 hr). ( E ) LDH assay of WT ESCs during differentiation (72 hr) after 48 hr KD of Bcl-xL or Mcl-1. ( F ) LDH assay of WT ESCs ± LIF (72 hr)±KD of Bcl-2. ( G ) RT-qPCR measuring the expression of lineage markers (trophectoderm: Cdx2 and Gata3 , ectoderm: Nes and Otx2 , endoderm: Gata4 , mesoderm: Gsc and T ) in WT and Yap1 KO cells in -LIF (72 hr, n = 3). Expression is indicated as a fold change in +Dox samples relative to -Dox. ( H ) Model proposing roles for Yap1 specific to the exit from self-renewal. In complex with Tead factors like Tead4, Yap1 co-activates anti-apoptotic genes and mildly co-represses pro-apoptotic genes to dampen mitochondrial priming, which thus prevents hyperactivation of the apoptotic cascade through Casp9. All data are expressed as mean ±standard deviation (n = 4 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Article Snippet: Cells were then resuspended in 200 μL 1X Annexin V binding buffer plus 5 μL of 0.2 mM NucView 488 caspase-3 substrate solution and 5 μL CF 594 annexin V solution (Biotium).

Techniques: Lactate Dehydrogenase Assay, Western Blot, Quantitative RT-PCR, Expressing, Standard Deviation, Two Tailed Test

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: 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