Journal: Journal of Nanobiotechnology

Article Title: Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation

doi: 10.1186/s12951-016-0171-3

Figure Lengend Snippet: SCBD-produced nanostructured zirconia induces neuronal differentiation in PC12 cells. a The phase contrast images demonstrate the biological responses of neuron-like PC12 cells after 24 h interaction with the different surfaces presented in Fig. in the absence or presence of nerve growth factor (NGF). White arrows indicated typical examples of neurite outgrowth of differentiated PC12 cells (in Additional file : Figure S1, a close up image of representative differentiated cells on ns-Zr15 is shown to illustrate more detailed the features of differentiated PC12 cells). b On the right the corresponding statistical quantification of the differentiation rate ( top ) and neurite outgrowth ( bottom ) is shown. A cell that developed at least one neurite with a length >10 μm was counted as differentiated, the quantification of neurite outgrowth are detailed in the “ ” section. The bars represent the change of differentiation and neurite outgrowth compared to the PLL condition in the absence of NGF. The bars represent the average and are shown with the SD, representing the global statistics of five independent experiments (n: >500 cells, >150 neurites)

Article Snippet: PC12 (PC-12 Adh ATCC Catalog no.CRL-1721.1TM) were cultured in RPMI-1640 Medium (Sigma-Aldrich) supplemented with 10 % horse serum (HS; Sigma-Aldrich), 5 % fetal bovine serum (FBS; Sigma-Aldrich), 2 mM l -glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mM pyruvic acid (sodium salt) and 10 mM HEPES.

Techniques: Produced

Journal: Journal of Nanobiotechnology

Article Title: Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation

doi: 10.1186/s12951-016-0171-3

Figure Lengend Snippet: Nanostructured zirconia surfaces alter the nanoscale adhesion site architecture. Representative TEM images of the cell/substrate interface on a flat-Zr, and b ns-Zr15 substrates. The scale bars equal 100 nm. c This image shows a close-up of a representative interaction zone between a PC12 cell and the ns-Zr15 surface. The cells interacted with the surfaces for 24 h. A description of experimental setting and quantification can be found in the “ ” section. In d, e the corresponding analysis of the width of the adhesion sites at the nanoscale are summarized. d The absolute values of all measurements for both substrates are shown. e The histogram of measured widths of adhesion sites are displayed here. The graphs represent the global statistics of images obtained from two independent experiments (fl-Zr: n = 120, ns-Zr15 = 164)

Article Snippet: PC12 (PC-12 Adh ATCC Catalog no.CRL-1721.1TM) were cultured in RPMI-1640 Medium (Sigma-Aldrich) supplemented with 10 % horse serum (HS; Sigma-Aldrich), 5 % fetal bovine serum (FBS; Sigma-Aldrich), 2 mM l -glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mM pyruvic acid (sodium salt) and 10 mM HEPES.

Techniques:

Journal: Journal of Nanobiotechnology

Article Title: Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation

doi: 10.1186/s12951-016-0171-3

Figure Lengend Snippet: NGF and TrkA activation are dispensable in nanostructure-induced neuritogenesis whereas β1 integrin activation/signaling are essential. a The scheme illustrates the interference points of the various inhibitors of proteins involved in the integrin and RTK signaling cascade which were used in the experiments. b–d The PC12 cells were plated on PLL (+NGF) or surfaces with a roughness R q of 15 nm rms. In case of inhibitor treatment, the inhibitors were preincubated for 15 min prior to cell plating and then present for the whole ongoing experimental period. The cell morphology was recorded by phase contrast microscopy 24 h after plating the cells. As biological read-out for the differentiation the quantification of the neurite outgrowth is shown (obtained with the help of ImageJ); representative images can be found in Additional file : Figure S1. The bars display the change of neurite outgrowth compared to the situation without treatment on the corresponding substrate. The bars are flanked by SD. The graph displays the results of an inhibitor treatment against b TrkA (GW441756 1 µM, from two independent experiments), c the incubation with the 4B4 inhibitory antibody (2.5 µg/ml) (or activity-neutral antibody K20 (2.5 µg/ml) as control) against β1 integrin (from three independent experiments) or d the inhibition of MEK 1/2 (U1026 10 µM, from two independent experiments), always both in the canonical (NGF-induced) and the nanostructure-induced condition (n: >500 cells, >150 neurites). Further inhibitions of major mediators and processes involved in integrin signaling and cytoskeletal organization are displayed in Additional file : Figure S3

Article Snippet: PC12 (PC-12 Adh ATCC Catalog no.CRL-1721.1TM) were cultured in RPMI-1640 Medium (Sigma-Aldrich) supplemented with 10 % horse serum (HS; Sigma-Aldrich), 5 % fetal bovine serum (FBS; Sigma-Aldrich), 2 mM l -glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mM pyruvic acid (sodium salt) and 10 mM HEPES.

Techniques: Activation Assay, Microscopy, Incubation, Activity Assay, Control, Inhibition

Journal: Journal of Nanobiotechnology

Article Title: Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation

doi: 10.1186/s12951-016-0171-3

Figure Lengend Snippet: Focal adhesion formation/dynamics and cytoskeletal organisation differ on flat or neuritogenesis-inducing nanostructured surfaces. a The graphic visualizes and summarizes the cytoskeletal and mechanobiological processes which are influenced by FA organization and dynamics. b In the panel representative images of the PC12 cells fixed with 4 % PFA after the indicated time periods on the different surfaces are demonstrated (vinculin staining recorded by TIRF microscopy, f-actin in epifluorescence). The white arrows indicate exemplary areas with focal complex ( pointed line ) or focal adhesion ( continuous line ) structures. The asterisks pinpoint to exemplary areas with strong actin fiber formation. c – e The graphs summarize the corresponding results (representing the global statistics of three independent experiments, vinculin clusters: n = 722–3678, cells: n = 16–34) of the quantifications (obtained with ImageJ) of c, d FA dynamics; c Vinculin cluster area, d Number of vinculin clusters per cell, and e cytoskeletal actin fiber organization. na not analyzable

Article Snippet: PC12 (PC-12 Adh ATCC Catalog no.CRL-1721.1TM) were cultured in RPMI-1640 Medium (Sigma-Aldrich) supplemented with 10 % horse serum (HS; Sigma-Aldrich), 5 % fetal bovine serum (FBS; Sigma-Aldrich), 2 mM l -glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mM pyruvic acid (sodium salt) and 10 mM HEPES.

Techniques: Staining, Microscopy

Journal: Journal of Nanobiotechnology

Article Title: Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation

doi: 10.1186/s12951-016-0171-3

Figure Lengend Snippet: Cellular rigidity is decreased on the neuritogenesis-inducing surface, being the decisive signal for the differentiation. a Representative morphological images ( left images ) and maps of the Young’s modulus of elasticity ( right images ) of living PC12 cells interacting with PLL (in the presence or absence of NGF), or with flat or the nanostructured neuritogenesis-inducing zirconia surfaces. b On the upper right , the graph displays the summary of the corresponding analysis of the biomechanical properties of the membrane/cytoskeletal layer. The bar represents the average of the global statistics obtained from two (flat-Zr, Glass-PLL ±NGF), respectively three (ns-Zr15) independent experiments (number of measured cells: flat-Zr: n = 6, ns-Zr15: n = 7, PLL −NGF: n = 8; Glass-PLL +NGF: n = 8), flanked with the error which was calculated as described in the “ ” section. YM young’s modulus. c Differentiation rate and neurite outgrowth of PC12 cells plated for 24 h on the neuritogenesis-inducing ns-Zr15 surface in the presence of isoosmotic medium or in medium with the indicated hypoosmolarity. The bars represent the average of two independent experiments and are flanked with the SD (n: >500 cells, >150 neurites). Representative images of all conditions can be found in Additional file : Figure S3

Article Snippet: PC12 (PC-12 Adh ATCC Catalog no.CRL-1721.1TM) were cultured in RPMI-1640 Medium (Sigma-Aldrich) supplemented with 10 % horse serum (HS; Sigma-Aldrich), 5 % fetal bovine serum (FBS; Sigma-Aldrich), 2 mM l -glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mM pyruvic acid (sodium salt) and 10 mM HEPES.

Techniques: Membrane

Journal: Journal of Nanobiotechnology

Article Title: Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation

doi: 10.1186/s12951-016-0171-3

Figure Lengend Snippet: Proteomic analysis confirms the differentiation and reveals alterations of the mechanotransductive cellular status upon nanostructure/cell interaction. a A shotgun proteomic analysis was carried out on PC12 cells on neuritogenesis-inducing ns-Zr15 or on flat-Zr or PLL in the presence of NGF (after 24 h cell/substrate interaction). An ANOVA test was performed in order to identify the proteins that were differentially expressed. In this report, only the data comparing ns-Zr15 and flat-Zr are presented. The colored data points in the volcano plot that are located above the p value line (t test value cut off is 0.0167) correspond to the proteins that were differentially expressed in these two conditions upon treatment with large magnitude fold changes and high statistical significance. In green are indicated proteins that are up regulated, in red are the down regulated. The proteins having a fold-change less than 1.5 are shown in gray. A complete list of these proteins can be found in Additional file : Table S1, Additional file : Table S2 in the supplementaries. b The cartoon summarizes and visualizes the sites of action and functions of adhesome- and mechanobiologically-relevant proteins found to be altered in their expression level upon interaction with the neuritogenesis-inducing nanostructured surface (for further details see text). Arrows indicate up- or downregulation compared to the flat zirconia condition

Article Snippet: PC12 (PC-12 Adh ATCC Catalog no.CRL-1721.1TM) were cultured in RPMI-1640 Medium (Sigma-Aldrich) supplemented with 10 % horse serum (HS; Sigma-Aldrich), 5 % fetal bovine serum (FBS; Sigma-Aldrich), 2 mM l -glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mM pyruvic acid (sodium salt) and 10 mM HEPES.

Techniques: Expressing

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: S-Nitrosoglutathione Reductase in Human Lung Cancer

doi: 10.1165/rcmb.2011-0147OC

Figure Lengend Snippet: Decreased activity of S-nitrosoglutathione (GSNO) reductase in non–small cell lung carcinoma. (A) Squamous-cell lung-cancer tissue homogenates were compared with homogenates of adjacent normal lung parenchyma for the activity of GSNO reductase ex vivo. (B) Cultured squamous-cell lung carcinoma cells (H226) were compared with nonmalignant human airway cell cultures (CFBE41o−) for the activity of GSNO reductase. *P < 0.05.

Article Snippet: Deparaffinized tissues were incubated in anti-GSNO reductase rabbit antibody (1:50; catalogue number 11051-1-AP; Protein Tech Group), and then with ABC reagents (Vector Laboratories, Burlingame, CA).

Techniques: Activity Assay, Ex Vivo, Cell Culture

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: S-Nitrosoglutathione Reductase in Human Lung Cancer

doi: 10.1165/rcmb.2011-0147OC

Figure Lengend Snippet: Decreased GSNO reductase immunoreactivity in lung-cancer specimens. (A) Airway tissue from healthy, nonsmoking adults expresses epithelial GSNO reductase (brown stain; ×20). (B and C) Wild-type (wt) mice (B), but not GSNO reductase−/− mice (C), express pulmonary GSNO reductase in their lungs. (D) A representative neutrophil is GSNO reductase–negative. (E) A representative macrophage is GSNO reductase–positive. (F) Inflammatory cells that immunostain positively serve as a positive internal control in lung-cancer specimens (in this case, squamous-cell carcinoma). (G) Examples of squamous-cell carcinoma specimens with reduced GSNO reductase immunoreactivity. (H) This decreased GSNO reductase expression is true for a range of cancers, including squamous-cell carcinoma, adenocarcinoma and large-cell (undifferentiated) carcinoma (top, ×40; bottom, ×10).

Article Snippet: Deparaffinized tissues were incubated in anti-GSNO reductase rabbit antibody (1:50; catalogue number 11051-1-AP; Protein Tech Group), and then with ABC reagents (Vector Laboratories, Burlingame, CA).

Techniques: Staining, Control, Expressing

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: S-Nitrosoglutathione Reductase in Human Lung Cancer

doi: 10.1165/rcmb.2011-0147OC

Figure Lengend Snippet: GSNO REDUCTASE EXPRESSION IN LUNG CANCER

Article Snippet: Deparaffinized tissues were incubated in anti-GSNO reductase rabbit antibody (1:50; catalogue number 11051-1-AP; Protein Tech Group), and then with ABC reagents (Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Staining, Negative Staining

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: S-Nitrosoglutathione Reductase in Human Lung Cancer

doi: 10.1165/rcmb.2011-0147OC

Figure Lengend Snippet: Confocal imaging shows altered distribution of GSNO reductase in squamous-cell lung carcinoma cells. (A) GSNO reductase is localized in punctate cytoplasmic structures and in mitotic spindles in nonmalignant bronchoepithelial cells (CFBE41o−). (B) GSNO reductase colocalization with mitotic spindles is inhibited by colchicine. (C) GSNO reductase (red) is colocalized with wt Ras (green) in CFBE41o− cells, but not (F) in squamous-cell carcinoma cells (H226). (D) The localization of GSNO reductase is perinuclear in primary squamous-cell lung carcinoma cells. (E) Background fluorescence (negative control).

Article Snippet: Deparaffinized tissues were incubated in anti-GSNO reductase rabbit antibody (1:50; catalogue number 11051-1-AP; Protein Tech Group), and then with ABC reagents (Vector Laboratories, Burlingame, CA).

Techniques: Imaging, Fluorescence, Negative Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: S-Nitrosoglutathione Reductase in Human Lung Cancer

doi: 10.1165/rcmb.2011-0147OC

Figure Lengend Snippet: Ras S-nitrosylation is increased in most lung-cancer cell lines, but is minimally affected by nitric oxide synthase inhibition or 2 days of exposure to NO. (A) The immunoprecipate (IP) of Ras was biotin-substituted from cell lines exposed with or without NO (30 ppm) for 2 days or to L-N-monomethyl arginine (L-NMMA) (100 μM). The biotin-substituted IP and—on a separate gel—total cell extract underwent immunoblotting for Ras and for actin (loading control). Densitometry revealed an increased SNO-Ras/Ras ratio in tumor cell lines (lanes 4–7) relative to nonmalignant cells (lanes 1–3), with a minimal effect of 3 days of NO or L-NMMA. Note that the molecular weight marker ran between lanes 6 and 7, but all bands are from the same gel. (B) The experiments in A were repeated without exposure to NO; again, SNO-Ras/Ras is increased in the cancer cells. (C) Lung-cancer cell lines in culture do not express NOS isoforms and express less GSNO reductase than nonmalignant CFBE41o− cells, with the exception of Calu-1 cells. (D) Our polyclonal antibody was highly reactive against human GSNO reductase, but much less so against the murine enzyme, and not at all against the GSNO reductase−/− mouse lung homogenates.

Article Snippet: Deparaffinized tissues were incubated in anti-GSNO reductase rabbit antibody (1:50; catalogue number 11051-1-AP; Protein Tech Group), and then with ABC reagents (Vector Laboratories, Burlingame, CA).

Techniques: Inhibition, Western Blot, Control, Molecular Weight, Marker

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: S-Nitrosoglutathione Reductase in Human Lung Cancer

doi: 10.1165/rcmb.2011-0147OC

Figure Lengend Snippet: Exposure to nitrosative stress for 5 days increases Ras S-nitrosylation, but does not alter the expression of S-nitrosoglutathione reductase (GSNOR). (A) In a sealed NO cell-exposure chamber, a steady concentration of NO at 30 ppm (400 nM in the gas phase) was measured using serial 0.2-ml gas samples from the chamber and was analyzed by chemiluminescence. A representative chemiluminescent signal is shown. (B) Nonmalignant epithelial cells (CFBE41o−) were incubated for 5 days in this chamber, and exhibited no change in the expression of GSNOR, but did exhibit increased Ras S-nitrosylation.

Article Snippet: Deparaffinized tissues were incubated in anti-GSNO reductase rabbit antibody (1:50; catalogue number 11051-1-AP; Protein Tech Group), and then with ABC reagents (Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Concentration Assay, Incubation

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: S-Nitrosoglutathione Reductase in Human Lung Cancer

doi: 10.1165/rcmb.2011-0147OC

Figure Lengend Snippet: The Ras denitrosylase function of GSNO reductase. (A–D) Immunoprecipitated H-Ras was S-nitrosylated with 100 μM S-nitroso-N-acetyl cysteine, and washed three times in PBS. SNO–H-Ras was exposed to glutathione (GSH) and nicotinamide adenine dinucleotide reduced (NADH), with (A and B) or without (C and D) GSNO reductase. SNO–H-Ras was measured by reduction chemoluminescence at 0 minutes (A and C) and 10 minutes (B and D). B and D represent identical conditions, except that GSNO reductase is present in B. The GSNO formed by transnitrosation from SNO-Ras requires GSNO reductase for its breakdown. (E) GSNOR−/− mice (lanes 1 and 2) had higher concentrations of SNO-Ras relative to total Ras than did background wt (lane 3) when wild-type mouse lungs were pretreated with 1 mM ethyl nitrite (EtONO; 5 μl). However, concentrations of SNO-Ras increased (lane 4). (F) Concentrations in E were quantitated by densitometry (background subtracted). (G) The activity of Ras was increased by S-nitrosocysteine (CSNO, 10 μM; hatched and solid bars) relative to baseline (open bars) in the presence of GSH and NADH in two non–small cell lung carcinoma cell lines. This effect tended to be augmented by the GSNO reductase inhibitor, C3 (solid bars; 100 μM; 26), in H226 cells, but the effect was not significant, consistent with the decreased activity of baseline GSNO reductase. HeLa cells stimulated with epidermal growth factor (gray bar) served as positive internal controls for the ELISA assay. *n = 3 each; P < 0.03, relative to baseline, according to ANOVA.

Article Snippet: Deparaffinized tissues were incubated in anti-GSNO reductase rabbit antibody (1:50; catalogue number 11051-1-AP; Protein Tech Group), and then with ABC reagents (Vector Laboratories, Burlingame, CA).

Techniques: Immunoprecipitation, Activity Assay, Enzyme-linked Immunosorbent Assay