Neural-like processes induced by NGLC films.

<p>Confocal immunofluorescence microscopy images showing morphological and structural effects of NGLC films on the SN4741 cells cultured on top of them. Nuclear marker DAPI (blue-label), synaptophysin (SYP: red-label) and G-protein-regulated inward-rectifier potassium channel 2 (GIRK2: green-label) proteins are shown as well as their colocalization (merge: yellow-label). The positive DAPI staining (<b>A1, B1, C1, D1</b>) showed a monolayer culture image without visible sign of differentiation. Cells cultured on BTTN films presented poor signal for SYP (<b>A2</b>) and GIRK2 (<b>A3</b>) markers. Cultures on NGLC 5-nm-thick films showed increasing neural-like processes with SYP/GIRK2 colocalization (<b>B1-B4</b>) where an axon-like process is measured (1843 μm) (<b>B5</b>). These processes were never seen in control cultures neither in BTTN cultures. For the measurement of the axon length Image J/Fiji software with the plugin ‘Microscope measurement Tools’ were used. It was observed with NGLC 20-nm-thick films (<b>C1-C5</b>) a greater synaptic capacity (SYP/GIRK2 signal) where a cell polarized and oriented growing towards the limit of the NGLC film (<b>C4</b>) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173978#pone.0173978.g007" target="_blank">Fig 7</a>, D4). Positive SYP highly expressed is a proof of a functional characteristic of the cells to address their processes to the outer limit. In the cell culture on NGLC 80-nm-thick films (<b>D1-D5</b>) axon-like processes (D3), arborization (D4) and denditric-like formation (D5) can be observed due to the cellular development and maturation and not to any artifactual folding of the film.</p

Cytotoxicity measurement via an apoptosis/necrosis assay.

<p>Values indicates apoptotic/necrotic cells expressed as percentage of positive-stained cells counted by flow cytometry after 24 hours, 3 days and 7 days of culture with nanocrystalline glass-like carbon (NGLC) microflakes at five different concentrations (1, 5, 10, 20 and 50 μg/ml) (<b>A</b>). Bright field (upper row) and immunofluorescence images (rows below) (<b>B</b>) showing the apoptosis/necrosis assay with SN4741 cells cultured in medium containing five different concentrations (1, 5, 10, 20 and 50 μg/ml) of NGLC microflakes. Calcein AM was used as vital immunofluorescence marker and EthD-1 as apoptosis marker, after 7 days of culture, studied also by merge images. For both experiments a positive control (C+) representing the use of 10% Triton X-100 in the culture was used to induce apoptotic processes, and negative control (C-) represents the culture without 10% Triton X-100 nor NGLC. The data are expressed as the mean± standard error of the means (SEM, n = 6). One-way ANOVA with Fisher’s post hoc test was used for comparison between groups; ***p<0.001 groups vs positive control (C+ = 10% Triton X-100), ^$ p<0.01 groups vs negative control (C- = without 10% Triton X-100 nor NGLC).</p

Proliferating cell nuclear antigen (PCNA) and thyroxin hydroxylase (TH) western-blots analysis.

<p>SN4741 cells were cultured for 7 days with a high concentration (50 μg/ml) of nanocrystalline glass-like carbon (NGLC) microflakes. PCNA (<b>A</b>) and TH (<b>B</b>) analysis for relative protein expressions were expressed as percentage. The data are shown as mean± standard error of the means (SEM, n = 3) and Student’s t tests were used for statistical significance between two groups; *p<0.05 and ***p<0.001.</p

MTT assay for cell metabolism and proliferation.

<p>SN4741 were cultured in media containing five different microflakes concentrations (0 as control, 1, 5, 10, 20 and 50 μg/ml) of nanocrystalline glass-like carbon (NGLC) for 24 h, 3 days and 7 days (<b>A</b>). Phosphorylate histone 3 (PH3) and SMP30 analysis by SDS-page western blot, shows the analysis at 24 h (<b>B</b>) and 7 days (<b>C</b>) using NGLC at 50 μg/ml vs control (without NGLC). Quantification of relative PH3 and SMP30 proteins expression (%) showing the normalized data with control sample at 24 hours and 7 days of culture (<b>D)</b>. Data are expressed as mean±standard error of the means (SEM, n = 6). One-way ANOVA with Fisher’s post hoc test was used for comparison between different groups; ***p<0.001 groups vs control.</p

Confocal immunofluorescence of functional and cell maturation markers in the SN4741 cells at long term in culture.

<p>Nuclear marker DAPI (blue-label), synaptophysin (SYP: red-label) and G-protein-regulated inward-rectifier potassium channel 2 (GIRK2: green-label) proteins immunofluorescence were localized in SN4741 cells cultured on plastic wells (<b>A1-A4</b>), baseline thermal-treated non-carbon (BTTN) film (<b>B1-B4</b>), NGLC 5-nm-thick film (<b>C1-C4</b>), NGLC 20-nm-thick film (<b>D1-D4)</b> and NGLC 80-nm-thick film (<b>E1-E4</b>). Colocalization (merge) as yellow-label and the specific protein markers related with functional differentiation is also quantifying (<b>F</b>). Brightfields images of BTTN (<b>B5</b>) and 5 nm (<b>C5</b>), 20nm (<b>D5</b>), 80nm (<b>E5</b>) NGLC films are located in the right column of the panel. Colocalization percentage of specific protein markers for functional differentiation SYP and GIRK2 was also calculated (<b>F</b>) by means of Pearson's correlation coefficient, as previously described by Dunn et al. (2011) using the Fiji-ImageJ software and the plugin ‘Colocalization Threshold’. Data are expressed as mean±standard deviation (SD, n = 6); **p<0.01 and ***p<0.001 vs control (cells cultured in plastic wells); ‘<i>f</i>’ = p<0,001 mark differences between NGLC samples and baseline thermal-treated non-carbon (BTTN) film.</p

Influence of the NGLC film thickness in the SN4741 cell culture on the films.

<p>The two left pictures columns of the panel (PAF-fixed cells) show the microscopy images for DAPI immunofluorescence (nuclei maker: blue label) at low magnification (5X) showing different patterns of growth after 2 weeks. The three right pictures columns (live cells) show images obtained in vivo at low magnification (5 X) after labeling the nuclei with Hoechst 33342 (blue-marker). The diagram to the right in the first row shows the photographed areas and the limits between the NGLC film and the PMMA substrate. In the Apical Zone it is shown film and PMMA limits; Bottom Zone shows the end of the film area and in the Internal Zone the view is exclusively film surface. Fixed cells grown in monolayer on a glass slide (<b>A1-A2</b>), in plastic surface of the well (<b>A3-A4</b>), or in a baseline thermal-treated non-carbon (BTTN) film (<b>B1-B5</b>; Fig <b>B3</b>: apical zone, <b>B4</b>: internal zone, <b>B5</b>: bottom zone) as experimental controls. NGLC 5-nm-thick film (<b>C1-C5</b>), NGLC 20-nm-thick film (<b>D1-D5</b> and <b>E1-E5</b>) and NGLC 80-nm-thick film (<b>F1-F5</b>) were used for cell culturing on top of them. Areas corresponding to PMMA and NGLC films are differentiated in the pictures (<b>C3-C5</b>). Also spontaneously cell linear arrangement together with directionality of cell growth and cells orientation (<b>D1;</b> arrows) is shown in the bottom area of the film meanwhile growing in the internal film zone has the same cell arrangement effect (<b>D2 and E1-E2</b>). Separation between PMMA and the Film (<b>D4</b>) and the internal zone of the film (<b>D5</b>) are shown demonstrating how the cells firmly adhere to the film. Linear cell growing is observed in the bottom zone of the film (<b>E3-E4</b>) meanwhile the internal zone of the film shows adhered cells (<b>E5</b>). Fixed cells grown on top of the NGLC 80-nm-thick film (<b>F1-F2</b>) showing the limits of PMMA substrate (<b>F3-F4</b> arrow) and film and a detailed film bottom is also shown (<b>F5</b>).</p

Images and characterization of nanocrystalline glass-like carbon (NGLC) films using different microscopy techniques (optical, electron microscopy and atomic force microscopy (AFM)).

<p>NGLC films with different thicknesses (~5, ~20 and ~80 nm) and baseline thermal treatment non-carbon film (BTTN) used in the experiments showing different degrees of transparency (<b>A</b>). The thinnest sample (~5 nm) shows the high transparency (86%) and moderately high electrical conductivity (sheet resistance: 7.8 kΩ/sq). They were obtained on a copper surface by carefully controlling the gas flow ratios used in the CVD procedure (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173978#sec002" target="_blank">Material and Methods</a>) (<b>B</b>). These nanostructured carbon thin films are composed of few-layer and curved graphene fragments of ~3 nm in average size joined by an amorphous carbon matrix (<b>C</b>), which replicates the structure of widely used glass-like carbons. Carbon-coated copper after CVD (<b>D</b>) and flakes of 82±37 μm length and approximately 300 nm thicknesses (<b>E</b>, <b>F</b>). Raman spectroscopy of NGLC films with different thicknesses (~5, ~20 and ~80 nm), microflakes and graphene, showing the broad spectra of amorphous carbons compared to highly crystalline graphene (<b>G</b>). Surface roughness of PMMA/Carbon film composites measured by AFM, showing increasing roughness on those with a 20- and 80-nm-thick carbon films (<b>H</b>). See also Romero et al., 2016 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173978#pone.0173978.ref015" target="_blank">15</a>] (Doi:<a href="http://dx.doi.org/10.1016/j.cej.2016.04.005" target="_blank">10.1016/j.cej.2016.04.005</a>).</p

Influence of the concentration of nanocrystalline glass-like carbon (NGLC) microflakes on cell growth.

<p>Changes in the cellular architecture in a monolayer SN4741 cells culture (<b>A-D</b>) after 7 days of culture with or without (control) NGLC microflakes at three different concentrations (10, 20 and 50 μg/ml). Control is shown in a monolayer culture with high confluence after 7 days (<b>A1</b>). An image analysis was performed using Image J/Fiji software and the pluging Canny Edge which inform about the outline structure (<b>A2</b>). A cellular architectural analysis was also done by using the plugin Skeletonized 2D/3D (<b>A3</b>). Brightfield of the same experiment at NGLC 10 μg/ml (<b>B1</b>) showing higher cellular network organization (<b>B2</b>) demonstrated by the skeletonized analysis (<b>B3</b>). Treatment with NGLC 20 μg/ml (<b>C1</b>) shows higher structural changes respect to the control (<b>C2</b>) producing greater networks (<b>C3</b>: see arrows). Cell culture with NGCL 50 μg/ml (<b>D1</b>) shows a cellular network with the greater branches among cell populations (<b>D2</b>) as it is indicated by the arrows (<b>D3</b>). Quantification of the branches length (<b>E</b>) was obtained by analysis of Skeletonized 2D/3D and Microscope Measurement Tools, ImageJ/Fiji software. Magnification = 5X. One-way ANOVA with Fisher’s post hoc test was used for comparison between different groups: *p<0.05 groups vs control.</p

List of TaqMan Gene Assay IDs.

<p>List of TaqMan Gene Assay IDs.</p

Descriptive statistics and tests for the investigated reference genes.

<p>Descriptive statistics and tests for the investigated reference genes.</p