Fig 8. Neural-like processes induced by NGLC films.

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 (A1, B1, C1, D1) showed a monolayer culture image without visible sign of differentiation. Cells cultured on BTTN films presented poor signal for SYP (A2) and GIRK2 (A3) markers. Cultures on NGLC 5-nm-thick films showed increasing neural-like processes with SYP/GIRK2 colocalization (B1-B4) where an axon-like process is measured (1843 μm) (B5). 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 (C1-C5) a greater synaptic capacity (SYP/GIRK2 signal) where a cell polarized and oriented growing towards the limit of the NGLC film (C4) (see also Fig 7, 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 (D1-D5) 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.