Figure 1.
Three different experimental PC12 cells culture conditions for DNA microarray study.
(A) laminin-coated PS cell culture dish. (B) laminin-coated 2-mm thick PMMA plate placed inside the cell culture dish. (C) laminin-coated 2-mm thick PMMA plate with through holes placed on the 2-mm thick PDMS layer. (D) A schematic view of a 2 mm thick PMMA plate with through holes placed on the PDMS substrate.
Figure 2.
The main experimental and analytical steps of the reported study.
The growth and differentiation of PC12 cells were investigated on 2 different polymeric materials: polystyrene (PS) and poly(methyl methacrylate) (PMMA). To determine trans acting effect of PDMS, differentiation was also initiated on PC12 cells seeded on perforated PMMA resting on a PDMS layer. In the next step, we examined whether the cell morphology changes between different polymeric materials can be identified (Fig. 4). Flow cytometry was employed to analyze cell cycle phases in all tested conditions (Fig. 5). For comprehensive examination of the possible effects of three different polymeric materials, DNA microarray based on transcription profiling was employed. PS.ND denotes cells growing on polystyrene, PS.D denotes cells differentiating on polystyrene, PMMA.ND denotes cells growing on PMMA, PMMA.D denotes cells differentiating on PMMA, PMMA-PDMS.ND denotes cells growing on PMMA resting on a PDMS slab and PMMA-PDMS.D denotes cells differentiating on PMMA resting on a slab of PDMS.
Figure 3.
A. Cell viability on the tested samples (Calcein/PI test).
B. Measurement of cells metabolic activity in response to the different substrates – MTT test. Error bars represent mean ± SD from three independent experiments.
Figure 4.
Morphological changes of PC12 cells cultured on: PS (green frame), PMMA (red frame), PMMA-PDMS (blue frame) after 4 days of NGF treatment.
Different types of neurites can be found on all polymeric materials used in the presented study: multiple neurites from the cell body with multiple branches, and multiple neurites from the cell body with a minor branch.
Figure 5.
A. The overlay histogram for PC12 cells treated with NGF for 24 hours (blank graph) and for 96 hours (solid, grey graph).
The Cyflogic program provided the estimate of percentage of cells in each of the intervals: sub-G1, G1, S, and G2/M. B. PC12 cells were collected after 24, 48, 72, 96 hours from cell seeding (left column, non-differentiated cells), and afterwards 24, 48, 72, 96 hours from NGF treatment (right column, differentiated cells). Samples were analysed by flow cytometry, as described in Materials and Methods. Error bars show mean ± SD from three independent experiments.
Figure 6.
The top10 differentially expressed genes between NGF-differentiated and non-differentiated PC12 cells similarly expressed in all culture conditions: PS, PMMA, PMMA-PDMS.
Notice: the magnitude of the fold-change is similar at all applied culture conditions.
Table 1.
Gene ontology processes and pathways associated with the similarly expressed genes at all tested conditions.
Figure 7.
Gene expression profiling of NGF-differentiated PC12 cells.
A Venn diagram was utilized representing each contrast as a circle enclosing the number of more than two fold regulated genes (up and down) relative to the PC12 cells cultured on PS. The number of genes similarly regulated on more than one contrast is presented in the overlapping region of the corresponding circles. Up to 10 top genes with their calculated logFC are listed for each contrast.
Table 2.
Functional classification clustering analysis presenting gene ontology processes associated with the 642 differentially expressed genes in NGF-differentiated PC12 cells cultured on PMMA-PDMS versus PS (Enrichment Score>0.5, Final group member threshold – minimum 10 genes).