Figure 1.
HepG2/C3A cells were grown using either classical 2D flat culture techniques or as 3D microgravity spheroid techniques and analysed by mass spectrometry, immunofluorescence or using standard assays for DNA, ATP or protein.
Table 1.
Summary of changes observed when 3D spheroid culture is compared to 2D flat culture.
Figure 2.
Immunohistochemical staining of actin and DAPI staining of DNA.
C3A cells were grown using either the classical cell culture techniques (2D) or grown as spheroids (3D). Cells from 2D cultures were fixed directly while cells grown in 3D were fixed and sectioned. a, c, e and g: HepG2/C3A exponentially growing cells (2D), b, d, f and h HepG2/C3A cells at dynamic equilibrium. a and b: phalloidin staining of filamentous actin, c and d: total actin staining; e and f same images as in a and b overlayed with DAPI staining for DNA; g and h DAPI same images as in a and b but with DAPI staining alone. All photographs were made at the same magnification: the bar in h indicates 25 µM.
Figure 3.
The log2 ratio change plotted against the statistical reliability (volcano plot).
All proteins are shown as grey dots in the graph. Particular groups of proteins are then highlighted in black: a) Actin and actin-associated structural proteins; b) Tubulin and tubulin-associated structural proteins; c) Keratins; d) Glycolytic enzymes; e) Pentose phosphate pathway enzymes; f) TCA cycle enzymes.
Figure 4.
Immunohistochemical staining of tubulin and keratin.
C3A cells were grown using either classical 2D flat culture techniques or as 3D microgravity spheroid techniques. Cells from 2D cultures were fixed directly while cells grown in 3D were fixed and sectioned. a, c, e and g: HepG2/C3A exponentially growing cells (2D), b, d, f and h HepG2/C3A cells at dynamic equilibrium. a and b: acetylated tubulin to highlight filaments, c and d: staining of α-tubulin; e and f keratin 8. All photographs were made at the same magnification: the bar in h indicates 25 µM.
Figure 5.
Dot plot of the ratios of protein abundance (dynamic equilibrium/exponential growth states) in various sub-cellular organelles.
a) Electron transport chain: complexes I, II, III and IV and the ATPase F1 and F0. b) HnRNP: core, E complex, H complex and scaffold. c) Ribosome: cytoplasmic large 60S and small 40S subunits, mitochondrial large 39S and small 28S subunits, ribophorin linker. d) Proteasome: core, lid-base, lid and ubiquitin proteins. Error bars indicate the standard deviation of the proteins in each group and the thick grey bar indicates the average. (n = 4). For a dot blot of the spliceosome, see File S1, tab P4_Dot-plot_Spliceososme.
Figure 6.
Determination of the degree of protein oxidation (carbonylation).
Proteins were extracted from cells grown using either classical 2D flat culture techniques or as 3D microgravity spheroid techniques. a) OxyBlot detection of carbonylated proteins; b) loading control visualised with Coomassie Brilliant Blue total protein stain; c) Levels of protein carbonylation expressed as average % optical density: *indicates statistically significant difference (t-test, p<0.05, n = 3).
Figure 7.
Relationship of the changes in the proteome following its adaptation from 2D to 3D culture with structural and physiological properties.