Figure legends
Figure 1 Schematic diagram of the sampling, quenching and extraction procedures for Hela cells from 2D monolayer cultures and 3D MTS.
Figure 2 Parameters evaluated during the optimization of metabolome extraction and analysis conditions. The cell collection procedure remains constant for all different combinations of metabolome quenching, extraction and GC/LC-MS analysis. Metabolic quenching is achieved by adding quenchers to the cells. Then, cell separation and metabolite extraction are performed at the same time by scraping the cells in different extraction solvents.
Figure 3 Comparison of the leakage degree of metabolites in different quenchers. (a) The leakage degree of amino acids in different quenchers; (b) The leakage degree of organic acids, phosphate sugars, adenosine nucleotide and coenzymes in different quenchers. Values were the mean of three biological replicates. Error bars represented standard deviation.
Figure 4 The relationship between the degree of leakage and molecular weight in 2D cells. The degree of leakage was calculated based on the level of metabolites in the quencher and the overall metabolite level (quencher + extractant).
Figure 5 The total number of metabolites obtained by each extraction method.
Figure 6 The sum of the different kind metabolites identified in cell extracts only in 2D cells. Significant analysis was performed against N2-A group.
Figure 7 Eighteen amino acids were identified in 12 quenching-extraction methods. Values were the mean of three biological replicates. Error bars represented standard deviation.
Figure 8 Eight organic acids, nine sugar phosphates, eight adenosine nucleotides and coenzymes were identified in twelve extraction methods. Values were the mean of three biological replicates. Error bars represented standard deviation.
Figure 9 The application of 12 intracellular extraction methods on 3D MTSs. (A) Heat map of the degree of leakage of the two quenchers in 3D MTSs; (B) 43 metabolites were identified in twelve extraction methods in 3D MTSs.
Figure 10 The measured concentrations of extracellular: glucose, glutamine, ammonium and lactate. The culture time of 2D cells and 3D MTSs was shown on the bottom and top axis, respectively.
Figure 11 Heat map of the absolute amount of metabolites. The 2D/3D cells were treated with a medium containing 1 μM of DOX for 48 h.
Figure 12 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways affected by DOX using pathway enrichment with the MetaboAnalyst pathway analysis module.