We believe that this cellular analysis is a step beyond and corroborates with the traditional kinome profiling data because it provides an unbiased read-out of the biological consequences of treatment with a small molecule inhibitor in live cells.
To evaluate the cellular distribution of the ubiquitylated proteins, Gene Ontology (GO) cellular compartment analysis was conducted using STRAP [ 35].
Since chemotherapy prior to surgery might have some effect on cellular nature, analysis was limited to cases without such treatment (pure-NAC cases), resulting in significantly higher expression in tumor (n = 18) than normal tissues (n = 9) (P = 0.012; Fig. 1D).
Sub-cellular localization analysis was performed using SignalP.
In addition, various standard biochemical tests and cellular fatty acids analysis was performed by the German Resource Centre for Biological Material (DSMZ, Braunschweig, Germany; http://www.dsmz.de/).
Propidium iodide staining and cellular DNA content analysis was as previously described.[ 7] Western blot analysis was as previously described.[ 15] Briefly, cells were lysed in M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) followed by centrifugation (16 000 g, 20 min, 4 °C).
DNA content and cellular granularity analysis were performed using FACSCalibur and CellQuestPro software as described by Korwek et al. (2012).
However, these molecules, and especially myo-inositol can take part in various cellular processes, and further analysis was therefore required to understand the cellular processes triggering PCD in atips1.
In addition, after completion of cellular analysis, plasma was obtained after centrifugation and stored at − 80 °C for subsequent quantification of following circulating cytokines: IL-6, IL-8, IL-10, IL-1RA, TNF-α, and IFN-γ.
By plotting cell index values over time, a precise real-time cellular analysis profile was generated.
