Fig. 3
From: MASTL overexpression promotes chromosome instability and metastasis in breast cancer
MASTL overexpression increases the phosphorylation of proteins involved in actin, stress kinase signalling pathways. a Experimental design, and SILAC workflow. MCF10A control and MASTL were grown for >6 doubling in heavy and light SILAC media. Heavy and light cultures were harvested reduced and alkylated, mixed 1:1, acetone precipitated and then digested with LysC/Trypsin. Phosphopeptides were enriched using the “EasyPhos” method, enriched peptides were then analysed on easy-nLC1200 coupled to a QExactive-HF mass spectrometer. b Volcano plot depicting the average Log2H/L ratio and –Log10P-value of the detected phosphopeptides. These were grouped into two populations according to significance (p < 0.05 cut-off, y~1.2 dotted line, n = 1961) and either; Log2H/L < −1 (DOWN; blue shading, x = −1 dotted line, n = 119), or Log2H/L > 1 (UP; red shading, x = 1 dotted line, n = 167). c KEGG pathway analysis of top UP and DOWN phosphosites. d Manual curation of top significant UP and DOWN phosphosites into an interconnected signalling network. The interaction between each protein is denoted by the arrow connecting the proteins. Sites that were close to significant indicated with * (p < 0.07). Specific phosphosites are colour-coded from blue (DOWN) to red (UP), based on observed Log2 ratios. Known functional phosphosites annotated in the PhosphoSitePlus database underlined in bold. e Western blot validation of the mass spectrometry results. MASTL and control lysates probed with total and phospho-specific antibodies (blots are representative of three biological replicates). f Heatmap from MagPix assay showing fold-change in phosphorylation of key proteins involved in AKT signalling between Control and MASTL cell lysates. Overexpressing cells normalised to the background signal, and relative to control (n = 3, unpaired t-test, *p < 0.05, ***p < 0.001)
