(A) Schematic representation of the experimental strategy to analyze the changes in transcriptome, proteome, and phosphoproteome profiles of MCF7 breast cancer cells after 24 hr of metformin treatment (10 mM). The coverage and overlap of the genes and gene products identified by the RNAseq and proteomic approaches are illustrated in the Venn diagram. Percentage and number of phosphorylation sites with protein expression levels quantified or not are shown in the pie chart.

(B) The correlation between the quantified proteome and transcriptome changes of each gene product after metformin treatment is shown as a density scatterplot.

(C) Protein expression and phosphorylation levels were compared and represented in the scatterplot. Proteins and phosphorylation sites were considered “regulated” by metformin according to the t test (FDR < 0.05). Comparison between biological replicates of MS-based phospho-proteomic experiments of metformin treated (Metf1-3) and untreated (NT1-3) cells. Each dot represents one protein.

(D) Metformin inhibits the mTOR pathway and hyperactivates AMPK. The metformin-induced change at the phosphorylation, protein, and transcript levels of mTOR direct and indirect substrates and AMPK direct substrates are shown as bar graph in a Log2 scale. Median and SD are shown.