Figure 2

Nucleolar proteome following senescence induced by sodium butyrate treatment in NIH3T3 cells (A) SILAC labeling of NIH3T3 cells to determine nucleolar protein dynamics in sodium butyrate induced senescent cells. The workflow shows SILAC labeling of cells in a heavy (R6K4) media and a light (R0K0) media for 5 days followed by treatment of cells with vehicle or sodium butyrate. The cells grown in the lighter media (R0K0) were treated with sodium butyrate for 4 days. Subsequently, the nucleolus from both control and sodium butyrate treated cells were isolated. (B) Coomasie staining of the SDS PAGE gels of control and sodium butyrate treated nucleolar lysates from cells grown in SILAC media. The protein nucleolar lysates were quantitated using Bradford-Lowry reagent and 50 ug of total nucleolar lysate were run on a SDS-PAGE. Note the changes in the protein composition of control and treated nucleolar lysate. (C) Dynamic regulation of nucleolar proteins identified from quantitative proteomics by using SILAC based LC MS/MS following senescence induced with sodium butyrate in NIH3T3 cells. SILAC ratio was defined by the log2 of the averaged R0K0/R6K4 ratios of the peptides identified for each protein. (D) Functional classification of 344 proteins identified from SILAC based mass spectrometry in sodium butyrate induced senescent NIH3T3 cell nucleoli. (E) Analysis of the fold change of the proteins in the nucleolus following sodium butyrate induced senescence based on their cellular function.