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Figure 5 | Molecular Cancer

Figure 5

From: A yeast-based genomic strategy highlights the cell protein networks altered by FTase inhibitor peptidomimetics

Figure 5

FTI-277 treatment reduces the phosphorylation of ribosomal protein S6 in HeLa and MCF-7 cells. The levels of RPS6 (p70) phosphorylation in HeLa and MCF-7 cells were measured using the ScanR analysis software based on the intensity of AlexaFluor 555-conjugated PhoS6 antibody (Ser 235/236: Cell Signalling) recorded from images acquired from samples treated with 5 μM FTI-277 (panel FTI-277) or not treated (panel NT) in the presence of 0.1% serum for 24 h. Five wells per condition were considered. Cells were seeded in a 96-well plate and fixed and stained as described in Methods. Sixteen images were acquired randomly per well with a 20× objective using the RFP filter. The mean intensity within the red channel was automatically calculated by ScanR analysis software considering at least 600 cells. A, Images representative of FTI-277-treated and untreated HeLa cells stained with PhoS6 antibody and Hoechst, single and merged pictures are shown. B, Images representative of FTI-277-treated and untreated (NT) MCF-7 cells, stained as in (A); C, Analysis of the distribution of the phosphorylated RPS6 population in HeLa and MCF-7 cell lines. Hoechst staining was used for cell identification (main mask), as described in Methods. The minimal threshold of the PhoS6 signal was set at mean intensity 43.8 for HeLa and at 63.8 for MCF-7 cells based on the background intensity. The percentage of cells having a PhoS6 signal localised within this gate was calculated and plotted in the graph.

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