Figure 5. Adding 0.1 µM H₂O₂ to FBS starved cells regulates p27Kip1 phosphorylation and localization, favoring proliferation.

Melanoma (A375) cells grown in complete medium with 10% FBS were arrested by FBS starvation (0% FBS) for a period of 24 h and then cells were incubated with different concentrations of H₂O₂ (0.01–10 µM) or to 10% FBS. (A) Intracellular ROS levels measured by DCFH-DA assay. (B) Cell proliferation rate evaluated by the MTT assay. (C) Representative images of p27Kip1 immunocytofluorescence showing the subcellular localization of the protein. DAPI: staining of nuclear DNA; p27Kip1: FITC staining of p27Kip1 protein. (D) Percentage of positive (□) cytoplasms and positive (■) nuclei for p27Kip1 relative to the total number of counted cells. (E) The expression of p27Kip1, p27pS10 and p27pT198 analyzed by western blot. (F) Relative densitometric values of () p27Kip1 levels, (□) p27pS10 and (■) p27pT198. Actin densitometric values were used to standardize for protein loading. Results are referred to control incubated with 10% FBS. (A, B, D and F) Data are expressed as mean ± SD. *p<0.05, **p<0.01 and ***p<0.001 vs. cells incubated with 10% FBS; ^#p<0.05, ^##p<0.01 and ^###p<0.001 vs. FBS-starved cells not-exposed to H₂O₂.