FIG. 5. Immunofluorescence staining of glutathionylated p53 in HCT116, HT29, and T47D cells upon treatment with oxidizing and DNA damaging agents.

Immunofluorescence showing the presence of p53-glut in untreated HCT116 cells (A); HCT116 cells treated with 0.6 mM diamide for 30 min (B); HCT116 cells treated as in (B) followed by 10 mM DTT treatment for 30 min (C); HCT116 cells treated with 0.4 mM H₂O₂ for 30 min (D); HCT116 cells treated as in (D) and postincubated in H₂O₂ free medium for 2h (E); HCT116 cells treated with 5 μM doxorubicin for 5h (F), and HCT116 cells treated with 10 μM cisplatin for 5h (G). Immunofluorescence showing the presence of p53-glut in untreated HT29 cells (H); HT29 cells treated with 0.6 mM diamide for 30 min (I); untreated T47D cells (J), and T47D cells treated with 0.6 mM diamide for 30 min (K). After the respective treatments, cells were fixed using 4% paraformaldehyde and incubated with p53-glut antibody (1:500 dilution) followed by goat anti rabbit Alexa Flour 594 secondary antibody. The cells were photographed using an Olympus IX 81 microscope. Similar patterns of staining were observed in three independent experiments.