Figure 5.

Signalling by apoptotic and oxidative stress-related pathways. The effects of copper and quercetin on the expression of p53, Bax, PUMAα/β, and NME1/NME2 in moderate oxidative stress were monitored for 24 h from the beginning of treatment with 0.5 mM CuSO₄ and 150 μM quercetin, and proteins of total cell extracts were separated on 10 or 11% SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Blots were probed with primary antibodies, followed by the appropriate horseradish peroxidase-labelled secondary antibody. Immunoreactivity was detected using enhanced chemiluminescence. β-Actin was used as loading control for normalization. Data are expressed as mean ± SD from up to five immunoblots of three independently prepared cell lysates. Exposure to copper and quercetin did not affect p53 and Bax expression. However, quercetin diminished copper-induced upregulation of PUMA and stimulated NME1 expression. Following densitometric analysis, obtained data were analyzed with one-way ANOVA followed by Dunnett's test (^aP < 0.05, ^cP < 0.001). Representative Western blots are also presented.