Quantitation of apoptosis and necrosis by flow cytometry in cisplatin-treated renal tubular cells: The effect of GADD45γ overexpression.

(A) Detection of overexpressed GADD45γ RNA by RT-PCR. HRE cells were infected with 50 MOI of adenoviral vectors for 24–48 hours. (B) Detection of overexpressed GADD45γ protein by Western blot. HRE cells were infected with a serial amount of Ad-GADD45γ for 48 hours. HRE cells were infected with 50 MOI of adenoviral vectors for 48 hours and then treated with 12.5 μM cisplatin for indicated time periods (panels C-F). (C) Representative scatter plots of flow cytometry. (D) Quantitation of apoptotic cells (PI negative and Annexin V positive, Q4). *p(E) Quantitation of necrotic cells (PI positive and Annexin V positive, Q2). *p(F) Quantitation of viable cells (PI negative and Annexin V negative, Q3). *p<0.05 vs control and Ad-GADD45γ/cisplatin. #p<0.05 vs all other corresponding values. Each bar represents mean ± SD of 3 experiments. *p < 0.05 vs corresponding Ad-LacZ transduced cells (One-way ANOVA followed by Bonferroni's post-hoc comparisons tests). MOI, multiplicity of infection. Ad-GADD45γ(G) represents adenoviral vectors expressing GADD45γ; Ad-LacZ, LacZ adenoviral vectors.