Cultured human podocytes (EV-shRNA and KLF6-shRNA) were treated with and without ADR for 24 hours. (A) Immunofluorescence images using Hoechst staining was performed to assess for apoptotic bodies. Representative images of six independent experiments are shown (original magnification, ×20). Arrows show apoptotic bodies. (B) To quantify apoptosis, annexin V/propidium iodide staining in combination with FACS was performed (n = 3). Kruskal-Wallis test with Dunn’s post-hoc test, *P < 0.05. (C) Immunofluorescence images of cytochrome c staining in EV-shRNA and KLF6-shRNA podocytes treated with and without ADR are shown. Representative images of six independent experiments are shown to demonstrate the distribution of cytochrome c staining (original magnification, ×20). Arrows show mitochondrial cytochrome c distribution. Arrowhead shows cytosolic distribution of cytochrome c. (D) Activation of the intrinsic apoptotic pathway was assessed using Western blot analysis for cleaved caspase-9, pro–caspase-3, and cleaved caspase-3 and is shown with β-actin as a loading marker. The representative images of six independent experiments are shown in the top panel. Quantification by densitometry (n = 6) is shown in the bottom panel. Kruskal-Wallis test with Dunn’s post-hoc test, *P < 0.05 vs. treated and untreated EV-shRNA, ^#P < 0.05 vs. all groups, †P < 0.01 vs. all groups. (E) To confirm whether the preservation of KLF6 prevents apoptosis, Western blot analysis was performed on human podocyte lysates from ADR-treated podocytes with (LentiORF-KLF6) and without (LentiORF-control) KLF6 overexpression. Representative images of three independent experiments are shown in the top panel (cleaved caspase-3 and pro–caspase-3 are from the same samples run on parallel gels). Quantification by densitometry (n = 3) is shown in the bottom panel. Mann-Whitney U test, ***P < 0.001, **P < 0.01.