Figure 4.

Effects of CRISPR/Cas9 knockout (KO) on the expression of mitochondrial proteins and cell death upon AT 101 treatment. (A) Immunoblotting analysis of ATG5 expression in MZ-54 ATG5 KO cells and U343 ATG5 KO cells compared to control. (B) Immunoblotting analysis of MAP1LC3B expression in MZ-54 ATG5 KO cells compared to control treated with AT 101 (15 µM), bafilomycin A₁ (BAF; 10 nM) or DMSO (Con). Bafilomycin A₁ was added 4 h before harvest. (C) Immunoblotting analysis of mitochondrial protein expression in MZ-54 ATG5 KO cells compared to control treated with AT 101 (15 µM) or DMSO (Con). The mean protein expression was quantified and normalized to the loading control (100%). Experiments were repeated 3–5 times. (D-H) Cell death was quantified by flow cytometric analysis of ANXA5/annexin binding and PI uptake after treatment with AT 101 (15 µM), staurosporine (STS; 1 µM, 24 h; 3 µM, 6 h) or DMSO (Con). Measurements were performed with U343 cells (D, F), MZ-54 cells (E, G) and MEF WT versus MEF bax bak1 DKO cells (H). Cell death includes ANXA5/annexin-positive only and PI-positive only as well as double-positive cells. Experiments were repeated at least 3 times. Data are mean + SEM from n = 9–12 samples (10,000 cells measured in each sample, 3–4 samples per experiment).