Western blot analyses and MTT assays of obatoclax effects on apoptosis, autophagy, and necroptosis by itself or combined with cell death pathway inhibition in MLL-AF4 ALL cell lines. (A) Time-course Western blot analysis of PARP cleavage using whole-cell lysates from SEM-K2 and RS4:11 cells after exposure to vehicle, or obatoclax at concentrations approximating 72-hour EC₅₀ or 3 × EC₅₀, or ADR (positive control for apoptosis) for indicated times. Increases in cleaved PARP by 72 hours with obatoclax treatment indicate apoptosis. (B) Time-course Western blot analyses of LC3-I to LC3-II conversion and p62 protein levels after exposure to vehicle or obatoclax at concentrations approximating 72-hour EC₁₀, EC₂₅, EC₅₀, or 3 × EC₅₀ for indicated times. Note time- and dose-dependent increases in LC3-I to LC3-II conversion and lack of p62 accumulation with obatoclax treatment. (C) Unchanged obatoclax-induced cytotoxicity with genetic autophagy inhibition in SEM-K2 cells. Representative Western blot depicting BECN1 protein knock down by BECN1 siRNAs #1 and #2 during obatoclax treatment compared with cells transfected with control siRNA. Cell lysates were prepared after obatoclax treatment of transfected cells for indicated times (left). Surviving fraction plots of cell viability measured by MTT assays performed after 72-hour obatoclax exposure (6 replicates/condition per experiment) in cells transfected with control siRNA or BECN1 siRNA 2 (right). The assay was performed 3 times. Bars indicate standard error. Transfection with BECN1 siRNA #1 gave the same result (not shown). (D) Unchanged obatoclax-induced cytotoxicity by chemical autophagy inhibition in SEM-K2 (left) and RS4:11 (right) cells. Surviving fraction plots show cell viability measured by MTT assays after 72-hour exposure to increasing obatoclax concentrations with indicated fixed 3-MA concentrations. Assays were repeated 4-7 times (6 replicates/condition per experiment). Bars indicate standard error. (E) Attenuation of obatoclax-induced cell death in SEM-K2 (left) and RS4:11 (right) cells by chemical necroptosis inhibition ± apoptosis and/or autophagy inhibition. Surviving fraction plots show cell viability measured by MTT assays 72 hours after treatment with increasing obatoclax concentrations ± 50 μM Nec-1 ± 20 μM zVAD-fmk and/or 0.5 mM 3-MA. MTT assays were performed 3-6 times (6 replicates/condition per experiment). Bars show standard error. * P ≤ .05 vs obatoclax alone; # P ≤ .05 vs obatoclax + Nec-1; ▲, P ≤ .05 vs obatoclax + Nec-1 + zVAD-fmk. Data in (D) and (E) were normalized to cells only treated with the relevant inhibitors alone or combined (supplemental Figure 1A). (F) Western blot analysis of SEM-K2 cells demonstrating dose-dependent increases in cleaved PARP indicative of more apoptosis with obatoclax treatment + Nec-1 for 72 hours compared with obatoclax alone, and reduction in obatoclax-induced PARP cleavage as well as attenuation of Nec-1 effect on PARP cleavage by zVAD-fmk (top). Also note the increase in LC3-I to LC3-II conversion induced by obatoclax in the presence of Nec-1 ± zVAD-fmk compared with obatoclax alone (bottom). Obatoclax concentrations are approximate 72-hour EC₅₀ (50 nM) or 3 × EC₅₀ (150 nM). Western blots are representative of 3 independent experiments. (G) Representative Western blot analyses of LC-I to LC-II conversion in RS4:11 cells treated with obatoclax at approximate 72-hour EC₅₀ (30 nM) or 3 × EC₅₀ (90 nM) ± Nec-1. Note increased (F) or unchanged (G) LC-I to LC-II conversion with Nec-1, indicating that obatoclax-induced autophagy was necroptosis-independent.