Gene expression changes in SEM-K2 and RS4:11 cells and changes in cell morphology in SEM-K2 cells indicative of triple death mode killing by obatoclax. Cells were treated for 6 hours in duplicate with vehicle or obatoclax at approximate 72-hour EC50 or 3 × EC50 concentrations, and respective cDNAs were used for Affymetrix U133_Plus_2 microarray analysis. (A) Venn diagrams showing numbers of probesets up- or downregulated by obatoclax and their overlap between different obatoclax concentrations for each cell line. Significant changes were determined using differences between mean log2 expression levels in cells treated with obatoclax at EC50 or 3 × EC50 and mean log2 expression levels in vehicle-treated cells (analysis of variance, P ≤ .05 and >50% up-/downregulation in at least a single mean log2 expression level comparison of obatoclax at EC50 or 3 × EC50 vs vehicle in either cell line). For a complete list of significantly up- or downregulated probesets, see supplemental Table 1. (B) Mini heatmaps of autophagy- and necroptosis-related probesets showing upregulation (red) or downregulation (green) in expression at or near significance (P ≤ .05) with obatoclax treatment at EC50 or 3 × EC50 in either cell line. Expression levels for all 3 conditions (vehicle, obatoclax EC50, obatoclax 3 × EC50) and 2 biologic replicates per condition (6 samples) were drawn for each cell line; mean log2 expression levels of respective vehicle treatments were used as reference. (C) SEM-K2 cells treated with vehicle, obatoclax, or ADR (positive control for apoptosis) for indicated times were harvested, stained, and sectioned for electron microscopy. Indicated arrowheads mark condensed chromatin and/or nuclear fragmentation of apoptosis, autophagic structures, or swollen Golgi (obatoclax; 24 hours) or endoplasmic reticulum (obatoclax; 48 hours, 72 hours) of necroptosis. Magnified insets are in boxes. Note morphologic changes of all 3 modes of death in single cell (obatoclax, 48 hours; right middle panel).
