Identification of cytoskeleton-associated proteins whose depletion induces non-apoptotic cancer cell death.

Abstract

<p>(A, B) MCF7 (A), HeLa (B) and U-2-OS (B) cells were left untreated, treated with Oligofectamine (Oligo) or transfected with control siRNA (CT) or three independent siRNAs against the indicated targets individually (8 nM; A) or in pools (3×6.67 nM; B). Cell density was measured after 72 h by the MTT reduction assay. (C) MCF7-Bcl-2 or MCF7-pCEP (control) cells were transfected as in (B). <i>Left bottom</i>, After 96 h, cell death was determined by counting Hoechst 33342-stained cells with condensed nuclei (three random fields of 100 cells). TNF (20 ng/ml, 24 h) served as a positive control for Bcl-2 sensitive apoptotic cell death. <i>Left top</i>, Western blot confirming overexpression of Bcl-2 in untreated MCF7-Bcl-2 cells. <i>Right</i>, Examples of images of Hoechst 33342-stained nuclei of MCF7-pCEP and MCF7-Bcl-2 cells 96 h after transfection with indicated siRNAs. (D) MCF7 cells were treated as in (B). <i>Left</i>, After 60 h, DNA was stained with propidium iodide and cell cycle distribution analyzed by flow cytometry (FL-2A). <i>Right</i>, Examples of histograms showing cell cycle distribution of cells 60 h after transfection with indicated siRNAs. Values represent means + SD of three independent experiments (A, C, D) or triplicates in one representative experiment (B, n = 3). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, vs. control siRNA-transfected cells or as indicated (C).</p