Journal: Molecular Cancer Therapeutics

Article Title: BH3-only proteins Mcl-1 and Bim as well as endonuclease G are targeted in spongistatin 1–induced apoptosis in breast cancer cells

doi: 10.1158/1535-7163.mct-08-1179

Figure Lengend Snippet: Figure 4. Spongistatin 1 induces the translocation of AIF and EndoG to the nucleus. A, cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 24 h. Nonnucleic and nuclear protein fractions were prepared and AIF and EndoG were detected by specific antibodies using Western blot analysis. Cytochrome c oxidase served as control for the quality of the extraction procedure. B, cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 24 h. Translocation of AIF and EndoG from mitochondria to the nucleus was analyzed by confocal microscopy. Blue, nuclei; green, AIF and EndoG. All experiments were done three times with consistent results. C, AIF and EndoG assume a functional role in spongistatin 1–induced apo- ptosis. MCF-7 cells were transfected or cotransfected with oligonucleotides encoding for either AIF siRNA and EndoG siRNA or a nonsense sequence. Cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 48 h. Apoptotic cells were quantified by flow cytometry. Results are percent specific apoptosis. Downregulation of AIF and EndoG protein levels was verified by Western blot. Equal protein loading was controlled by staining membranes with β-actin. Columns, mean of three independent experiments done in triplicate; bars, SE. ***, P < 0.001 (ANOVA/Bonferroni).

Article Snippet: Cells were blocked with 0.2% bovine serum albumin and incubated with specific antibodies against AIF (rabbit polyclonal IgG; Upstate) and EndoG (rabbit polyclonal antibody; Prosci).

Techniques: Translocation Assay, Western Blot, Control, Extraction, Confocal Microscopy, Functional Assay, Transfection, Sequencing, Flow Cytometry, Staining

Journal: Molecular Cancer Therapeutics

Article Title: BH3-only proteins Mcl-1 and Bim as well as endonuclease G are targeted in spongistatin 1–induced apoptosis in breast cancer cells

doi: 10.1158/1535-7163.mct-08-1179

Figure Lengend Snippet: Figure 6. Bim functions as a proapoptotic regulator upstream of mitochondria. A, MCF-7 cells were transfected with oligonucleotides encoding for either Bim siRNA or nonsense sequence. Cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 24 h. Nonnucleic and nuclear protein fractions were prepared and AIF and EndoG were detected by specific antibodies using Western blot analysis. Cytochrome c oxidase served as control for the quality of the extraction procedure. As a control, Bim protein level in cell lysates from nonsense and Bim siRNA cells was analyzed by Western blot. B, Bim and EndoG are the major regulators of spongistatin 1–induced cell death. MCF-7 cells were cotransfected with oligonucleotides encoding for either Bim siRNA or EndoG siRNA or nonsense sequence. Cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 48 h. Apoptotic cells were quantified by flow cytometry. Results are percent specific apoptosis. Downregulation of Bim and EndoG protein levels was verified by Western blot. Equal protein loading was controlled by staining membranes with β-actin. All experiments were done three times with consistent results. Columns, mean of three independent experiments done in triplicate; bars, SE. ***, P < 0.001 (ANOVA/Bonferroni). C, proposed mechanism of spongistatin 1–induced apoptosis. Thick arrows, main signaling pathway of spongistatin 1. The tubulin depolymerizing agent spongistatin 1 frees Bim from its sequestration both by the microtubule network and by the antiapoptotic protein Mcl-1. Bim triggers the translocation of AIF and EndoG from mitochondria to the nucleus leading to caspase-independent apoptosis. Thin arrows, inferior role of the caspase-dependent cell death induced by spongistatin 1.

Article Snippet: Cells were blocked with 0.2% bovine serum albumin and incubated with specific antibodies against AIF (rabbit polyclonal IgG; Upstate) and EndoG (rabbit polyclonal antibody; Prosci).

Techniques: Transfection, Sequencing, Western Blot, Control, Extraction, Flow Cytometry, Staining, Translocation Assay