American Society of Hematology

Figure 3.

$Figure 3. Translocation of caspase-dependent and caspase-independent death effectors from mitochondria of P39 cells treated with NF-κB inhibitors. Cells were treated with standard doses of BAY11-7082, bortezomib, and/or z-VAD-fmk for 12 hours and then stained with the chromatin dye Hoechst 33342 (blue), with an antibody specific for the mitochondrial antigen Hsp60 (red), as well as antibodies specific for Cyt c (A,D), EndoG (B,E), or AIF (C,F) (all green). Images were acquired as described for Figure 1C. Representative confocal immunofluorescence pictures are shown (A-C), as well as the percentage of cells demonstrating the mitochondrial release of Cyt c, EndoG, or AIF (D-F). Translocation of Cyt c and EndoG from mitochondria of P39 cells, as determined by subcellular fractionation and immunoblot (G-H). Cells were treated with BAY11-7082, bortezomib, and/or z-VAD-fmk for 12 hours and then subjected to subcellular fractionation to obtain cytosolic fractions and heavy membrane fractions enriched in mitochondria (mito), followed by immunoblot detection of Cyt c (G) or EndoG (H), with GAPDH, actin, or Hsp60 serving as loading controls. This experiment was repeated 3 times yielding similar results.$

Translocation of caspase-dependent and caspase-independent death effectors from mitochondria of P39 cells treated with NF-κB inhibitors. Cells were treated with standard doses of BAY11-7082, bortezomib, and/or z-VAD-fmk for 12 hours and then stained with the chromatin dye Hoechst 33342 (blue), with an antibody specific for the mitochondrial antigen Hsp60 (red), as well as antibodies specific for Cyt c (A,D), EndoG (B,E), or AIF (C,F) (all green). Images were acquired as described for Figure 1C. Representative confocal immunofluorescence pictures are shown (A-C), as well as the percentage of cells demonstrating the mitochondrial release of Cyt c, EndoG, or AIF (D-F). Translocation of Cyt c and EndoG from mitochondria of P39 cells, as determined by subcellular fractionation and immunoblot (G-H). Cells were treated with BAY11-7082, bortezomib, and/or z-VAD-fmk for 12 hours and then subjected to subcellular fractionation to obtain cytosolic fractions and heavy membrane fractions enriched in mitochondria (mito), followed by immunoblot detection of Cyt c (G) or EndoG (H), with GAPDH, actin, or Hsp60 serving as loading controls. This experiment was repeated 3 times yielding similar results.

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