RAS and BCL-2 status of AML mice. (A) Percentage of peripheral blood BCL-2–positive cells in 4-week-old single transgenic (n = 10), untreated at 4-weeks old (day 0, n = 11), and posttreatment mice (postday 33, n = 14) showing a significant increase in BCL-2–positive cells with disease progression and after treatment (P < .0001). (B) Representative spleen cells from WT FVB/N, single and double MRP8[NRASD12/hBCL-2] AML transgenic mice assessed for total RAS and total BCL-2 expression by western blot analysis. Active RAS-GTP levels were measured via a sensitive RAF1-RBD pull down assay followed by western blot analysis with an anti-RAS antibody. Blots were reprobed with anti β-actin antibody to assess protein loading (n = 3). (C) Double MRP8[NRASD12/hBCL-2] AML untreated (day 0) and treated (+ABT-737) mice assayed (postday 33). Mice were assessed, as in (B). Histograms of RAS-GTP and hBCL-2 normalized to actin show an increase of the hBCL-2:RAS-GTP complex. The relative values were presented as fold increase over control samples as indicated (error bar = SD; n = 3). (D) Western blot analysis showing threonine 56 and serine 70 phosphorylations of hBCL-2 of spleen cells from untreated (day 0) and treated (postday 33), with β-actin showing equal protein loading (n = 2 mice). (E) MMP measurements by DiOC₂(3) show a reduction of dye uptake in ABT-737–treated WT and AML mice relative to untreated samples (minimum in each group, n = 3 mice). (F) Confocal microscopy of stained cells of BM showing subcellular localization of RAS:BCL-2 complex from the AML mice shifting from mitochondrial localization (Mito, stained with Tom20⁺ antibody) in untreated (−) mice to plasma membrane (PM) localization (wheat germ agglutinin (WGA) after treatment (+) on postday 33. Rows 1 and 2 show pretreatment BM samples with RAS and BCL-2 co-localizing with the mitochondria (pale), whereas the pink coloration with the WGA antibody is consistent with co-localizations of RAS and BCL-2 with virtually no plasma membrane staining; rows 3 and 4 are 2 independent mice posttreatment probed with either Tom 20 (rows 3a and 3b) or WGA (rows 4a and 4b) showing the RAS:BCL-2 complex localizing in the plasma membrane with a white color, whereas with the mitochondrial antibody, green patches are visible with pale pink coloration in row 3a consistent with RAS and BCL-2 co-localizing in the absence of mitochondrial staining (in each group, n = 2 mice).