American Society of Hematology

Figure 1

$Figure 1. Inhibition of MEK/ERK activity strikingly enhances perifosine-mediated apoptosis in human leukemia cells. (A) U937 cells were exposed for 24 hours to the designated concentration of perifosine (P) alone (○) or in conjunction with 10μM PD184352 (PD, ●), after which the percentage of apoptotic cells was determined by annexin V analysis, as described in “Assessment of apoptosis.” (B) U937 cells were exposed to the designated concentration of PD184352 alone (□) or in combination with 2.5μM perifosine (■), after which apoptosis was determined, as above. (C) Cells were exposed to perifosine (2.5μM) and PD184352 (10μM) alone or in combination for the indicated intervals, after which the percentage of apoptotic cells was determined as above. (D) Median dose effect analysis of apoptosis induction by perifosine and PD184352. U937 cells were exposed to various concentrations of PD184352 and perifosine at a fixed ratio (4:1), after which apoptosis was monitored at 24 hours by annexin V/PI analysis. Combination index values were determined in relation to the fractional effect using a commercially available software program, as described in “Statistical analysis.” Combination index values less than 1.0 correspond to a synergistic interaction. (E) U937 cells were exposed to perifosine (2.5μM) and U0126 (10μM), PD184352 (10μM), LY294002 (LY; 20μM), and PI-103 (3μM) either individually or in the designated combinations for 24 hours, after which the percentage of apoptotic cells was determined by annexin V analysis. (F-G) U937 cells were exposed to PD184352 (10μM) and perifosine (2.5μM) alone or in combination for the indicated intervals, after which protein lysates were prepared and subjected to Western blot analysis using designated antibodies. For this and all subsequent Western blot analysis, blots were subsequently reprobed with anti-tubulin (Tub) antibodies to document equivalent loading and transfer. The results of a representative study are shown; 2 additional experiments yielded equivalent results. (H) Cells were treated with PD184352 (10μM) and perifosine (2.5μM) alone or in combination for 16 hours, after which mitochondria-free cytosolic fractions were obtained, as described in “Subcellular fractionation,” and subjected to Western blot analysis to monitor release of cytochrome c, AIF, and Smac/DIABLO into the cytosol.$

Inhibition of MEK/ERK activity strikingly enhances perifosine-mediated apoptosis in human leukemia cells. (A) U937 cells were exposed for 24 hours to the designated concentration of perifosine (P) alone (○) or in conjunction with 10μM PD184352 (PD, ●), after which the percentage of apoptotic cells was determined by annexin V analysis, as described in “Assessment of apoptosis.” (B) U937 cells were exposed to the designated concentration of PD184352 alone (□) or in combination with 2.5μM perifosine (■), after which apoptosis was determined, as above. (C) Cells were exposed to perifosine (2.5μM) and PD184352 (10μM) alone or in combination for the indicated intervals, after which the percentage of apoptotic cells was determined as above. (D) Median dose effect analysis of apoptosis induction by perifosine and PD184352. U937 cells were exposed to various concentrations of PD184352 and perifosine at a fixed ratio (4:1), after which apoptosis was monitored at 24 hours by annexin V/PI analysis. Combination index values were determined in relation to the fractional effect using a commercially available software program, as described in “Statistical analysis.” Combination index values less than 1.0 correspond to a synergistic interaction. (E) U937 cells were exposed to perifosine (2.5μM) and U0126 (10μM), PD184352 (10μM), LY294002 (LY; 20μM), and PI-103 (3μM) either individually or in the designated combinations for 24 hours, after which the percentage of apoptotic cells was determined by annexin V analysis. (F-G) U937 cells were exposed to PD184352 (10μM) and perifosine (2.5μM) alone or in combination for the indicated intervals, after which protein lysates were prepared and subjected to Western blot analysis using designated antibodies. For this and all subsequent Western blot analysis, blots were subsequently reprobed with anti-tubulin (Tub) antibodies to document equivalent loading and transfer. The results of a representative study are shown; 2 additional experiments yielded equivalent results. (H) Cells were treated with PD184352 (10μM) and perifosine (2.5μM) alone or in combination for 16 hours, after which mitochondria-free cytosolic fractions were obtained, as described in “Subcellular fractionation,” and subjected to Western blot analysis to monitor release of cytochrome c, AIF, and Smac/DIABLO into the cytosol.

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