Figure 5.
Figure 5. PI3 kinase inhibitor Ly294002 causes apoptosis in the presence or absence of IL-3 in all cell lines except those lacking both Bax and Bak. (A) Cells of the indicated genotypes were cultured for 24 hours with or without IL-3 with or without the addition of Ly294002 (25 μM). Cell viability was determined by staining with FITC-coupled annexin V and PI followed by flow cytometric analysis. Data show the mean ± SEM of 3 independent experiments using 2 independent lines of each genotype. (B) Ly294002 inhibits proliferation in Bax-/-;Bak-/- cells. Cells were stained with CSFE and cultured in the presence of IL-3 alone (left panel), IL-3 plus Ly294002 (middle panel), or without IL-3 (right panel). At 0 (a), 24 (b), and 48 hours (c), the cells were counted and CFSE fluorescence determined by flow cytometric analysis. The histograms for each time point are overlaid. CFSE fluorescence declines (shifts to the left) as cell number increases. The results shown are from one experiment that is representative of 3 independent experiments with different Bax-/-;Bak-/- clones. (C) Bax-/-;Bak-/- cells were cultured in the presence or absence of IL-3 and plus or minus Ly294002 (25 μM). Lysates from these cells were run on SDS-PAGE and immunoblotted with antibodies recognizing only phosphorylated AKT (S473) or total AKT. (D) Bad phosphorylated on S112 and total Bad decline following IL-3 withdrawal. Western blot of lysates harvested from Bax-/-;Bak-/- cells cultured with IL-3 (+), or 24 or 48 hours following IL-3 withdrawal (-), and from wild-type cells cultured with IL-3, or 24 hours following IL-3 withdrawal, were probed with antibodies to S112 phospho-Bad or total Bad. Arrows indicate Bad and asterisks mark nonspecific bands. Lysates from equal numbers of cells were loaded in each lane. (E) Ly294002 reduces the level of both phosphorylated Bad and total Bad when combined with IL-3 withdrawal. Western blot of lysates from Bax-/-;Bak-/- cells cultured for 24 hours with or without IL-3 and Ly294002 (25 μM) for 24 hours were probed with antibodies to S112 or S136 phospho-Bad or total Bad. Probing with an antibody to Hsp-70 was used as a loading control. Lysates from equal numbers of cells were loaded in each lane. Arrows indicate Bad and asterisks mark nonspecific bands.

PI3 kinase inhibitor Ly294002 causes apoptosis in the presence or absence of IL-3 in all cell lines except those lacking both Bax and Bak. (A) Cells of the indicated genotypes were cultured for 24 hours with or without IL-3 with or without the addition of Ly294002 (25 μM). Cell viability was determined by staining with FITC-coupled annexin V and PI followed by flow cytometric analysis. Data show the mean ± SEM of 3 independent experiments using 2 independent lines of each genotype. (B) Ly294002 inhibits proliferation in Bax-/-;Bak-/- cells. Cells were stained with CSFE and cultured in the presence of IL-3 alone (left panel), IL-3 plus Ly294002 (middle panel), or without IL-3 (right panel). At 0 (a), 24 (b), and 48 hours (c), the cells were counted and CFSE fluorescence determined by flow cytometric analysis. The histograms for each time point are overlaid. CFSE fluorescence declines (shifts to the left) as cell number increases. The results shown are from one experiment that is representative of 3 independent experiments with different Bax-/-;Bak-/- clones. (C) Bax-/-;Bak-/- cells were cultured in the presence or absence of IL-3 and plus or minus Ly294002 (25 μM). Lysates from these cells were run on SDS-PAGE and immunoblotted with antibodies recognizing only phosphorylated AKT (S473) or total AKT. (D) Bad phosphorylated on S112 and total Bad decline following IL-3 withdrawal. Western blot of lysates harvested from Bax-/-;Bak-/- cells cultured with IL-3 (+), or 24 or 48 hours following IL-3 withdrawal (-), and from wild-type cells cultured with IL-3, or 24 hours following IL-3 withdrawal, were probed with antibodies to S112 phospho-Bad or total Bad. Arrows indicate Bad and asterisks mark nonspecific bands. Lysates from equal numbers of cells were loaded in each lane. (E) Ly294002 reduces the level of both phosphorylated Bad and total Bad when combined with IL-3 withdrawal. Western blot of lysates from Bax-/-;Bak-/- cells cultured for 24 hours with or without IL-3 and Ly294002 (25 μM) for 24 hours were probed with antibodies to S112 or S136 phospho-Bad or total Bad. Probing with an antibody to Hsp-70 was used as a loading control. Lysates from equal numbers of cells were loaded in each lane. Arrows indicate Bad and asterisks mark nonspecific bands.

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