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. 2000 Mar;20(5):1626–1638. doi: 10.1128/mcb.20.5.1626-1638.2000

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Copyright © 2000, American Society for Microbiology

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FIG. 6 — The inhibition of endogenous Akt activity sensitizes cells to H-Ras(V12)-induced apoptosis. (A) Expression of H-Ras(V12) is associated with an increase in apoptosis in cells expressing the dominant negative Akt protein (DN Akt). Rat-1:iRasV, Rat-1:iRas–dominant negative Akt clones (.5, .7, and .15), and Rat-1:iRas–dominant negative Akt-P cells (pooled clone) were grown overnight in medium containing a reduced concentration of serum (2% FBS). Eighteen hours later, cells were treated in either the absence or presence of IPTG (5 mM). Apoptotic cells were harvested from the supernatants 48 h after IPTG addition, fixed in paraformaldehyde, and stained with Hoechst dye, and cells displaying nuclear fragmentation and condensation were counted, as described in Materials and Methods. Results are expressed as numbers of apoptotic cells (means ± standard deviations). Assays were repeated at least three independent times. (B) Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells were cultured and stimulated with IPTG as described above. Cell morphologies were analyzed 48 h after IPTG addition. Paraformaldehyde-fixed cells were analyzed for apoptosis by performing TUNEL analysis (Boehringer Mannheim). The upper four images show phase-contrast microscopy (magnification, ×20). The bottom four images show fluorescence microscopy of TUNEL-positive cells (magnification, ×40). (C) H-Ras(V12) no longer stimulates the p65 transactivation domain in cells stably expressing the dominant negative Akt protein. Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells (pooled clone) were transfected with the Gal4-luciferase reporter (100 ng) and with constructs bearing the gene encoding the Gal4-p65 fusion protein (100 ng). Eighteen hours following transfections, cells were stimulated with IPTG (5 mM). Twenty-four hours following IPTG addition, cell extracts were isolated and luciferase activities were determined. Results are the averages ± standard deviations from three independent experiments performed in triplicate. (D) H-Ras(V12) stimulates Gal4–Elk-1 in the vector control and cells expressing dominant negative Akt [Akt(K179A)]. Cells were transfected with Gal4-luciferase and Gal4–Elk-1 (100 ng each). Twenty-four hours posttransfection, cells were incubated in either DMEM containing 10% serum and IPTG (5 mM) or DMEM plus 10% serum alone for an additional 24 h. Whole-cell extracts were isolated and assayed for luciferase levels. Results are expressed as multiples of the level of activation obtained with the vector control or dominant negative Akt-P without IPTG incubation and are the averages ± standard deviations of results of three independent experiments performed in triplicate.

FIG. 6 — The inhibition of endogenous Akt activity sensitizes cells to H-Ras(V12)-induced apoptosis. (A) Expression of H-Ras(V12) is associated with an increase in apoptosis in cells expressing the dominant negative Akt protein (DN Akt). Rat-1:iRasV, Rat-1:iRas–dominant negative Akt clones (.5, .7, and .15), and Rat-1:iRas–dominant negative Akt-P cells (pooled clone) were grown overnight in medium containing a reduced concentration of serum (2% FBS). Eighteen hours later, cells were treated in either the absence or presence of IPTG (5 mM). Apoptotic cells were harvested from the supernatants 48 h after IPTG addition, fixed in paraformaldehyde, and stained with Hoechst dye, and cells displaying nuclear fragmentation and condensation were counted, as described in Materials and Methods. Results are expressed as numbers of apoptotic cells (means ± standard deviations). Assays were repeated at least three independent times. (B) Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells were cultured and stimulated with IPTG as described above. Cell morphologies were analyzed 48 h after IPTG addition. Paraformaldehyde-fixed cells were analyzed for apoptosis by performing TUNEL analysis (Boehringer Mannheim). The upper four images show phase-contrast microscopy (magnification, ×20). The bottom four images show fluorescence microscopy of TUNEL-positive cells (magnification, ×40). (C) H-Ras(V12) no longer stimulates the p65 transactivation domain in cells stably expressing the dominant negative Akt protein. Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells (pooled clone) were transfected with the Gal4-luciferase reporter (100 ng) and with constructs bearing the gene encoding the Gal4-p65 fusion protein (100 ng). Eighteen hours following transfections, cells were stimulated with IPTG (5 mM). Twenty-four hours following IPTG addition, cell extracts were isolated and luciferase activities were determined. Results are the averages ± standard deviations from three independent experiments performed in triplicate. (D) H-Ras(V12) stimulates Gal4–Elk-1 in the vector control and cells expressing dominant negative Akt [Akt(K179A)]. Cells were transfected with Gal4-luciferase and Gal4–Elk-1 (100 ng each). Twenty-four hours posttransfection, cells were incubated in either DMEM containing 10% serum and IPTG (5 mM) or DMEM plus 10% serum alone for an additional 24 h. Whole-cell extracts were isolated and assayed for luciferase levels. Results are expressed as multiples of the level of activation obtained with the vector control or dominant negative Akt-P without IPTG incubation and are the averages ± standard deviations of results of three independent experiments performed in triplicate.

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