Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

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

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 2000, American Society for Microbiology

PMC Copyright notice

FIG. 7 — M-Akt-transformed cells require NF-κB to block etoposide-induced apoptosis. (A) Generation of H-Ras(V12)- and M-Akt-transformed Rat-1 cells. Rat-1 cells stably expressing activated H-Ras(V12) or M-Akt were generated as described in Materials and Methods. Total protein (50 μg) was isolated from Rat-1:Hygro, Rat-1:H-Ras(V12), and Rat-1:M-Akt cells, resolved by performing PAGE, and transferred to nitrocellulose membrane. Immunoblot analysis was performed to detect transgenic expression of p21^ras and M-Akt using the pan-Ras antibody or the hemagglutinin antibody, respectively. (B) The p65 transactivation domain is activated in Rat-1 cells transformed with either H-Ras(V12) or M-Akt. Rat-1:Hygro, Rat-1:H-Ras(V12), and Rat-1:M-Akt cells were transiently transfected with a Gal4-luciferase reporter (100 ng) and with constructs bearing the genes encoding Gal4-p65 (100 ng). Forty-eight hours following the start of transfection, cell extracts were harvested and luciferase activities were determined. Data are the averages ± standard deviations of results of three experiments performed in triplicate. (C) Rat-1:M-Akt cells are resistant to apoptotic induction agents. Rat-1:Hygro and Rat-1:M-Akt cells were either left untreated (No Add) or given etoposide (15 μM) or staurosporine (50 μM). Apoptotic cell numbers were determined 18 h following the addition of either etoposide or staurosporine (Stauro). Results presented here are the means ± standard deviations of results of two independent experiments performed in duplicate. (D) M-Akt requires NF-κB to overcome etoposide-induced apoptosis. Rat1:M-Akt cells were cultured overnight in medium containing 2% FBS, after which cells were infected with either Ad-CMV or Ad-SRIκBα (50 PFU/cell). Six hours following adenovirus-mediated gene delivery, cells were either left untreated (No Add) or treated with either etoposide (5 μM) or staurosporine (25 μM). Apoptotic cell numbers were analyzed in etoposide-treated Rat-1:M-Akt cells over the time course indicated, while the apoptotic cell numbers detected for staurosporine were analyzed 60 h after the drug addition. Data presented are the averages ± standard deviations of results of two different experiments where the numbers of apoptotic cells were counted in triplicate. (Gel) Immunoblot analysis demonstrating that Ad-SRIκBα is effectively expressed in the Rat-1 cell lines. IκBα proteins were detected using a rabbit polyclonal antibody (C-21; Santa Cruz Biotech). Protein samples analyzed in lanes 1, 3, 5, and 7 are from Rat-1:M-Akt cells infected with Ad-CMV, while those in lanes 2, 4, 6, and 8 are from cells infected with Ad-SRIκBα.

FIG. 7 — M-Akt-transformed cells require NF-κB to block etoposide-induced apoptosis. (A) Generation of H-Ras(V12)- and M-Akt-transformed Rat-1 cells. Rat-1 cells stably expressing activated H-Ras(V12) or M-Akt were generated as described in Materials and Methods. Total protein (50 μg) was isolated from Rat-1:Hygro, Rat-1:H-Ras(V12), and Rat-1:M-Akt cells, resolved by performing PAGE, and transferred to nitrocellulose membrane. Immunoblot analysis was performed to detect transgenic expression of p21^ras and M-Akt using the pan-Ras antibody or the hemagglutinin antibody, respectively. (B) The p65 transactivation domain is activated in Rat-1 cells transformed with either H-Ras(V12) or M-Akt. Rat-1:Hygro, Rat-1:H-Ras(V12), and Rat-1:M-Akt cells were transiently transfected with a Gal4-luciferase reporter (100 ng) and with constructs bearing the genes encoding Gal4-p65 (100 ng). Forty-eight hours following the start of transfection, cell extracts were harvested and luciferase activities were determined. Data are the averages ± standard deviations of results of three experiments performed in triplicate. (C) Rat-1:M-Akt cells are resistant to apoptotic induction agents. Rat-1:Hygro and Rat-1:M-Akt cells were either left untreated (No Add) or given etoposide (15 μM) or staurosporine (50 μM). Apoptotic cell numbers were determined 18 h following the addition of either etoposide or staurosporine (Stauro). Results presented here are the means ± standard deviations of results of two independent experiments performed in duplicate. (D) M-Akt requires NF-κB to overcome etoposide-induced apoptosis. Rat1:M-Akt cells were cultured overnight in medium containing 2% FBS, after which cells were infected with either Ad-CMV or Ad-SRIκBα (50 PFU/cell). Six hours following adenovirus-mediated gene delivery, cells were either left untreated (No Add) or treated with either etoposide (5 μM) or staurosporine (25 μM). Apoptotic cell numbers were analyzed in etoposide-treated Rat-1:M-Akt cells over the time course indicated, while the apoptotic cell numbers detected for staurosporine were analyzed 60 h after the drug addition. Data presented are the averages ± standard deviations of results of two different experiments where the numbers of apoptotic cells were counted in triplicate. (Gel) Immunoblot analysis demonstrating that Ad-SRIκBα is effectively expressed in the Rat-1 cell lines. IκBα proteins were detected using a rabbit polyclonal antibody (C-21; Santa Cruz Biotech). Protein samples analyzed in lanes 1, 3, 5, and 7 are from Rat-1:M-Akt cells infected with Ad-CMV, while those in lanes 2, 4, 6, and 8 are from cells infected with Ad-SRIκBα.

HHS Vulnerability Disclosure