|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 4. Methotrexate and docetaxel activate Akt and induce constitutive nuclear Akt translocation. (A) MCF7 cells were treated with methotrexate (10 µM) (upper panel) or docetaxel (0.05 µM) (lower panel) for the indicated time, then phosphorylated Akt and total Akt were detected by immunoblotting with the respective antibodies. (B) The localization of Akt in the nucleus in either control untreated cells or the drug-treated cells (as indicated) was assessed by immunostaining followed by confocal microscopy. Six hours after docetaxel treatment, or 3 hours after methotrexate treatment, cells were fixed, and the immunostaining was performed with anti-Akt antibodies followed by secondary Cy-3 conjugated antibody. DAPI was used as nuclear counterstain. The superimposed micrographs of Akt- and nuclear staining visualize changes in the cellular localization of Akt. (C) 293T cells were left untreated, treated with methotrexate or docetaxel alone, or in combination with either adenoviral vector coding for nuclear Akt, or with wortmannin. Twenty-four hours later, the cells were stained using the Nicoletti method, and the percentage of cell death was assessed by flow cytometry. (D) 293T and PC-3 cells were transiently transfected with Akt-CA coding vector and the cell death was then assayed by flow cytometry as described above. Comparison was made between the Akt-CA-transfected cells and the non-transfected control cells. The amount of cell death also confirmed using MTT assay in an independent experiment (data not shown).
|
