Article Figures & Data
Figures
- Figure 1.
Enzastaurin suppresses cell signaling downstream of the AKT/mTOR pathway. Cancer cells were incubated with 0 to 4μmol/L enzastaurin in media supplemented with 1% HI FBS for 16 and 24 hours. Western blot analyses were done for total 4E-BP1, 4E-BP1Thr37/46, 4E-BP1Ser65, 4E-BP1Thr70, AKTSer473, AKTThr308, mTOR, mTORSer2448, p70S6KThr389, rpS6Ser240/244, rpS6, AKT, and β-actin. A, HCT116 colon cancer cells; B, CRL2611 glioblastoma cells; C, Farage B-cell lymphoma cells.
- Figure 2.
Enzastaurin decreases eIF4F complex levels. Cancer cells were incubated with 0 to 4 μmol/L enzastaurin in media supplemented with 1% HI FBS for 24 hours. Western blot analyses were done for total eIF4G, eIF4E, and 4E-BP1 from m7GTP pull-down lysates and whole cell lysates. Whole lysates were additionally probed for β-actin to control for loading and transfer. Graph represents eIF4G normalized to eIF4E divided by 4E-BP1 normalized to eIF4E [(eIF4G/eIF4E)/(4E-BP1/eIF4E)]. Data represent the mean of 3 separate experiments (±SEM). A, HCT116 colon cancer cells; B, Farage B-cell lymphoma cells; C, CRL2611 glioblastoma cells; D, percentage of apoptotic cells following enzastaurin treatment in HCT116 cells and in HCT116 colon cancer cells selected for reduced sensitivity (HCT116res) to enzastaurin as measured by TUNEL staining. Inset shows the levels of eIF4E and 4E-BP1 by Western blot from whole cell lysates. β-Actin was assessed to control for loading and transfer.
- Figure 3.
4E-BP1 depletion reduces enzastaurin-induced apoptosis. A, Western blot analyses for total 4E-BP1 and β-actin 72 hours after 4E-BP1 siRNA transfection in HCT116 colon cancer cells and CRL2611 glioblastoma cells. Apoptosis after 72 hours 4E-BP1 siRNA treatment followed by incubation with 0 to 2 μmol/L enzastaurin in media supplemented with 1% HI FBS for 48 hours HCT116 (B) or 72 hours CRL2611 (C). Apoptosis was determined using the Cell Death Detection ELISA (Roche Applied Science). Graph represents mean (±SEM); *, P < 0.05 by unpaired t test.
- Figure 4.
4E-BP1/2 knockout increases eIF4F complex levels and reduces enzastaurin-induced apoptosis. Wild-type (WT) and 4E-BP1/2 knockout (KO) murine embryonic fibroblasts were incubated with 1 to 2 μmol/L enzastaurin in media supplemented with 1% HI FBS. A, apoptosis was determined 72 hours after enzastaurin treatment using the Cell Death Detection ELISA (Roche Applied Science). Graph represents mean (±SEM); *, P < 0.05 by unpaired t test. B, Western blot analyses from m7GTP pull-down lysates were done for total eIF4E and 4E-BP1 after 24 hours enzastaurin treatment. C, graph represents eIF4G normalized to eIF4E divided by 4E-BP1 normalized to eIF4E [(eIF4G/eIF4E)/(4E-BP1/eIF4E)]. Data represent mean of 3 separate experiments (±SEM).
Volume 9, Issue 12
