Article Figures & Data
Figures
- Fig. 1.
PP2A activity is enhanced in stable E1A-expressing cells through up-regulation of PP2A/C. A, Protein expression of PP2A/A, PP2A/C, E1A, and actin in stable E1A-expressing cells (E) and corresponding vector controls (V). B, PP2A activity was measured using a protein phosphatase assay kit. The substrate phospho-peptide concentrations were 20, 40, 100, 200, and 400 μmol/L, respectively. Protein concentrations include 0.1, 1.0, and 10 μg, respectively. V, 231-Vector. E, 231-E1A. Results shown here are from free independent experiments. C, Dephosphorylation of Akt by purified hPP2A (1 milliunit = 1 × 10−3 units) and inhibition of by okadaic acid (1 nmol/L) in vitro. Aliquots of immunoprecipitated endogenous Akt were incubated with purified human PP2A enzyme (rhPP2A); relative phosphatase activities were measured by anti-phospho-Akt (T308) antibody. Total Akt was used as a loading control. D, Blocking PP2A activity by exposing 231-E1A cells to okadaic acid (10 nmol/L) increased Akt phosphorylation and inhibited p38 phosphorylation.
- Fig. 2.
Up-regulation of PP2A/C is required for E1A-mediated chemosensitization. A, PARP cleavage and PP2A/C activation in 231-Vect and 231-E1A cells after treatment with paclitaxel. Cyt. c, cytochrome c. B, Western blot analysis of PP2A and the catalytic subunit PP2A/C in Vect (V)- or E1A (E)-transfected MCF-7 and MDA-MB-453 cells after exposure to 0.01 and 1.0 μmol/L paclitaxel, respectively. C, a dose-finding test of specific PP2A/C small interfering RNA on PP2A/C expression in 231-E1A cells. Relative intensity of PP2A/C was shown in the bottom. D. Cells were transfected with small interfering RNA using JetSI cationic transfection reagent (Obiogene, Inc., Carlsbad, CA) for 16 hours and replaced with fresh medium before addition of 0.01 μmol/L paclitaxel and were incubated for another 24 hours before harvesting. Relative intensities of PARP and PP2A/C in specific small interfering RNA (PP2A/C) and nonspecific control small interfering RNA (Control) protected cells were shown in the bottom of each band. E, For nuclear fragmentation analysis, cells were grown in Lab-Tek Chamber Slides (Nunc, Inc., Naperville, IL) and were treated by the same procedure as described above. Cells were then washed with PBS twice, fixed with 70% alcohol, and stained with Hoechst 33342 (0.5 μg/mL; Sigma). Events of apoptotic nuclei were counted under a fluorescence microscope, and the mean values in every 200 cells in each field were plotted.
- Fig. 3.
Enhanced PP2A phosphatase activity in TNF-α induced apoptosis. PP2A phosphatase activity and Western blot analysis of the expression of phospho-Akt, phospho-p38, and PARP cleavage in MDA-MB-231 cells with treatment of insulin-like growth factor-1 (50 ng/mL), TNF-α (50 ng/mL), the phosphatidylinositol 3′-kinase inhibitor Wortmannin (WORT; 0.5 μmol/L), and the MEK1/2 inhibitor PD98058 (PD; 20 μmol/L).
- Fig. 4.
Conserved domains of E1A required for up-regulation of PP2A/C. A, a domain structure and map for deletion mutation of CR1 and CR2. B, Western blot analysis of PP2A/C in vector-transfected, or wild-type (WT) E1A and mutant E1A (ΔCR1, ΔCR2, or ΔCR3). Actin was used as a loading control.
Volume 64, Issue 17
