Protein phosphatase 2A regulates life and death decisions via Akt in a context-dependent manner

Andrabi et al. 10.1073/pnas.0706696104.

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SI Figure 6

Fig. 6. Polyoma small T induced apoptosis. (A) DNA laddering in cells growing in 10% calf serum. DNA was extracted from control (CON), wild-type small T (POLST), or mutant small T that does not associate with PP2A (POLST-). Equal amounts of DNA were loaded on a 2% gel and stained with ethidium bromide. (B) U2OS cells were transiently transfected with EF-GFP and vector, wild-type, or mutant small T constructs. Nuclei were stained with Hoechst 33342 and observed by fluorescence microscopy. (C) The extent of apoptosis was estimated by the number of cells with condensed nuclei in comparison with the total number of cells transfected as assessed by GFP expression. Results are expressed as a percentage. (D) The extent of AIF translocation in control and small T-expressing cells in Fig. 2D shown as a percentage. (E) Western blot showing the expression of AIF, p38, and small T in control and small T cell extracts.

SI Figure 7

Fig. 7. Effects of passaging small T-expressing cells. Polyoma small T and its effect on p38 were lost upon passaging. Cell extracts from the passages indicated were blotted for phospho p38 (A) and p38 and Small T (B).

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Fig. 8. LY294002 and DPQ protected from small T-induced apoptosis. (A) Small T-expressing cells growing in 10% serum were treated with either the PI3 kinase inhibitor LY294002, (20 mM, 20-24 h) or PARP-1 inhibitor DPQ (30 mM, 20-24 h). DPQ as well as LY294002 reduced ADP ribosylation as shown by blotting of cell extracts with anti-PAR antibody (Upper). Inhibition of PI3 kinase by LY294002 was confirmed by inhibition of S6 kinase phosphorylation seen by the disappearance of slower migrating bands (Lower). (B) DNA was extracted from cells treated as in A and resolved on a 2% agarose gel stained with ethidium bromide. DPQ as well as LY294002 reduced apoptosis as measured by the extent of DNA fragmentation. (C) Western blots of extracts from control or small T cells showed the activation of p38 by small T as indicated by slower migration of its downstream target MAPKAP-K2 (MK2). Successful inhibition of p38 by its inhibitor (SB203580) was indicated by the faster migrating bands.

SI Figure 9

Fig. 9. Serum starvation induced apoptosis. (A) The percentage of apoptotic cells estimated by Hoechst staining is shown in control (CON), small T (POLST), or mutant small T (POLST-)-expressing cells. (B) PARP-1 cleavage of NIH 3T3 cells in the presence of serum (G) and serum starvation (S/S) cells for 4 h. The lanes on the right indicate the addition of PI3 kinase inhibitor (LY294002, 10 or 20 mM) in serum starvation. Enhancement of apoptosis upon serum starvation is indicated by cleaved PARP-1.

SI Figure 10

Fig. 10. Akt, but not small T, enhanced phosphorylation of BAD. Cells (3T3) were transfected with BAD and control vector (C) or with increased dosages of small T or Akt plasmids. Cell extracts made 48 h after transfection were blotted with phospho BAD (Upper) or BAD (Lower) antibodies.