FIGURE 6.

Acetylated survivin 129K inhibits STAT3 enhanceosome activity. A, HEK293T cells were cotransfected with cDNAs encoding MYC-STAT3 and the three survivin variants, in the presence or absence of CBP, as indicated. STAT3/Survivin complexes were affinity-purified using agarose beads conjugated with the SIE oligonucleotide and blotted with anti-MYC. B, HEK293T cells were cotransfected with survivin acetylation mutants, STAT3, CBP, and a STAT3-dependent luciferase activity reporter. The data are means ± S.E. for triplicate experiments (*, p < 0.005, two-tailed t test). EV, empty vector. C, HEK293T cells were cotransfected with cDNAs encoding STAT3 and CBP, with or without survivin, as indicated. RNA was purified, reverse transcribed into cDNA, and Q-RTPCR performed with primers to human BCLXL and MCL. ΔΔCts were calculated. The data are means ± S.E. for triplicate experiments (*, p < 0.005 in each comparison). D, HEK293T cells were transfected with EV or with cDNAs encoding MYC-STAT3, with or without the addition of IL-6 or CBP. For comparison, cells were cotransfected with cDNAs encoding MYC-survivin and either wild-type MYC-STAT3 or the phosphorylation-defective (Y705F), or acetylation-defective (K685R) MYC-STAT3 mutants. Lysates were affinity purified using agarose beads conjugated with SIE oligonucleotides then immunoblotted with anti-MYC, anti-STAT3-pY705, or a pan-acetylation (Ac) antibody. EV, empty vector; IB, immunoblot.