The wt peptide blocks SALL4 repression of PTEN. (A) Flow cytometric analysis on SNU-398 cells treated with Pep-1 carrier peptide alone (top panel), wt peptide alone (middle panel), or Pep-1 carrier + peptide (bottom panel). More than 90% of cells showed FITC-labeled peptide uptake after the use of Pep-1 carrier. (B) Confocal images of the distribution of FITC-labeled peptide in SNU-398 cells treated as shown in panel A. (C) The endogenous SALL4 interaction with HDAC is blocked by wt (wt(H): 20μM; wt(L): 10μM), but not scr peptide. SALL4 was immunoprecipitated from SNU-398 nuclear extracts (1 × 10⁶ cells) pretreated with wt or scr peptides as indicated using an anti-SALL4 antibody. Immunoprecipitates were analyzed by Western blot using HDAC1 (top panel) or SALL4 (bottom panel) antibodies. The interaction was completely abrogated by pretreatment with 20μM wt peptide (lane 2) but not scr peptide (lane 3). An equal amount of SALL4 protein was present in all samples. In lane 4 (10% input), 10% of the amount of nuclear extract used in lanes 1 through 3 was subjected to Western blot analysis without immunoprecipitation. (D) Western blot showed increased PTEN expression after wt peptide treatment compared with scr. α-tubulin was used as a loading control. (E) ChIP-quantitative RT-PCR showed increased H3 acetylation markers at the PTEN promoter in SNU-398 cells on wt peptide treatment. Enrichment with control IgG was set at 1.