Figure 7. HDAC inhibitors induce the β-catenin signaling pathway to increase CSC population.

(A&B) Western blot analysis showed upregulation of β-catenin in SUM159 ALDH⁻ cells pretreated with 1mM VA, 1μM SAHA or vehicle for 7 days. (C) Percentage of GFP⁺ cells in SUM159 cells transfected with 7TGP, an eGFP expressing WNT activity reporter construct ²⁶ and treated with 1mM VA, 1μM SAHA or vehicle for 7 days. Bar graph shows mean ± SEM of 3 biological replicates. Untransfected cells served as controls.

(D) WNT/beta-catenin reporter activity measured through GFP expression in single cell derived clones. GFP⁻ or GFP⁺ single cells were FACS deposited into each well in 24-well culture plate with or without 1mM VA, expanded and GFP percentage evaluated in randomly selected five clones from each group (P1). The same clones were passaged subsequently to obtain P2.

(E) Western blot showing overexpressionn of TCF4 in SUM159 cells transfected with EdTP, a TCF4 dominant negative construct ²⁶. (F) Flow cytometry analysis of ALDH activity indicates that SUM159 cells transfected with EdTP have reduced ALDH% and the expansion of the ALDH⁺ population was abolished in transfected cells treated with HDAC inhibitors. Bar graph shows the ALDH ratio to the control and is mean ± SEM of 3 biological replicates.