Inhibition of β-catenin expression by CTNNB1 shRNAs dramatically reduces the increase in the active pool of β-catenin and β-catenin/TCF-regulated target gene expression seen following <i>APC</i> inhibition in HCECs.

<p>HCECs stably transduced with a lentiviral vector (ShRNA V1) expressing an <i>APC</i> shRNA or a non-silencing scramble shRNA (Scrmbl) were further transduced with lentiviral vectors (ShRNA V2) driving expression of two different shRNAs targeting <i>CTNNB1</i> (<i>CTNNB1</i>-1 and <i>CTNNB1</i>-2) or a non-silencing scramble shRNA (Scrmbl). Protein and RNA were collected from the HCECs following a 3-day induction of shRNA expression by addition of doxycycline (DOX, “+”) at 2 μg/ml or no DOX-mediated induction (i.e., solvent control “-“). (A) ShRNA-mediated changes in the expression of the total and active (un- or hypophosphorylated) pools of β-catenin in the HCECs were assessed by Western blot analysis for active β-catenin and total β-catenin, with β-actin as a loading and transfer control (left) and by qRT-PCR for <i>CTNNB1</i> transcripts normalized to <i>U6</i> expression (right). The two <i>asterisks</i> denote <i>P</i> < 0.01 in Student's <i>t</i> test, and <i>error bars</i> denote S.D. Analysis of the expression of selected Wnt/β-catenin/TCF-regulated target genes was undertaken in the cell lines with or without DOX-mediated induction of the indicated shRNAs—<i>AXIN2</i> (B), <i>BMP4</i> (C), <i>NKD1</i> (D) and <i>IRS1</i> (E), as assessed by qRT-PCR and normalized to <i>U6</i> expression. In panels (<i>B</i>), (C) and (E), **<i>P</i> < 0.01. In panel (D), *<i>P <</i> 0.05.