(A) Two parallel approaches were used to understand whether inhibition of UNG activity or depletion of the entire enzyme molecule produced similar effects in syngeneic mouse models of colorectal cancer. Inhibition of UNG catalytic activity was achieved through inducible expression of the potent small UNG inhibitor (UGI) protein in the MC38 murine colon cancer cell line using lentiviral transduction methods. This maintains scaffolding functions of the protein and abrogates enzymatic activity. Depletion of the total UNG protein was achieved through doxycycline-inducible expression of UNG-specific shRNA (shRNA^UNG sequence A) using lentiviral transduction methods. This inhibits both scaffolding and enzymatic functions. Depletion may not be equivalent to inhibition because the UNG N-terminal domain (NTD) remains intact in the context of UGI inhibition, which could still allow UNG to localize to replication forks through its known protein-protein interactions (see text). (B) To establish the inducible inhibition by UGI, we collected cell extracts from induced and noninduced MC38 cell cultures and measured the UNG activity using a 5′-FAM–labeled 19mer DNA substrate containing a single uracil. Excision of uracil from this substrate results in a 9mer 5′-FAM–labeled product that can be resolved from the substrate by denaturing polyacrylamide gel electrophoresis. Greater than 97% inhibition of UNG activity was achieved through induction of UGI. Controls included the addition of purified UNG enzyme to the substrate in the absence of extract, and a no-extract negative control. (C) The same activity assay was used to test the efficacy of inducible shRNA^UNG in MC38 cells (70% knockdown), and (D) CT-26 cells (90% knockdown). Two biological replicates of each UNG activity experiment were performed.