(A) Scatter plot correlating the relative level of SNON expression (qPCR) and nuclear SNON staining (IHC) from human CRC samples. Patients with high SNON stability highlighted in black were selected for deep sequencing. (B) Protein alignment of the C-terminus of AKD from human (Q6ZNA4), mouse (Q99ML9), chick (Q90ZT8), Xenopus (Q0V9R0), Drosophila (Q9VGI6) and C.elegans (Q9XUM8). The grey lines indicate the NRG (NRGASQG), TIER (TIERCTY) and NLS (PHKYKKV) domains and the black line highlights the RING domain. Numbers indicate four key mutations identified. (C) Luciferase CAGA12 reporter assay values from three biological repeat experiments, each in quadruplicate. The Q899STOP mutation exhibits a dominant-negative effect, similar to the W972R control. (D) Immunoblot shows relative stability of GFP-AKD proteins (top bands) compared to GFP control (bottom bands). GFP-Q899STOP is stable at approximately 140kDa. H3 was used as a loading control. 40μg of protein extract was loaded in each lane.