Given that EVO downregulates ASS1 expression via the Wnt/β-catenin/c-MYC pathway, to further clarify the critical role of ASS1 in EVO-mediated anti-CRC effects, we first established an ASS1 knockdown model in CRC cell lines, validated the efficiency of gene silencing at both mRNA and protein levels (Figure 5a–c), and selected ShASS1#1 for subsequent experiments. Here, MYC is linked to colorectal carcinoma.