Dissection of RAC1 and ODC1 DDX3X sensitivity. (A) Schematic of the HART construct with deletions tiling DDX3X-sensitive 5′ UTRs. (B) Diagram of the structure of RAC1 with the deletions highlighted. (C) HART data for B. HART constructs containing RAC1 5′ UTR or five deletion mutants were transduced via lentivirus into HCT116 degron cells. The cells were treated with auxin to induce loss of endogenous DDX3X or DMSO control for 48 h, and the HART ratio (mCherry/eGFP) was measured by flow cytometry and presented as an absolute ratio (upper panel) or normalized to DMSO (lower panel). Data were obtained in three separate experiments for a total of nine replicates. Statistical significance was determined by unpaired t-test: (ns) P > 0.05, (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001, (****) P ≤ 0.0001. (D) Diagram of the structure of ODC1 with the deletions highlighted. (E) Same experiment as in C, but with HART-ODC1 constructs from D. Data were obtained in three separate experiments for a total of nine replicates. Statistical significance was determined by unpaired t-test: (ns) P > 0.05, (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001, (****) P ≤ 0.0001.
