Fig. 3: Fluorinated ribosomes display altered translational properties.

a HCT116, HT29 and SW480 cells were treated for 24 h with either 10 µM or 50 µM 5-FU, and translationally active ribosomes were purified from the polysomal fraction. Incorporation of 5-FUrd was measured by LC-HRMS. Data are expressed as mean + /- s.d of independent experiments (n = 3). b Schematic representation of the hybrid in vitro translation assay used in c and d. c and d Ribosomes were purified from HCT116 cells treated with 10 µM 5-FU for 24 h or 48 h, and their translational activity was evaluated using the hybrid in vitro translation assay. Translation efficacy was evaluated on luciferase reporter mRNA containing the 5’-untranslated region (5’-UTR) of the indicated gene. CDS = coding sequence. Values are units of Renilla luciferase activity normalized against the untreated (NT) condition. c, Evaluation on capped mRNA containing the 5’UTR of actin, GAPDH, IGF-1R and c-Myc genes. d Evaluation on uncapped mRNA containing the IRES element from the cricket paralysis virus (CrPV) and the encephalomyocarditis virus (EMCV). Data are expressed as mean + /- s.d. of independent experiments (n = 3). e IRES activity of IGF-1R 5’UTR and deletions was evaluated by transfection of a bicistronic reporter vector represented on top of the figure. IRES activity is monitored as the ratio of firefly luciferase (Fluc) activity over Renilla luciferase (Rluc) activity. HCT116 cells were transfected for 24 h and then treated for 24 h with either 10 µM or 50 µM 5-FU. Data are expressed as mean + /- s.d. of independent experiments (n = 4). f Translation initiation activity of IGF-1R 5’UTR and deletions and of GAPDH 5’UTR was evaluated by co-transfection of a reporter vector represented on top of the figure with a renilla reporter vector for normalization. Translation activity is monitored as the ratio of firefly luciferase (Fluc) activity over Renilla luciferase (Rluc) activity. HCT116 cells were transfected for 24 h and then treated for 24 h with either 10 µM or 50 µM 5-FU. Data are expressed as mean + /- s.d. of independent experiments (n = 4). Results of unpaired two-tailed t test are indicated as nonsignificant (ns) p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Source data are provided as a Source Data file.