FIGURE 1.

Incorporation of 5-FU into 5-FU-resistant cell line RNA. (A) Chemical structure of 5-fluorouracil (5-FU). The position numbers are indicated in blue. (B) Chemical structure of 5-fluorouridine (5-FUrd). (C) Cell growth of SAS (left) and FR2-SAS cells (right) cultured in the medium containing 0, 5, or 15 µM 5-FU. Cell growth was quantified by performing WST assay and measuring absorbance at 450 nm. The values are shown as the relative levels versus the mean of 0 h cells. Mean ± standard error of the mean (SEM) from n = 8 biological replicates. (D) 5-FUrd level in cellular RNA from SAS cells and FR2-SAS cells upon addition of 5-FU into cell culture medium. After 48 h of 0 or 10 µM 5-FU addition in the medium, total RNA was collected and digested into nucleosides for LC-MS analysis. Peak areas were normalized by uridine peak areas of the same samples and are shown as the relative levels versus SAS 5-FU 10 µM samples. (E) DPYD mRNA levels in SAS cells and FR2-SAS cells. DPYD mRNA levels were quantified by RT-qPCR and normalized by GAPDH mRNA levels, and are shown as the relative levels versus SAS cells. (F,G) 5-methyluridine (m⁵U) and pseudouridine levels in cellular RNA of SAS cells and FR2-SAS cells upon addition of 5-FU into cell culture medium. After 48 h of 0 or 10 µM 5-FU addition in the medium, total RNA was collected and digested into nucleosides for LC-MS analysis. m⁵U (F) or pseudouridine (G) peak area was normalized by uridine peak area of the same sample and are shown as the relative levels versus SAS 0 µM samples. Mean ± SEM from n = 4 biological replicates in D–G. (*) P < 0.05 by Mann–Whitney test.