FIGURE 5.

Reduced pseudouridine modification in cells and sEVs after 5‐FU treatment. LC–MS/MS approaches were used to quantify RNA base modifications in RNA from cells and sEVs. (a) Nucleoside content in 5‐FU treated DLD‐1 cells showed a modest but significant increase (p < 0.05) in uridine content when compared to untreated cells. No changes were observed for the other RNA nucleosides. (b) No changes in nucleoside levels were detected in sEVs purified from cells treated or not with 5‐FU. (c) Pseudouridine levels were significantly decreased in both cells and sEVs after 5‐FU treatment. (d−l) Quantitation of the effect of the indicated base modifications in cells and sEVs treated or not with 5‐FU. Base modifications include dihydrouridine (DHU), methylated cytosine (m⁵C and m³C), methylated adenosine (m¹A), methylated inosine (m¹I), and methylated guanosine (m¹G, m⁷G, m²G) and demethylated guanosine (m² ₂G). To compare modification levels across conditions, modified nucleoside values were normalized to the total levels for each nucleoside. For all graphs, the error bars indicate average and standard deviation.