Fig 2: Protein and mRNA levels of 5-FU metabolic enzymes. (A) Protein levels. The whole cell lysates of sh-control, sh-IFIT2-1 and sh-IFIT2-1 cells were immunoblotted for TS, TK1, OPRT and TP. Bar graph indicates quantitative densitometry of Western blot analysis of three independent experiments relative to sh-control. The symbols ** and *** denote statistically significant differences compared to sh-control at p < 0.01 and p < 0.001, respectively. (B) mRNA levels. The relative mRNA expression levels of 5-FU metabolic enzymes in sh-control, sh-FIT2-1 and sh-IFIT2-2 cells were determined by Q-PCR. GAPDH mRNA was used as an internal control. * p < 0.05, ** p < 0.01 denotes a statistically significant difference compared to sh-control. (C) Immunostaining. The intracellular distribution of TS in sh-control, sh-FIT2-1 and sh-IFIT2-2 cells with or without 5-FU treatment (10 µM for 48 h) was determined by immunostaining (red). The nuclei were counterstained with DAPI (blue). Quantitative analysis of the mean fluorescence intensity is shown in bar graph. The symbols ** and *** denote statistically significant differences compared to non-treated sh-control at p < 0.01 and p < 0.001, respectively. (D) Formation of the TS/5-FU complex. As described above, the cells were treated with 10 µM 5-FU for various time periods. The upper band (red arrow) denotes the TS/5-FU complex, and the lower band (black arrow) represents free TS. GAPDH was used as a loading control. The numbers represent the relative expression level of Free TS protein in compared to control cells. Quantitative densitometry of Western blot analysis of three independent experiments. *** p < 0.001 denotes a statistically significant difference compared to sh-control at 0 h.