Figure 2.

Characterization of FaDu and THP-1 derived resistant cell lines. A. Detailed growth curves for FRI and FRII resistant cells. All cell lines have indistinguishable doubling times in the absence of ribavirin (far right panel). B. THP-1 resistant (TR) cell lines are not sensitive to treatment with ribavirin at the doses and times used. Ribavirin no longer targets eIF4E activity (i.e. Mcl-1) in resistant cells (far right panel). There were no changes in eIF4E levels between resistant and parental cell lines (and Figure 1C). Actin provides a loading control. C. Resistance is retained after 6 months growth in the absence of ribavirin. D. Incubation of live cells with ³H-ribavirin indicates that THP-1 resistant cells have impaired uptake of ribavirin similar to FRI cells. E. eIF4E cap binding and eIF4G binding activity are retained in FRII cells. F. FRII cells are sensitive to eIF4E knockdown measured by cell growth. G. Effects of RNAi mediated knockdown of Gli1 or eIF4E on UGT1A levels. Western blots were probed as indicated. RNAi mediated knockdown of Gli1 led to reduced levels of UGT1A whereas knockdown of eIF4E did not. For UGT1A, a pan-UGT1A antibody was used. Antibody controls for UGT1A and Gli1 are shown in Extended Data Figure 8. Results are representative of at least three independent experiments. Average values are reported and error bars indicate standard deviations. Experiments were carried out in triplicate, three independent times. Western blots are representative of at least three independent experiments.