Fig. 3.
Fusion cell lines exhibit dose-dependent response to RET inhibition. a Dose−response curves after 72 h of drug treatment with cabozantinib or sorafenib in NIH/3T3 cells expressing RETamp, ΔRET, NCOA4-RET, and vector. Cell viability normalized to vehicle (DMSO)-treated cells. Error bars indicate s.d. of three replicates and are representative of three independent experiments (n = 3). b Western blot indicating inhibition of RET fusion kinase, MEK and P70 S6 signaling with increasing concentration of cabozantinib in NIH/3T3 cells transiently expressing NCOA4-RET or ΔRET. Measurements were made after overnight serum starvation and 1 h of incubation with cabozantinib in the absence of serum. Graphs represent image densitometry analysis of western blots from three independent experiments (n = 3). Ratio of phosphorylated to total proteins is measured at each concentration and mean values with error bars indicating s.d. are plotted relative to DMSO-treated control. c Dose-dependent reduction in ΔRET and NCOA4-RET colony numbers upon treatment with increasing concentrations of cabozantinib in NIH/3T3 transduced cells for 14 days. 0 represents DMSO-treated controls. All cells were plated at equal numbers per well in triplicates per experiment. Error bars indicate s.d. of three replicate measurements per condition (n = 3) and are representative of experiments performed three times. p ≤ 0.05 (*), ≤0.01 (**) and ≤0.001 (***) by ANOVA (one-way for (b) and two-way for (c)) with Tukey’s multiple comparisons test. Open-ended brackets depict comparison between the indicated group and each of the groups under the bracket. Where brackets are absent, comparison is with DMSO control