Figure 3.

RET is an important driver of OS tumorigenesis and confers resistance against BTZ. A, Three individual siRNAs targeting RET were introduced into U-2 OS/BTZ and HOS/BTZ sublines by Lipofectamine transfection for 24 h. Lysates of control and RET knockdown BTZ-resistant cells were blotted to confirm RET suppression efficiency. B, Colony formation-promoting activity of RET. Control and RET knockdown (siRET#1) or RET-overexpressing OS or OS/BTZ cells were cultured for 2 weeks with BTZ (100 nM) treatment for the first two days of the experiment. Then, the cells were fixed and stained. C, RET protects cells against BTZ-induced apoptosis. U-2 OS_siRET#1 and U-2 OS_siCon cells or U-2 OS_RET and U-2 OS_vector cells were challenged by either vehicle or BTZ (100 nM), stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI), and analysed by flow cytometry to evaluate the role of RET in the prevention of apoptosis. D, RET blocks BTZ-induced activation of the caspase cascade. OS_siRET#1 and OS_siCon cells or OS_RET and OS_vector cells were incubated with BTZ (100 nM) for 24 h to determine the levels of molecules involved in caspase pathways. E, Interference of RET with the antitumor effects of BTZ is connected with ATF4 inhibition as shown via western blot analysis of ATF4 expression. F, RET activation in BTZ-resistant OS cell lines. U-2 OS/BTZ OS_siCon and U-2 OS/BTZ_siRET#1 cells cultured in serum-free medium overnight were stimulated for 30 min with or without 20 ng/mL GDNF and subjected to western blotting using the indicated antibodies. G, Kaplan-Meier curves showing survival benefits of high RET expression in patients with sarcoma (KM plotter database).