Toyocamycin

Cytarabine (AraC) (77 (link)), trans-ISRIB (4 (link), 63 (link)), FX-1 (49 (link)), and toyocamycin (64 (link), 65 (link)) were obtained from Cayman Chemicals. FXR1 OE and control cells were treated with 1 μM trans-ISRIB for 24 hours. For cell viability that was measured by trypan blue staining and cell counts (1 (link)), FXR1 OE and control cells were treated individually or with a combination of 5 μM AraC and 1 μM trans-ISRIB for 24 hours, 100 nM toyocamycin and 1 μM AraC, and 10 μM FX-1 and 5 μM AraC.

Cells were grown in 96‐well plates (As One) at 1000 cells/well for 12 h. In 3D culture conditions, cells were grown in round‐bottomed ultra‐low attachment 96‐well plates at 1000 cells/well for 4 days. To evaluate the growth of different clones, cells were cultured for 0–4 days. To examine their sensitivity to Raf kinase inhibitor V (Merck), MEK inhibitor I (Merck), tunicamycin (Cayman Chemical), and toyocamycin (Cayman Chemical), cells were treated with these drugs at different concentrations for 48 h in 2D culture and for 10 days in 3D culture. Then cells were incubated with 0.5 mg/mL MTT (Nacalai Tesque) for 4 h, followed by incubation with 5% SDS in 5 mM HCl for 12 h. Absorbances at 550 and 690 nm were measured using SpectraMax® M2e (Molecular Devices).