Thalidomide

HCT116 WT or trRpn13 cells were seeded at 4000 cells/well whereas RPMI 8226 WT, trRpn13-MM1, or trRpn13-MM2 cells were seeded at 8000 cells/well with RPMI 1640 medium (no phenol red, Thermo Fisher Scientific 11835030) containing 2% fetal bovine serum in 96-well plates. After 24 h, cells were treated with 0.4% DMSO (as a control) and this concentration was maintained with XL5, XL5-PROTACs XL5-VHL, XL5-VHL-2, XL5-CRBN, XL5-IAP, E3 ligand VHL-ligand, thalidomide (Selleckchem, catalog NO. S1193), or IAP-ligand at 10 µM, 20 µM, 30 µM, or 40 µM concentration. RPMI 8226 WT, trRpn13-MM1, trRpn13-MM2, or MM.1S cells were treated similarly but with XL5-VHL-2 at 2.5 µM or 5 µM concentration. Each condition was performed in sextuplicate. After 48 h, 0.35 mg/mL MTT was added for 4 h of incubation. Stop solution (40% DMF, 10% SDS (W/V), 25 mM HCl, 2.5% acetic acid in H₂O) was added to the cells and incubated overnight. Absorbance at 570 nm was measured by using CLARIOstar (BMG LABTECH).

Panc02 cells, derived from C57BL/6J mice, were kindly provided by Dr. M. Li (Baylor College of Medicine, Houston, Texas 77030, USA). The Colo357 cell line was provided by prof. Helmut Friess (Technical University Munich, Munich, Germany). PC cells were maintained in low glucose (5 mM; LG) Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin and incubated at 37°C in 5% CO₂. Various amounts of glucose were added to the culture medium to create cell growth environments containing different concentrations of glucose. In addition, 10 μM SB203580 (Sigma-Aldrich, St. Louis, MO, USA) or 10 ng/ml TGF-β (PeproTech, Rocky Hill, USA) was added to the culture medium. Caffeic acid phenethyl ester (CAPE) and thalidomide were purchased from Selleck Chemicals (Houston, TX, USA).