Silmitasertib

Human normal colon cells CCD 841 (CoN) and colon cancer cell lines (SW-480, DLD-1, HT-29 and HCT-116) were purchased from ATCC. Cells were maintained at 37 °C and 5% CO₂ in RPMI-1640 medium (Invitrogen, Paisley, UK) supplemented with 10% Fetal Bovine Serum (HyClone, Logan, UT), antibiotics (10000 U/ml penicillin and 10 μg/ml streptomycin) and eventually treated with silmitasertib (ApexBio Technology LLC, Houston, TX).

Protein stability assays were performed as described elsewhere [23 (link),24 (link)]. Briefly, cells (1 × 10⁶ cells/well) were seeded into 12-well plates, cultured overnight at 37 °C and 5% CO₂ in DMEM-F12 medium supplemented with 10% FBS, and incubated overnight with 20 μg/mL cycloheximide (CHX, Tocris, Bristol, Avon, UK) to inhibit protein synthesis. Then, cells were incubated in the presence of 25 μM silmitasertib (a selective CK2 inhibitor, formerly known as CX-4945; Apexbio, Houston, TX, USA) or vehicle only (0.001% DMSO) for 6 h. Cells were then harvested and lysed, and the total protein was analyzed using Western blots.

Cells (5 × 10⁵) were seeded into P60 plates and cultured for 36 h in complete medium under normal conditions, with 20 μg·mL⁻¹ cycloheximide (CHX) in the absence or presence of 25 μm silmitasertib (ApexBio Technology LLC, Houston, TX, USA). At different time points, cells were harvested and lysed and 30 μg of total protein was separated by SDS/PAGE. ECE1c was detected by western blot using a mouse anti‐FLAG antibody (1 : 2000; Sigma‐Aldrich).