Fig. 1.

Bortezomib and Everolimus show synergistic antitumor effects in mutant KRAS CRC cells. Mutant KRAS HCT116, DLD1, SW480 and LS180 cells were treated with vehicle (untreated, Un), Bortezomib (B), Everolimus (R), or their combination (BR, 5 nM and 10 µM or specified ranges). a Cell growth at 48 h was assessed using MTS assay. b Calculated combination index (CI). The CI presents synergism (CI < 1), additive effect (CI = 1), and antagonism (CI > 1). Red area presents synergism, and green area presents antagonism. Fa represents fraction affected. c Attached cells were visualized by crystal violet at 48 h. (D) Colony formation assay. Cells were treated for 24 h and replated in drug free medium for 14 days before crystal violet staining. e The relative number of colonies as in (d) was normalized to untreated group (100%). f Apoptosis was quantitated by Annexin V + cells using flow cytometry. g Indicated proteins at 24 h was detected using western blotting. ACTIN is used as loading control. The status of major drivers is shown below. e, f, values are mean + s.d. (n = 3). ***p < 0.001 (Student’s t-test, two tailed). B, R vs. BR