IC50 Calculation of naïve DU-145, DU-145 RB60, and DU-145 RB60U cells.

The data from crystal violet assays were analyzed using GraphPad Prism 8, and the calculated IC50 values are presented here. The resistant cells exhibited a 5-fold increase in Bortezomib tolerance after 24 weeks of Bortezomib presence, which was augmented more following another 24 weeks of drug presence, reaching a more than 10-fold increase compared to the naïve clone. Additionally, to some extent, cross-resistance to Carfilzomib was observed; the DU-145 RB60 clone achieved 4-fold Carfilzomib resistance compared to the naïve clone after 24 weeks and an almost 6-fold change after another 24 weeks. The long-deprived clone maintained its acquired resistance to both inhibitors during the 24-week monitoring. Regarding resistance to doxorubicin, all three cell clones tested exhibited a similar response to doxorubicin, regardless of resistance to Bortezomib. The experiments were repeated in triplicate, and for the IC50 calculation, the built-in model from GraphPad Prism 8 was used.