FIGURE 3.

(a) LMP2 inhibitory activity of DB-60 (20) and other compounds in ABCB1-overexpressing RPMI-8226 cells. ABCB1-overexpressing RPMI-8226 cells or the parental RPMI-8226 cells were incubated with varying concentrations of respective compounds (n=3 replicates per concentration). After 72 hours, cell lyses were prepared to measure proteasome catalytic subunit activities (LMP2 and LMP7). Curve fitting analysis was performed to calculate IC₅₀ values reported as the mean ± SD. *Due to carfilzomib's cytotoxic effect (a dual inhibitor of cP and iP), cells were incubated for 3 hours, followed by the measurement of chymotrypsin-like (CT-L) activity. (b) Lack of interactions between DB-60 (20) and ABCG2. The inhibitory interactions with ABCG2 were assessed by measuring the changes in the cellular accumulation of pheophorbide A (PhA, a fluorescent ABCG2 substrate) using MDCKII cells stably expressing ABCG2 (established in our previous study41) and parental cells. The known ABCG2 inhibitor Ko143 was used as a positive control. After cells were preincubated in the complete medium containing PhA (1 μM) with Ko143 (0.2 μM) or DB-60 (20) (2 or 5 μM) at 37°C for 30 min, cells were then washed with ice-cold medium and incubated again with Ko143 or DB-60 (20) for 45 min at 37°C. Subsequently, the fluorescent signal was measured via flow cytometry using the excitation and emission wavelengths at 635 and 670 nm, respectively. Colored lines represent the following groups: grey, PhA only; orange, PhA in the presence of 20 (2 μM); red, PhA in the presence of 20 (5 μM); purple, PhA in the presence of Ko143 (0.2 μM) (n=2 per group).