Figure 4

Inhibition of 26S proteasomal activities of human malignant melanoma cells treated with derivatives 2, 5 and 6. Human melanoma cells HTB68 were plated (1 × 10⁶ cells/well) in 12 well plates and incubated 24 h at 37°C in CO₂ incubator. Cells were then treated with 2 (1.5 mg/mL), 5 (2.6 mg/mL) and 6 (1.75 mg/mL) or epoxomicin (50 ng/mL) as a positive control for 24 h. Cells were harvested and cytosolic fractions were prepared using nuclear/cytosol fractionation assay kit (Bio-Vision incorporated). The chymotrypsin-like activity, PGPH activity and trypsin-like activity of 26S proteasome was monitored using fluorogenic substrates in fluorometer with an excitation filter of 360 nm and emission filter of 460 nm.