Figure 4

Longer treatment increased proteasome inhibition and cytotoxicity. (a) RS4;11 cells were continuously treated with indicated concentrations of ONX-0914. At times indicated inhibition of active sites was determined with either substrates (combined ß5 activity on the left panel, LMP7 and LMP2 activity on the right panel) or a mixture of BODIPY(FL)-NC-001 and BODIPY(TMR)-NC-005 activity-based probes (all others, n = 2). Suc-LLVY-amc was used to measure total ß5 activity on the left panel, the residual activity at 4 h is due to ß5c. (b) RS4;11 cells were treated for times indicated, followed by culture in drug-free media. 48 h after the start of the treatment viability was measured with Alamar Blue (top panel, n = 2). (c) Caspase-3,7 activity was measured in extracts of RS4;11 cells continuously treated with 100 nM ONX-0914 (n = 3).