Figure 3.

MPT0G413 inhibited the binding of histone deacetylase (HDAC6) to dynein and induced the significant accumulation of polyubiquitinated proteins when combined with bortezomib. (A) Hypothetical rationale of proteasome and aggresome pathway inhibition by bortezomib and MPT0G413. (B) RPMI-8226 and NCI-H929 cells were treated with MPT0G413 (1 and 2.5 μM) for 6 h, and total cell lysates were subjected immunoprecipitation with 1 μg of an anti-dynein or anti-ubiquitin antibody, followed by immunoblotting with an anti-HDAC6 antibody. (C) RPMI-8226 and NCI-H929 cells were cultured with MPT0G413 (2.5 μM), bortezomib (2.5 nM), or combined therapy for 12 h. Whole cell lysates were subjected to Western blotting with antibodies specific for ubiquitin and LC3. (D) NCI-H929 cells were treated with MPT0G413 (2.5 μM), bortezomib (2.5 nM), or combination therapy for 6 h and stained by anti-LC3B (green) and anti-ubiquitin (red) antibodies and DAPI (blue) prior to confocal microscopy analysis. Scale bar = 50 μm.