Figure 5.

Bortezomib-induced aggresome formation and LBH589-induced α-tubulin hyperacetylation. RPMI8226 cells were treated for 24 hours with 16 nM LBH589, 16 nM bortezomib, or both, stained with anti-acetyl-α-tubulin and DAPI, then examined by inverted microscopy. (A) In untreated RPMI8226 cells, acetylated α-tubulin showed a reticulated appearance (left panel). The center panel shows the DAPI stain. In the merged image in the right panel, a normal mitotic figure (arrow, right panel) is shown, as well as other nondividing cells. (B) HDAC inhibitor LBH589 induced diffuse or segmented α-tubulin hyperacetylation. Hyperacetylated-α-tubulin (arrow, left panel); diffuse acetyl-α-tubulin (arrowhead, left panel); segmented α-tubulin hyperacetylation (center panel); mitotic figure (arrow, right panel). (C) Proteasome inhibitor bortezomib induced aggresomes in the presence of normal reticulated α-tubulin (left panel). Focal aggresome (arrow, right panel); apoptotic nuclei (arrowheads, center and right panels). (D) LBH589 and bortezomib induced large bundles of hyperacetylated α-tubulin (arrowheads, left and center panels) that were not seen in cells treated with LBH589 alone. Diffuse hyperacetylated α-tubulin was also observed (arrow, right panel). Although aggresome formation was observed (arrow, left panel), here they were smaller than when treated with bortezomib alone, and were associated with a diffuse cytoplasmic background and apoptotic nuclei.