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. 2017 Jan 19;24(1):9–23. doi: 10.1016/j.chembiol.2016.11.009

Figure 4.

Figure 4

p300/CBP-Specific BDIs Reduce the Formation of Aggregates upon Hyperacetylation

(A) U2OS cells were treated with SAHA (5 μM) or DMSO (−) with or without the indicated BDIs (2.5 μM). After 24 hr the cells were fixed, permeabilized, and stained with Proteostat (red), anti-CBP (green), and DAPI (blue) as a nuclear counterstain, and analyzed by confocal microscopy. Panels on the right of each pair represent the enlarged area shown by a dashed square on the left panels. Aggregates stained with Proteostat that co-localize with CBP appear as yellow/orange in the images. BSP, bromosporine; 33, compound 33.

(B) U2OS cells were treated with CXD101 (5 μM) or DMSO (−) with or without the respective BDIs (2.5 μM). After 24 hr the cells were fixed, permeabilized, and stained with Proteostat (red), anti-CBP (green), and DAPI (blue) as a nuclear counterstain, and analyzed by confocal microscopy. Panels on the right of each pair represent the enlarged area shown by a dashed square on the left panels. Aggregates stained with Proteostat that co-localize with CBP appear as yellow/orange in the images. BSP, bromosporine; 33, compound 33.

(C) SH-SY5Y cells were treated with SAHA (5 μM) or DMSO (−) with or without the respective BDIs (2.5 μM). After 24 hr the cells were fixed, permeabilized, and stained with Proteostat (red), anti-CBP (green), and DAPI (blue) as a nuclear counterstain, and analyzed by confocal microscopy. Panels on the right of each pair represent the enlarged area shown by a dashed square on the left panels. Aggregates stained with Proteostat that co-localize with CBP appear as yellow/orange in the images (see also Figure S5). BSP, bromosporine; 33, compound 33.

(D) Corresponding immunoblot measuring acetylated histone H3Ac, which demonstrates that the pan-HDAC inhibitor was active, the appearance of hyperacetylation of proteins, and the influence of two CBP/p300-specific BDIs. U2OS cells were treated with SAHA (5 μM) and the respective BDIs (2.5 μM) and then harvested after 24 hr. β-Actin was used as a loading control.

See also Figures S3 and S5.