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

Figure 5.

Figure 5

Depletion of CBP/p300 Reduces the Formation of Aggregates upon HDI Treatment

(A) U2OS cells were transiently transfected with either a control siGFP or sip300 for 4 days. Twenty-four hours before fixation, cells were treated with SAHA. Cells were stained with Proteostat (red channel) and anti-p300 (green channel), and DAPI was used as a nuclear counterstain (blue). Arrowheads indicate Proteostat-positive aggregates. Co-localization of Proteostat-positive aggregates with p300 appears orange/yellow in the overlay. Panels on the right of each pair represent the enlarged area shown by a dashed square on the panels to the left.

(B) U2OS cells were transiently transfected with either a control siGFP or siCBP for 4 days. Twenty-four hours before fixation, cells were treated with SAHA. Cells were stained with Proteostat (red channel) and anti-CBP (green channel), and DAPI was used as a nuclear counterstain (blue). Arrowheads indicate Proteostat-positive aggregates. Co-localization of Proteostat-positive aggregates with CBP appears orange/yellow in the overlay. Panels on the right of each pair represent the enlarged area shown by a dashed square on the panels to the left.

(C) U2OS cells were treated with siRNAs for 4 days (control siGFP, sip300, or siCBP) and subsequently with SAHA (5 μM), and 24 hr later fixed, permeabilized, and stained with Proteostat. At least 20,000 cells were measured by FACS and the mean fluorescence recorded, which was normalized to the siGFP/DMSO-only (−) treated control. The data were derived from six independent biological replicates and the aggregation propensity factor (APF) was calculated. SAHA-treated cells are depicted in red and DMSO-treated cells in black. Level of statistical significance is indicated (**p ≤ 0.01). Error bars denote SD.

(D) Immunoblot showing the protein level of CBP and p300 in siRNA-treated cells. Cells were treated for 4 days and 24 h before lysis, DMSO (−) or SAHA was added. β-Actin was used as a loading control, and an antibody against acetylation of the N terminus of histone 3 served as a control for the effect of SAHA.