Figure 1.

Amyloid-like Protein Aggregates in HDAC Inhibitor-Treated Cells

(A) U2OS cells were treated with either DMSO (control, −) or SAHA (5 μM) for 24 hr and stained with the dye X-34 (blue), which specifically recognizes amyloid-like structures. The cells were then fixed, permeabilized, and counterstained with propidium iodide (PI) to visualize the nuclei (red). Nuclei are indicated with white dashed circles. White arrowheads indicate the position of X-34-positive aggregates. Scale bar represents 10 μm.

(B) U2OS cells were treated with either DMSO (−) or etoposide (5 μM) for 24 hr and stained with X-34 (blue). The cells were then fixed, permeabilized, and counterstained with PI to visualize the nuclei (red). Scale bar represents 10 μm.

(C) U2OS cells were treated with either DMSO as negative control (−) or SAHA (5 μM) for 24 hr, fixed, and permeabilized. The cells were then stained with Proteostat, recognizing aggregated (not necessarily amyloid-like) structures (red), indicated by white arrowheads. Cells were counterstained with DAPI (blue) to visualize their nuclei, indicated here by white circles. Scale bar represents 10 μm.

(D) U2OS cells were treated with either DMSO (−) as negative control or SAHA (5 μM) for 24 hr, fixed, and permeabilized. The cells were then stained with thioflavin S solution, which can specifically stain amyloid structures in in situ specimens (blue). Scale bar represents 10 μm.

(E) U2OS cells were treated with SAHA inducing the formation of Proteostat-positive aggregates, as shown for instance in (C). Cells were measured by flow cytometry in the FL3 channel and the aggregation propensity factor (APF) was calculated. From at least five independent biological replicates APFs were averaged and APF_max½ was calculated, indicating that at half-maximal concentration aggregation occurs. Error bars denote SD.

(F) Immunoblot corresponding to (A) and (B) measuring acetylated histone H3Ac, demonstrating that the pan-HDAC inhibitor was active and hyperacetylation of proteins appeared. Increasing levels of p53 give a measure for the successful application of etoposide (Eto). β-Actin was used as a loading control.

DIC, differential interference contrast. See also Figures S1 and S2.