384 well black flat bottom microplate

The fluorescence polarization (FP)-based binding assay for hERG was measured according to the protocol of the Predictor™ hERG FP kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA). The membrane fraction containing the hERG channel protein (Predictor™ hERG membrane) and tracer (Predictor™ hERG tracer red) was prepared with dilution in the binding buffer provided by the manufacturer. The binding assay was conducted in a final volume of 20 μL with a 10 μL membrane, 5 μL of a 4 nM tracer, and 5 μL of test compounds. The assays were conducted in 384 well black flat-bottom microplates (Corning Life Sciences, Lowell, MA, USA). After incubation for 4 h at room temperature, the FP was determined using a multimode reader (Infinite M1000PRO; Tecan, Mannedorf, Switzerland) in the FP detection mode, with excitation and emission filters of 535 and 590 nm, respectively.

Aliquots of Hsf1 were thawed and immediately centrifuged (4°C, 15 min, 15000 rpm). In order to capture any occurring trimeric Hsf1, the supernatant was incubated for 20 min on ice with DNA containing three HSEs coupled to magnetic beads (5’-CCCCTTCCCGAATATTCCCCC-3’, 0.5 mg per aliquot, Dynabeads M-280 by Invitrogen). The supernatant concentration was determined by absorbance at 280 nm. Subsequently, four discrete concentrations of Hsf1 (5 µM, 1 µM, 300 nM, 100 nM) were prepared with Hsf1-buffer and heat-shocked for 10 min at five different temperatures (30°C, 35°C, 37°C, 39°C and 42°C) using a temperature-controlled water bath. Additionally, 300 nM Hsf1 was kept on ice for the same period of time as a control. Fluorescence anisotropy measurements were performed with a CLARIOstar microplate reader (BMG Labtech) and 384-well black flat-bottom microplates (Corning) in a final sample volume of 30 µL. Samples were serial diluted 1:2 until concentrations were below 1 nM. 10 nM of Alexa Fluor 488-labelled DNA containing three HSEs (5’-[A488]CCCCTTCCCGAATATTCCCCC-3’ (Sigma-Aldrich) was added by the injection system of the plate reader to start the measurement.

ThT fluorescence assay. A Corning 384-well black flat-bottom microplate was used. Each well contained 20 µL of glycine buffer 0,1 M pH 8,4, 5 µL of treatment (AuNPs@POM@PEG) in PBS, 20 uL Aβ monomer, and 5 µL ThT 100 µM. Before the experiment, ThT 100 µM was prepared and filtered through 0.22 um. The glycine and the treatment were first added into the wells. Then, previously prepared Aβ aliquots (0.1 mg) were thawed and diluted with HFIP and PBS 1×, to obtain Aβ_1–42 44.3 µM. Then, the Aβ solution was added in the wells. Just before measurement, the ThT solution was added, reaching a final volume of 50 µL in each well. The data were read every 5 min using a microplate reader (Infinite M200 PRO). After two hours of incubation at 37 °C, untreated Aβ samples was established as 100%, and the rest of the percentages were calculated with respect to this value. In this way, we normalized the variability in the ThT assay, to be able to combine the results. These experiments were performed N = 5.