Proteostat dye

Zeiss LSM 710 inverted confocal microscope was used to obtain all the confocal microscopy images. SMB and CAD cells were grown on cover slips in 24 well plates (at a concentration of 4 × 106). In general cells were fixed for 20 min in ice cold 4% PFA at 4 °C. Fixed cells were washed and permeabilised with 0.1% triton X100 for 10 min at 4 °C and blocked with 2% FCS for 30 min at RT before staining with primary antibodies. To observe PrP^Sc aggregates an additional step of treating the cells with 5M guanidine isothiocynate for 5–10 min at room temperature after permeabilising them with 0.5% tritonX-100 in PBS was employed. Live imaging for lysosomes was done using LYSO-ID red dye (Enzo life sciences, ENZ-51005-0100) (1:1000) by incubating the cells for 30 min at 37 °C. Proteostat dye (Enzo life sciences, ENZ-51035) was used to visualise the aggregates, cells were washed and fixed and permeabilised as above and incubated with Proteostat dye and Hoechst (1:100) and incubated in dark for 30 min at RT, and cells were later washed and mounted on glass slides.

The Proteostat Dye (Enzo ENZ-51023-KP050) was used according to manufacturer’s protocol. For microscopy, Lab-Tek dishes were coated with poly-L-lysine-hydrobromide (Sigma P6282) before adding cells.

Cells were fixed and permeabilized by BD Fixation/Permeabilization Solution kit (BD Biosciences). The protein aggregation level in the cells were analyzed by FACS using ProteoStat Dye (Enzo Life Sciences).

Adenine hemisulfate salt ≥99%, TMAO, DMSO, DCA, UDCA, sulfolane ≥99%, benzophenone, diphenyl sulfoxide, and ThT were purchased from Sigma-Aldrich (Rehovot, Israel). 1,4-Thioxane-1,1-dioxide ≥98% and tetrahydrothiophene 1-oxide were purchased from Tzamal D-Chem (Petah Tikva, Israel). ProteoStat dye was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). DCA, UDCA, 1,4-Thioxane-1,1-dioxide, benzophenone and diphenyl sulfoxide stocks were prepared and diluted in pure DMSO. TMAO and tetrahydrothiophene 1-oxide stocks were prepared and diluted in PBS.

One milliliter of logarithmic cells were washed with PBS buffer and sonicated using 15 s pulses at 20% power. For each sample, 2*10⁶ cells were resuspended with ProteoStat dye (Enzo Life Sciences) diluted 1:3000 in ProteoStat assay buffer. Cells were incubated for 15 min at room temperature protected from light. Flow cytometry was preformed using Stratedigm S1000EXi and the CellCapTure software (Stratedigm, San Jose, CA). Live cells were gated (P1) by forward scatter and side scatter. Fluorescence channels for FITC (530/30) and PE-Cy5 (676/29) were used utilizing a 488 nm laser source. A total of 50,000 events were acquired for each sample. Analyses were performed using FlowJo software (TreeStar, version 10). The results displayed are representative of three biological experiments performed in triplicate.