Figure 2.

Therapeutic treatment reduces prion seeding activity. (A) A schematic representation of infection and treatment timeline. COs were inoculated with sCJD brain homogenate or NBH and therapeutically exposed to DMSO (vehicle) or PPS (PPS-T) from 63 to 91 DPI. (B,C) Log SD₅₀s per mg tissue of RT-QuIC seeding activity from COs treated therapeutically with DMSO or PPS and collected at 91 (B), or 120 dpi (C). Each data point represents a single organoid with mean + /− SD from three COs represented. The dashed line indicates the lower detection limit of the assay. Data points colored gray indicate an over-estimation of log SD₅₀ values as these samples did not have a tested dilution where all replicate wells had positive reactions. Western blots showing protease-resistant PrP (PK + ; D), total PrP (PK−; E), and total protein stain for the PK− blot (F) for 120 dpi organoids. Letters a, b, and c denote individual organoids. Samples were run over several blots and spliced for simplicity. Uncropped blots can be found in Supplementary Fig. S4. To adjust for the use of multiple blots samples were quantified (G). Total PrP, relative to the total protein, was normalized to the maximum value (G; PK−). Proteinase K digested samples were quantified by subtracting the baseline of the average NBH values and then normalized to the maximum value (G; PK +). The dashed line denotes “0”. Error bars represent standard deviation. (E) PrestoBlue analyses were performed to monitor the cellular metabolism and viability of the COs over time. (F) In parallel, cultures were monitored for cellular breakdown in COs by measuring LDH levels in the media. The same three organoids were monitored throughout the study starting just prior to the addition of PPS. The control values (gray) are the same traces from Fig. 1. Error bars represent standard deviation from triplicate reading of three pooled organoids. DMSO and PPS represent treatments on sCJD infected organoids. NBH was similarly treated with DMSO.