Figure 5: Tolcapone prevents TTR-induced cytotoxicity of TTR variants and inhibits L55P-TTR aggregation under physiological conditions.

(a) Tolcapone protects human cardiomyocytes from the toxic effect of V122I-TTR and WT-TTR. AC16 cells were incubated for 24 h with WT or V122I-TTR in the presence or absence of 2 molar equivalents of tolcapone or tafamidis. Resazurin reduction assay was used to measure cell viability. Percentage of cell viability was calculated compared with cells treated with vehicle only. Both compounds rescued the cells from the toxicity induced by TTR. Error bars indicate s.e.m. (n=8). (b) Tolcapone lowers cytotoxicity of Y78F-TTR aggregates. RN22 cells were exposed to aggregates generated in the absence or presence of tolcapone or tafamidis for 24 h. Caspase-3 activation on cell lysates was used to probe cytotoxicity. Both compounds lowered caspase-3 activation promoted by the aggregates. Error bars indicate s.e.m. (n=4). (c) Tolcapone prevents aggregation of L55P-TTR under physiological conditions. RN22 cells expressing L55P-TTR under the control of methallothienin promoter were incubated in the presence or absence of tolcapone or tafamidis (1 μM). Secreted L55P-TTR aggregation was monitored by a filtration assay followed by a dot blot (representative blot triplicates are shown on the right panel). Please note that the same control was published in a previous article⁶⁵ that reported the results obtained for other inhibitors of L55P-TTR aggregation that were tested in the same dot blot assay. Uncropped images of the blots are shown in Supplementary Fig. 8. Filtration of the medium retains high molecular mass aggregates that are detected using a rabbit anti-human TTR antibody. Intensities of the dots were measured, percentage of aggregation was calculated with respect to cells treated with vehicle, and plotted as bars (left panel). Error bars indicate s.e.m. (n=3).