Fig. 6: Amyloid-binding dyes suppress crystal formation.
From: A rapid in vivo pipeline to identify small molecule inhibitors of amyloid aggregation
A Six-day C. elegans larval viability assays showing that amyloid binding dyes (CR and ThS) can suppress wact-190-induced lethality, but that chitin/chitosan-binding dyes (CFW and EY) cannot. Synchronized first larval stage worms were added to each well that contained the indicated concentration of dye and crystalizing wact-190 small molecule. Six days later, the viability of worms in each well was assessed (N = 3, n = 4). B Representative birefringence images that show that amyloid-binding dyes, but not chitin/chitosan-binding dyes, can suppress crystal formation. All images show the heads of individual worms similar to Fig. 2B. On the top row, animals were pre-incubated in the indicated dye or solvent control for 3 h. Then, the right-most five samples were incubated in 60 μM wact-190 for three hours without the initial dyes present. On the bottom row, animals were incubated in the indicated dye or solvent control, and for the five right-most five samples, simultaneously incubated with wact-190 (60 μM) for 3 h. The bright signal shows where crystals have formed (the blue arrows highlight a few examples). C The quantification of wact-190 birefringent (crystal) signal from worms either pre-incubated with dyes before adding 60 μM of the crystallizing wact-190 molecule (left panel) or co-incubated with dyes and 60 μM of the crystallizing wact-190 molecule (right panel). In both panels, the grey bars are controls, with the first grey bar having only DMSO solvent added without wact-190 added, which shows the background birefringence signal from worms without crystals. The two other grey control bars show that worms were either preincubated (>) for 3 h with solvent and then wact-190 (w190) was added, or that DMSO and wact-190 were co-incubated (+). The red bars show the results of preincubation (left) or coincubation (right) with Congo Red (CR) and wact-190; the dark blue bars show the same with ThioflavinS (ThS) and wact-190; the light blue bars show the same with Calcofluor White (CFW) and wact-190; and the yellow bars show the same with eosin Y (EY) and wact-190. Nearly all experiments had N = 3 and n ≥ 10) (see the Source Data file for details). Two-sided Student’s t-tests were used to assess differences. No adjustments for multiple comparisons were made. Standard error of the mean is shown. The p-values for the dye pre-incubation experiments are as follows: DMSO > DMSO vs DMSO>w190, 4.5E-19; 60 μM CR>w190 vs DMSO>w190, 4.3E-13; 1.2 mM CR>w190 vs DMSO>w190, 7.7E-14; 60 μM ThS>w190 vs DMSO>w190, 8.1E-02; 1.2 mM ThS>w190 vs DMSO>w190, 1.2E-05; 60 μM CFW>w190 vs DMSO>w190, 8.8E-01; 1.2 mM CFW>w190 vs DMSO>w190, 2.7E-01; 60 μM EY>w190 vs ETOH>w190, 1.0E-01; 1.2 mM EY>w190 vs ETOH>w190, 6.4E-01. The p values for the dye co-incubation experiments are as follows: DMSO > DMSO vs DMSO>w190, 4.5E-05; 60 μM CR>w190 vs DMSO>w190, 9.5E-03; 1.2 mM CR>w190 vs DMSO>w190, 8.7E-12; 60 μM ThS>w190 vs DMSO>w190, 9.1E-01; 1.2 mM ThS>w190 vs DMSO>w190, 1.7E-08; 60 μM CFW>w190 vs DMSO>w190, 5.3E-02; 1.2 mM CFW>w190 vs DMSO>w190, 7.4E-02; 60 μM EY>w190 vs ETOH>w190, 8.4E-01; 1.2 mM EY>w190 vs ETOH>w190, 9.8E-02.
