Fig. 4: A hanging drop method and mRNA transfections to assess WT:mutant ratios required for protein aggregation.
From: Transcript errors generate amyloid-like proteins in human cells
A 4 × 6 screening tray was set up with a 1 ml reservoir that contains protein buffer and an increasing concentration of NaCl. B A 10 µl WT TP53 solution (60 µM) was then added to a siliconized coverslip and a 1 µl drop of TP53S149F seed particles was placed immediately adjacent at decreasing concentrations. C If the mutant seed particles have amyloid potential, this event will trigger conversion of WT proteins at the drop-drop interface and lead to localized fiber growth D If no TP53S149F is provided as seeding material, no fiber-like material forms in the WT TP53 solution. E However, if TP53S149F seeding material is provided, fiber-like material grows out of the WT solution. F These fibers show strong birefringence under polarized light, suggestive of amyloid structures. G–J. Primary fibroblasts were transfected with 90% WT and 10% mutant SOD1G142E transcripts display co-localized WT and mutant proteins inside protein aggregates G DAPI. H SOD1G142E-mCherry. I WT SOD1-eGFP. J Overlay of G–I. These experiments were performed 3 times with similar results. K Quantitation of G–J (n = 3 biological replicates), data is presented as mean values ± SEM. Source data are provided as a Source Data file.
