Fig. 6

An HSF1-dependent transcriptional program suppresses TDP-43 acetylation-mimic-induced aggregation. a Schematic diagram of HSF1 constructs highlighting active and inactive mutations. The regulatory domain (RD) was deleted and a single L395E was introduced to generate active HSF1, while R71G represents inactive HSF1. DBD DNA-binding domain, HR hydrophobic heptad repeat domain. b Cells were transfected with TDP-43-ΔNLS-K145Q in combination with the various HSF1 constructs (or control pCDNA vector) followed by biochemical fractionation and immunoblotting using the indicated TDP-43, P409/410, HSF1-HA, or GAPDH antibodies. Shown is a representative image containing duplicate samples from N = 3 independent experiments. c TDP-43 and phospho-TDP-43 levels from b were quantified by densitometry. d Cells expressing constructs similar to c above were analyzed by immunofluorescence using GFP and P409/410 antibodies. White arrows highlight phosphorylated TDP-43 aggregates. e Inclusion formation in d was quantified. f A sequential transfection was performed first with TDP-43-ΔNLS-K145Q followed by subsequent transfection of HSF1 for another 48 h. Soluble and insoluble fractions were analyzed by immunoblotting using the indicated antibodies. g Insoluble TDP-43 and phospho-TDP-43 were quantified, showing reduction of pre-formed TDP-43 aggregates by active HSF1. h Cells expressing TDP-43-ΔNLS-K145Q aggregates were exposed to DMSO or HSF1A and analyzed by immunofluorescence. i Aggregates were quantified and normalized to the number of total TDP-43 expressing cells. Error bars indicate SEM, and the asterisk indicates statistical significance with ***p-value < 0.001 and **p-value < 0.01, as measured by Student’s t-test from N = 3 biological replicates. ns = not significant. Scale bar = 50 μm