Fig. 3: Lipoamide interacts with disordered proteins in cells.
From: Small-molecule dissolution of stress granules by redox modulation benefits ALS models
a, TPP scheme for testing the effect of lipoamide on protein thermal stability. Soluble protein quantity was measured by mass spectrometry, normalized to 0.1% DMSO-treated controls, of HeLa cells treated with 100 µM lipoamide and/or 1 mM arsenate at ten temperatures. Heating caused decreased detection through protein denaturation and precipitation, modulated by lipoamide. b, Volcano plot of thermal stabilization z scores (mean, n = 3 experiments) and FDRs following treatment with lipoamide and arsenate. Broken vertical and solid horizontal lines show the z score (±1.5) and FDR (<0.05) cutoffs, respectively, classifying stabilized (green) and destabilized (blue) proteins. The positions of DLD, SFPQ, SRSF1 and several stress granule proteins are indicated. c, Violin and box plots showing the proportions of IDRs in each protein, categorized into stabilized (z > 1.5, FDR < 0.05; 70 proteins), destabilized (z < 1.5, FDR < 0.05; 144 proteins) and unaffected (5,811 proteins). Box plots show the median (bold bar), 25th and 75th percentiles and outliers (closed dots), and whiskers extend to the most extreme values. P values were determined by a two-tailed Wilcoxon signed-rank test, followed by a Holm’s test. d, Mean ± s.d. enrichment (>0) or depletion (<0) of each amino acid in the IDRs of stabilized (green) and destabilized (blue) proteins compared to IDRs from all 6,025 detected proteins. P values were determined by unpaired t-test, followed by a Bonferroni test. e, Representative G3BP1 immunofluorescence images of HeLa cells (more then three independent experiments) depleted of SFPQ or SRSF1 and treated with 10 µM lipoamide or 0.1% DMSO for 1 h, followed by 1 mM arsenate for 1 h in the presence of lipoamide. f, Mean ± s.d. of the percentage of stressed HeLa cells with three or more G3BP1+ stress granules (SGs); n = 292–615 cells from three independent experiments. Dots indicate the mean values from each experiment. P values were determined by Tukey’s test. g, Representative immunofluorescence images of HeLa cells (more than three independent experiments) treated with 1 mM arsenate for 1 h. Broken lines indicate the edges of the cytoplasm and nucleus of one example cell.
