Fig 1: Effects of AuNCs on TFEB nucleocytoplasmic distribution and lysosomal activity. (A) U251N cells were treated with vehicle, 1 µM Au15SG13 or 1 µM Au15PEG in DMEM for the times indicated. Panels depict the results for cells incubated for 30 min with vehicle or AuNCs. TFEB was detected by immunocytochemistry, phalloidin stained F-actin, and Hoechst 33342 demarcated nuclei. Whole cell and nuclear fluorescence signals were quantified, and the nuclear/cytoplasmic ratio was calculated. The percentage of cells with elevated nuclear TFEB signals was determined for two independent experiments. For each condition and time point 80 to 101 cells were scored; bars show averages ± SEM. (B) AuNCs increase cathepsin B activity. Cathepsin B activity was measured in U251N cells treated with vehicle, 1 µM Au15SG13 or 1 µM Au15PEG. Cells were incubated with Magic Red as described in the Methods section. Hoechst 33342 identified nuclei. For quantification, Magic Red fluorescence intensity was normalized to the vehicle control. Bars depict average ± SEM for at least two independent experiments. Between 34 and 92 cells were analyzed per condition and time point; **, p<0.01; ***, p<0.001.
Fig 2: Autophagy-lysosomal response in fibroblasts from CMT1A patients. A: Western blots for LAMP1, LC3-II, p62, and TFEB on whole-cell lysates from the indicated individuals. Each membrane was reprobed with antibodies for a constitutive marker, tubulin, actin, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B–E: Quantification of LAMP1, LC3-II, p62, and TFEB protein levels after immunoblotting with the respective antibodies. F: The detection of lysosomes in fibroblasts after labeling with LysoTracker. G and H: Confocal images of cells from CMT1A patients (P3 and P4) after double immunolabeling with anti-PMP22 (red) and anti-cathepsin D (G), or anti-LC3 (H) antibodies. In F–H, nuclei are stained with Hoechst dye (blue). *P < 0.05, **P < 0.01 (t-test). Scale bars: 50 µm (F); 10 µm (G and H). CatD, cathepsin D; Tub, tubulin.
Fig 3: Lysosomal changes in GRLysMcre mutant microglia. a Confocal fluorescent images of Lysotracker uptake in living control and mutant primary microglial cells. Bar = 5 µm. The mean fluorescence intensity of Lysotracker was quantified using ImageJ, n = 3 separate experiments. *p < 0.05, control vs. mutant cells. b A representative western blot experiment shows an increase in nuclear fraction of TFEB in mutant microglial cultures, mirrored by a decrease in the cytoplasmic fraction of TFEB. Full gel blots in Supplementary Fig. 5. c In vitro RT-qPCR analysis of expression of lysosomal genes Clcn7, mcoln1, HEXA, ATP6v1h, and lamp1, in microglial cultures prepared from control and GRLysMCre pups. ß-microglobulin gene was used as internal control. *p < 0.05; control vs. GRLysMCre mutant cultures, n = 3 independent experiments. The data are presented as mean with error bars as s.e.m. and Mann–Whitney test for statistical significance. d Representative EM images of a control and a mutant cell. Red arrows point to lysosomes. e Number of lysosomes per 100 µm2 in 21 control and 20 mutant cells. Data are expressed as Tukey boxplot where the median is indicated by a horizontal line, the bottom of the box represents the 25th percentile, the top the 75th percentile, whiskers represent minimum and maximum values. f Distribution of the lysosome surface (µm2). 1386 and 1737 lysosomes were measured, respectively, in control or mutant cells using ImageJ. ?2 test of independence: ?2 = 84.81, df 10, p < 5.10–14
Fig 4: Simplified model depicting the cellular components that are affected, at least transiently, by AuNCs. AuNCs in this study were surface-modified with GSH (Au-SG) or PEGylated (Au-PEG). Lysosomes with more acidic pH (light green) are perinuclear, whereas peripheral lysosomes are less acidic (dark green). AuNCs stimulate the transient nuclear accumulation of transcription factors TFEB and Nrf2. PEGylated AuNCs may increase protein aggregation. See text for details.
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