Trehalose increases autophagy activity in CHIP-mutant human fibroblasts.

<p>After 5 DIV, fibroblasts were pre-treated with trehalose (50 or 100 mM) for 15 minutes followed by addition of epoxomicin or solvent for another 24 h. <b>A</b>) LC3 staining (green) and nuclei (Hoescht, blue) immunofluorescence in CHIP-mutant fibroblast treated with trehalose (100 mM) and their quantification <b>B</b>) in percentage of cells with more than 50 autophagic vesicles (Scale bar = 20 µm). <b>C</b>) Representative microphotographs of Lysosomal-associated membrane protein, LAMP-2A, present in lysosomes and endosomes, (Scale bar = 20 µm) and <b>D</b>) quantification in percentage of cells with LAMP-2A positive vesicle distribution in the perinuclear region, around the nucleus. <b>E</b>) Images of fibroblasts expressing CD63, as a late endo/lysosomal marker (Scale bar = 30 µm) and <b>F</b>) quantification in percentage of optical intensity for CD63 inmunoreactivity. The insets show boxed regions at higher magnification (Scale bar = 15 µm). <b>G</b>) Immunofluorescence for HSC70 and LAMP-2A in CHIP-mutant cultures treated with trehalose 100 mM for 48 h (Scale bar = 20 µm) and <b>H</b>) percentage of co-localization LAMP-2A/HSC70 positive cells. <b>I</b>) Percentage of LAMP-2A positive cells. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean ± SEM. Values in 5B, 5D, 5H and 5I are the mean ± S.E.M. of two experiments with six cover slips, from 150 to 250 cells of every group were counted. In 5F, in CD63 immunostaining data are the mean of 4 independent dishes of cells for 2 control individuals (n = 2), in the CHIP-mutant group 6 independent dishes of cells of the only patient (pseudo-replicates, n = 6). Statistical analysis was performed by Student's t-test. *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> solvents; δp<0.05, δδδp<0.001 trehalose + epoxomicin <i>vs</i> epoxomicin treated cultures.