<p>The cells were treated for 24 h with o without trehalose 50 mM in the presence or absence of 10 µM chloroquine (CQ), a lysosomotropic agent widely used to block lysosomal degradation. <b>A</b>) Fluorescence microscopy images showing autophagosome marker LC-3 (green), lysosomal marker Lamp-2A (red) and LC3-LAMP-2A colocalization (yellow). (Scale bar = 20 µm). <b>B</b>) Integrated optical density (IOD) quantification of LC3 expression. <b>C</b>) The lysosomal degradation was measured by IOD quantification of colocalization LC-3-LAMP-2A (yellow). <b>D</b>) Western blot showing LC3-II accumulation. 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. In 7B and 7C values are the mean ± S.E.M. of six independent coverslips (each value represents the mean of 20 field for coverslip). Three individuals with the mean of 2 independent coverslips (n = 3) in the control group and in the CHIP-mutant group 6 independent coverslips of the only patient (pseudo-replicates, n = 6). In 7D values represents the mean of 2 independent dishes of cells of 2 controls (n = 2) and for the CHIP-mutant group one patient with four independent dishes (pseudo-replicates, n = 4). Statistical analysis was performed by Student's t-test. *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> solvents without chloroquine; δδp<0.01, δδδp<0.001 trehalose -CQ <i>vs</i> trehalose + CQ treated cultures.</p
