Proliferation and apoptosis of human fibroblasts with compound heterozygous CHIP mutations.

<p>Trehalose protects from apoptosis and epoxomicin-induced cell death in CHIP-mutant human fibroblasts. <b>A</b>) Photomicrographs of dose-dependent effects of epoxomicin and trehalose pre-treatment 15 min before epoxomicin for 24 h on the expression of cleaved caspase-3 positive cells in control and CHIP-mutant fibroblasts (scale bar = 30 µm). <b>B</b>) Percentage of cleaved caspase-3 positive cells in control and CHIP-mutant fibroblasts treated with 0, 2 and 10 nM epoxomicin for 24 h and trehalose 50 mM pre-treatment after 5 days in vitro. One hundred fifty to two hundred cells per coverslip cells were counted. <b>C</b>) Photomicrographs of dividing BrdU⁺ cells and total nuclei stained with bis-benzimide. (Scale bar = 30 µm). <b>D</b>) Percentage of BrdU⁺ cells with respect to the total number of fibroblasts with 7 and 12 passages, respectively. Sixty to eighty cells were counted per coverslip to obtain the percentages of BrdU⁺ 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 are the mean of two experiments of 6 independent cell dishes (pseudo-replicates, n = 6) of the patient group and in control group: the mean of the 3 different controls with 3 dishes of cells each one (n = 3). Statistical analysis was performed by Student's t-test. **p<0.01, ***p<0.001 <i>vs</i> solvents; δδδ p<0.001 trehalose + epoxomicin <i>vs</i> epoxomicin; ∧∧∧p<0.001 fibroblasts with 12 <i>vs</i> 7 passages.