Analysis of autophagy process in patients-derived fibroblasts with homocystinuria.

<p>(A) Analysis of protein levels by immunoblotting. Equal amounts from control and the five patients were loaded (50 μg of total cell lysates) and subjected to Western Blot with anti-LAMP1 antibody. We used anti-GAPDH antibody to ensure equal amounts of protein loaded in each lane. This result is representative of three independent experiments. (B) Protein quantification was performed by laser densitometry. The ratios between proteins/GAPDH for each cell line were calculated to determine the expression fold-change relative to control. Data represent mean ± standard deviation of three independent experiments. (C) Analysis of autophagosomes formation by fluorescence microscopy. Representative images of the autophagosome monitorization in control and patients-derived fibroblasts. (D) Quantification of autophagosome number per area in patients´cells compared to control. Results were obtained from three independent experiments. Data represent mean ± SEM (standard error of the mean = SD/) of positive fluorescence points of LC3B in each mm² of 80 cells analysis. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.