Lysosomal alterations in NCL fibroblasts.

<p>(A) Representative electron micrographs of control, CLN2 and CLN3 fibroblasts incubated in high proteolysis medium to activate autophagy. Arrows indicate lysosomal vacuoles. Bar: 0.5 µm. (B) TPP1 activity measurements. Control (black bars), CLN2 (white bars) and CLN3 (gray bars) fibroblasts were incubated under high (H) and low (L) proteolysis conditions as indicated. TPP1 activity and total protein were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055526#s4" target="_blank">Materials and Methods</a>. Results are expressed as RFU values per minute and µg protein, and are the mean and S.D. from three separate experiments with triplicated samples. (C) Pulse-chase experiments. Labelling of human fibroblasts with [³H] valine in high proteolysis medium, measurements of degradation of long-lived proteins and the contribution of lysosomal (total and macroautophagy) and proteasomal pathways were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055526#s4" target="_blank">Materials and Methods</a>. Results are presented as the percentage of the labelled protein that is degraded per hour, and are the mean and S.D. from eight to fifteen separate experiments with duplicated samples. In B and C, stars immediately on top of bars indicate statistically significant differences from control values (*p<0.05 and ***p<0.005).