Reduced autophagosome formation in CLN2 and CLN3 fibroblasts and decreased autophagosome maturation in CLN3 cells.

<p>(A) Total extracts from control (CTRL), CLN2 and CLN3 fibroblasts incubated under high (H) and low (L) proteolysis conditions and with or without lysosomal inhibitors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055526#s4" target="_blank">Material and Methods</a>) were analyzed by western-blot using LC3 and, as a loading control, actin antibodies. The position of LC3-I, and LC3-II bands are indicated on the left. Densitometric measurements from three different experiments are shown below. Results are presented as LC3-II/actin ratios and are expressed as percentages of the control values with inhibitors under high proteolysis conditions. Stars indicate statistically significant differences under high proteolysis conditions from the corresponding control values (**p<0.01). (B) Representative fluorescence images of control, CLN2 and CLN3 fibroblasts incubated in high proteolysis medium for 2 h at 37°C. Upper micrographs: immunocytochemistry with anti-LAMP1. Lower micrographs: control, CLN2 and CLN3 fibroblasts, 48 h after transfection with pEGFP-LC3. Bar: 20 µm.