Figure 1

GSK257815A activates autophagy in SH-SY5Y cells. (a) Confocal images of SH-SY5Y cells transfected with GFP-LC3 vector. Twenty-four hours after transfection, cells were treated with 1 nM GSK2578215A for 12 h in the presence or absence of CQ (50 μM). Untreated control cells showed diffuse cytosolic GFP-LC3 distribution, whereas those treated with GSK2578215A, CQ, or both presented increased numbers of GFP-LC3 dots per cell. (b) Histogram showing the percentage of autophagic cells (cells with more than six GFP-LC3 dots) at 3, 6, 9 and 12 h after 1 nM GSK2578215A addition. (c) Western blot analysis of p62 and LC3 protein levels using lysates of SH-SY5Y cells, which were treated or not with 1 nM GSK257815A for 3 or 12 h. Tubulin was used as a loading control. All data are representative of three independent experiments. (d) Histogram representing the effects of LRRK2 knockdown (siRNA) and LRRK2-IN-1 (5 μM, 12 h) in SH-SY5Y cells on autophagy. (e) Dot graph representing the number of GFP-LC3 dots per cell, treated or not with GSK2578215A in the absence or presence of CQ. Autophagic flux values for synthesis calculated as detailed in the Materials and Methods section. (f) Confocal microscopy pictures of SH-SY5Y cells transfected with mRFP-GFP-LC3. Twenty-four hours after transfection, cells were treated with 1 nM GSK2578215A. (g) Quantification of autophagosomes (yellow dots) and autolysosomes (red dots). Rapamycin (10 nM) was used as a positive control. Hoechst staining was used to show the chromatin state. Data shown in histograms are the means±S.E.M. of at least three independent experiments, each performed in triplicate. Statistical significance was determined by a two-tailed Student’s t-test: *P<0.05 and **P<0.01. Scale bars, 10 μm