Figure 6.

Inhibition lysosome fusion with autophagosome impaired clearance of the Q79-EGFP aggregates.

Notes: (A) Puncta determination by confocal microscopy. The induced Q79 cells were incubated with 200 nM insulin, 20 µM 3-MA, 1 nM Baf A1, and 10 µM Sp600125, respectively, for 1 h before being treated with DMSO as vehicle control (top panel) or 20 µM of OC-13 (bottom panel) for 12 h. The cells were fixed, incubated with LC3 (red) and EGFP (green) antibodies and counterstained with DAPI (blue). The slides were analyzed by confocal microscopy (scale bar =10 µm, and applied to all images). (B) Quantitative determination of LC3 puncta per three cells. To determine puncta in three cells, the numbers in the induced Q79 clone with pretreatment of various inhibitors were counted as specified before treatment with OC-13 (20 µM) or DMSO for 12 h. The total puncta in three cells by OC-13 treatment were compared with DMSO (**P<0.01, *P<0.05, Student’s t-test).

Abbreviations: Baf A1, bafilomycin A1; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescence protein; 3-MA, 3-methyladenine; h, hour; W/O, without.