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Fig. 1. Effect of N-terminal truncation in LC3 on its processing and autophagosomal localization. (A) 3D structure of LC3 and its different N-terminal truncations (illustrated in red). PyMol molecular visualization system was used for presentation of 1UGM:A file from RCSB PDB (Sugawara et al., 2004). LC3^N28 contains the ubiquitin core alone. (B) CHO cells stably expressing GFP-LC3, GFP-LC3^N10, GFP-LC3^N28 or GFP-LC3^N40 were cultured for 2 hours in MEM (control) or EBSS (starvation) medium, fixed and analyzed by fluorescence microscopy. (C) Stably expressing CHO cells were starved in the presence of 100 nM bafilomycin A (Baf A), lysed with RIPA extraction buffer and cell lysates were analyzed by immunoblotting using anti-GFP antibodies (right). Asterisk labels the lipidated form (LC3-II). Quantification of GFP-LC3 lipidation (left) was performed as described in Materials and Methods. Data are means ± s.d. (D) Left, extracts of GFP-LC3 and GFP-LC3^N28 expressing cells were subjected to western blot analysis using anti-GFP or anti-LC3 antibodies. Middle panels, cells expressing GFP-LC3^N28 were incubated in MEM or EBSS medium in the presence or absence of Baf A, fixed and immunostained with anti-LC3 antibodies. Colocalization was analyzed by confocal microscopy. The boxed areas shown are enlarged on the right. Right, colocalization analysis of GFP-LC3^N28 or GFP-LC3^{N28 G120A} with endogenous LC3 in starved cells was performed using the JACoP plugin in ImageJ (NIH Image) as described in the Materials and Methods. (E) Cells expressing GFP-LC3, GFP-LC3^N28 or GFP-LC3^N40 were incubated for 2 hours under starvation conditions in the presence of 100 nM Baf A. Lysosomes were labeled with monoclonal antibodies against LAMP-1 and colocalization was analyzed by confocal microscopy (left panels). The areas shown in white boxes are enlarged on the right. Colocalization analysis of GFP-LC3^WT, GFP-LC3^N28 or GFP-LC3⁴⁰ with LAMP-1-labeled lysosomes (right) was performed as described in the Materials and Methods. Scale bars: 5 µm.

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