Fig. 4. Overexpression of LC3 mutants inhibits incorporation of p62 into autophagosomes and its degradation within lysosomes. (A) HeLa cells were transfected with GFP fused to LC3^WT or to its mutants and after 24 hours were cultured for 3 hours in EBSS medium, fixed, immunostained with anti-Atg16 antibodies and analyzed by confocal microscopy. The boxed areas are enlarged on the right. (B) Cells transfected as in A were starved in the presence of 0.1 µM Baf A, fixed, immunostained with monoclonal anti-LAMP-1 antibodies and analyzed by confocal microscopy. The areas shown in white boxes are enlarged on the right. Colocalization analysis of GFP fused proteins with LAMP-1 (right panel) was performed as described in the Materials and Methods. (C) Cells transfected as in A were starved in the presence of 0.1 µM Baf A, fixed, immunostained with monoclonal anti-p62 antibodies and analyzed by confocal microscopy. The boxed areas are enlarged on the right. Arrows indicate nontransfected cells; arrowheads indicate cells overexpressing GFP-LC3 proteins. Quantitative colocalization analysis of GFP fused proteins with p62 (right panel) was performed as described in the Materials and Methods. Mean ± s.d. of five independent experiments is presented below. ^**P<0.001. (D) Cells transfected as in A were starved in the presence or absence of 0.1 µM Baf A, lysed with RIPA extraction buffer and analyzed by western blot with anti-p62, anti-actin and anti-GFP antibodies (left panel). Quantification of relative p62 degradation (right panel) was calculated as described in Materials and Methods, and mean ± s.d. of four independent experiments is presented below. ^*P<0.05. (E) The rate of degradation of long-lived proteins was measured in HeLa cells incubated in either MEM (control) or EBSS (starvation) medium, 24 hours following transfection of GFP-fused proteins. Values expressing the percentage of cellular proteins degraded in 4 hours are represented as the means ± s.d. of six determinations. Scale bars: 5 µm.