Figure 3

The Atg5/12-LC3 pathway is indispensable for PCAF-mediated δ-catenin degradation. (A) Atg5 is necessary for PCAF-mediated δ-catenin degradation. Tet-off Atg5−/− mouse embryonic fibroblasts m5-7 were cultured in the presence or absence of 10 ng/ml doxycycline (Dox) for 4 days. Then, cells were transfected as indicated for 24 h, and cell lysates were subjected to immunoblotting. (B) Overexpression of LC3 further decreases δ-catenin levels. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoblotting. Actin was used as a loading control. Relative values of δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. *p < 0.05; **p < 0.01; NS, no significant difference compared with the control group. (C) The cellular localization of δ-catenin depends on autophagic activity and PCAF. HEK293T cells were transiently transfected with a plasmid encoding GFP-LC3 and RFP-δ-catenin for 12 h, and incubated in the absence (a) or presence of 3-MA (b) or transfected with flag-PCAF (c) for 24 h. (a) Arrows indicate δ-catenin located at the plasma membrane or cell-cell contact region. (b) Arrows indicate cytoplasmic δ-catenin accumulation. 3-MA treatment dispersed LC3 signals and no colocalization with δ-catenin was observed. (c) Arrows indicated δ-catenin colocalization with LC3. PCAF overexpression decreased the accumulation of plasma membrane or cell-cell contact region and increased the colocalization with LC3. “−”, Mock transfection and/or vehicle treatment.