Figure 7: In vivo aggregation of polyubiquitin chains and degradation by macroautophagy.

(a) MEFs were transiently transfected with Ub^VV₆-EGFP (top), Ub^VV-EGFP (middle) or EGFP (bottom) and imaged 48 h after transfection. No ubiquitin-positive aggregates were detected in cells expressing Ub^VV-EGFP and EGFP, whereas ubiquitin-positive aggregates of up to 2 μm in diameter were observed in cells expressing Ub^VV₆-EGFP. Scale bar, 20 μm. Data are representative of three independent experiments. (b) Immortalized wild-type MEFs harbouring two regulator gene cassettes, CAG-rtTA and either TRE-Ub^VV₆-EGFP or TRE-Ub^VV-EGFP, were cultured for 24 h in the presence of Dox to induce expression of Ub^VV₆-EGFP or Ub^VV-EGFP. Subsequently, the cells were cultured in the absence of Dox for 24 h. E64d and pepstatin were added as indicated. The cell lysates were prepared and immunoblotted with the indicated antibodies. Data are representative of two independent experiments. (c) The MEFs shown in b were cultured for 24 h in the presence of Dox to induce expression of Ub^VV₆-EGFP or Ub^VV-EGFP, and then immunostained with p62 and LC3 antibodies or p62 and Ser351-phosphorylated p62 antibodies. Scale bar, 20 μm. Data are representative of three independent experiments. (d) Immortalized wild-type and Atg7-deficient MEFs were stably transfected with two regulator gene cassettes, CAG-rtTA and TRE-Ub^VV₆-EGFP. The cells were cultured for 24 h in the presence of Dox to induce expression of Ub^VV₆-EGFP. Subsequently, the cells were cultured in the absence of Dox for 24 h. The cell lysates were prepared and immunoblotted with the indicated antibodies. Data are representative of two independent experiments. (e) After induction of Ub^VV₆-EGFP in the Atg7-knockout MEFs as shown in d, Atg7 or Atg7 C567S was expressed using an adenovirus system. At the indicated time points, cell lysates were prepared and immunoblotted with the indicated antibodies. Data are representative of two independent experiments. (f) MEFs treated as shown in e were immunostained with p62 and Ser351-phosphorylated p62 antibodies, 36 h after infection with the indicated adenovirus vectors. Scale bar, 20 μm. Data are representative of three independent experiments. A.V., adenovirus; w.t., wild type.