Dissection of Sqstm1-GFP^KI/+ MEFs under starvation conditions. (A) Immunoblot analysis. Sqstm1-GFP^KI/+ MEFs were cultured in regular or starvation medium for 2 h (stv.) or regular medium with E64d and pepstatin A for 24 h (EP). Cell lysates were prepared, followed by immunoblot analysis with the specified antibodies. The data shown are representative of three separate experiments. (B) Immunofluorescence staining. Sqstm1-GFP^KI/+ MEFs were cultured in regular (non-deprived) or starvation medium for 2 h (deprived), and then immunostained with anti-LC3 and anti-GFP antibodies. Each inset is a magnified image. Scale bar: 10 µm. (C) Sqstm1-GFP^KI/+ MEFs were cultured in starvation medium for 1 h and directly observed by time-lapse video microscopy. Scale bar: 1 μm. The duration of each spot of Sqstm1–GFP was measured for 26 cases, which is represented graphically underneath the images. (D) Structured Illumination Microscopic (SIM) analysis. Sqstm1-GFP^KI/+ MEFs under starvation conditions were immunostained with anti-LC3 antibody, and then observed by SIM. Scale bar: 1 µm. (E) Sqstm1-GFP^KI/+ MEFs were cultured in amino-acid-free medium for 1 h, and then fixed for immunoelectron microscopy using anti-GFP antibody as described in the Materials and Methods. Four representative profiles of autophagosomes are shown. Colloidal gold particles indicating Sqstm1–GFP (arrowheads) and high-density structures positive for Sqstm1–GFP (arrows) are labeled. Scale bars: 200 nm.

Dissection of Sqstm1-GFP^KI/+ MEFs under stress conditions. (A) Relative mRNA levels of Sqstm1, GFP, and Nqo1 and Ho-1. Total RNAs were prepared from Sqstm1-GFP^KI/+ MEFs cultured for 12 h in the presence or absence of 10 µM sodium arsenite (As[III]) and then reverse-transcribed into cDNAs, which were used as templates for real-time quantitative PCR analysis. Values were normalized to the amount of each mRNA in the non-treated MEFs. The experiments were performed three times; data are mean±s.e.m. **P<0.01; ***P<0.001 (Welch test). (B) Immunoblot analysis. Sqstm1-GFP^KI/+ MEFs were cultured as described in A. Total cell lysates and nuclear fractions were prepared and subjected to immunoblot analysis with the specified antibodies. Data are representative of three independent experiments. (C) Immunoprecipitation (IP) analysis. Sqstm1-GFP^KI/+ MEFs were cultured as described in A. Immunoprecipitates obtained with anti-GFP antibody were analyzed by immunoblotting with the specified antibodies. Data are representative of three independent experiments. (D) Immunoblot analysis. Sqstm1-GFP^KI/+ and Atg7^−/−;Sqstm1-GFP^KI/+ MEFs were challenged by As[III]. After removal of As[III], cells were cultured in regular medium for the indicated time. Cell lysates were prepared and subjected to immunoblot analysis with the specified antibodies. Data are representative of three independent experiments. (E) Immunofluorescence staining. Sqstm1-GFP^KI/+ MEFs were cultured as described in D, and then immunostained with anti-LC3 antibody. Each inset is a magnified image. Scale bar: 10 µm. (F) Time-lapse video microscopic analysis with Sqstm1-GFP^KI/+ MEFs cultured as shown in D. Scale bar: 1 μm. (G) Immunoelectron microscopy. Sqstm1-GFP^KI/+ MEFs were cultured as described in D, and fixed 1 h (d) or 3 h (a,b, and c) after removal of As[III]. They were then immunolabeled with anti-GFP antibody, followed by secondary antibody conjugated to colloidal gold particles (indicated by arrowheads for c and d). The boxed region in a was magnified and is shown in the inset. For better recognition of colloidal gold particles on dark background area, gamma correction was performed for c and d. Scale bars: 100 nm.