Figure 1. SQSTM1 is restored during prolonged starvation. (A) Wild-type, Rb1cc1 KO and Atg5 KO MEFs were cultured in starvation medium lacking amino acids and serum for 1, 2, 4, 6, and 8 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies. Densitometric quantification of SQSTM1 protein levels using ImageJ software is shown on the graph. Data represent the mean ± SEM of 4 independent experiments (including the data in , , and ) for wild-type cells. A representative result is shown for Rb1cc1 KO and Atg5 KO cells. (B) HepG2, HeLa and HEK293 cells were analyzed as in (A). (C) Wild-type immortalized and primary MEFs were analyzed as in (A). (D) Wild-type MEFs were cultured in starvation medium lacking amino acids and serum for 2, 4, and 8 h. Cells were fixed and stained with anti-SQSTM1/p62 antibody. Scale bar: 5 µm.

Figure 2. SQSTM1 restoration requires de novo protein synthesis but is independent of MTORC1. Wild-type MEFs were cultured in starvation medium lacking amino acids and serum for 1, 2, 4, 6, and 8 h. At 3 h after starvation, Torin1 (250 nM) or cycloheximide (CHX, 50 μg/ml) was added. Cell lysates were analyzed by immunoblotting using the indicated antibodies. Densitometric quantification of SQSTM1 protein levels using ImageJ software is shown on the graph.

Figure 3. Amino acid starvation-induced upregulation of Sqstm1 transcription is required for SQSTM1 restoration during prolonged starvation. (A) Wild-type MEFs were cultured in DMEM lacking both amino acids and serum, or either amino acids or serum alone, or regular DMEM containing 250 nM Torin1 for 2, 4, and 8 h. Relative Sqstm1 mRNA is estimated by quantitative PCR using Actb as an internal control. Data represent the mean ± SEM of 3 independent experiments. (B) HepG2, HeLa and HEK293 cells were cultured in DMEM lacking both amino acids and serum and analyzed as in (A). (C) Wild-type MEFs were cultured as in (A). The SQSTM1 protein level was analyzed by immunoblotting. ACTB was used as a loading control. Densitometric quantification of SQSTM1 protein levels using ImageJ software is shown on the graph. Data represent the mean ± SEM of 3 independent experiments. (D) Wild-type MEFs stably expressing GFP-SQSTM1 were cultured in DMEM without amino acids and serum. Expression levels of endogenous SQSTM1 and GFP-SQSTM1 were analyzed as in (C). (E) Wild-type MEFs were pretreated with or without 20 µg/ml α-amanitin for 4 h, and then cultured in DMEM without serum and amino acids for 4 or 8 h in the presence or absence of 20 µg/ml α-amanitin. Sqstm1 mRNA was quantified as in (A). (F) Wild-type MEFs were treated as in (E) and the SQSTM1 protein level was analyzed by immunoblotting. ACTB was used as a loading control. Densitometric quantification of SQSTM1 protein levels using ImageJ software is shown on the graph.

Figure 4. Inhibition of lysosomal function abolishes SQSTM1 restoration. (A) Wild-type MEFs were cultured in DMEM lacking both amino acids and serum in the presence of 20 µM chloroquine (CQ) or 0.1 µM bafilomycinA₁ (Baf) for 1, 2, 4, 6, and 8 h. Densitometric quantification of SQSTM1 protein levels is shown on the graph. (B) Wild-type MEFs were cultured in regular DMEM medium. These MEFs were pretreated with 1 µM LysoTracker for 30 min and washed with PBS twice. Pretreated MEFs were treated with 20 µM CQ or 0.1 µM Baf for indicated times. Scale bar: 5 µm. (C) Wild-type and Rb1cc1 KO MEFs were cultured in regular DMEM medium or starvation medium (DMEM without amino acids and serum) for 4 h. Wild-type MEFs were also treated with 20 µM CQ or 0.1 µM Baf during the 4 h of starvation. Relative Sqstm1 mRNA was estimated by quantitative PCR. Data represent mean ± SEM of 3 independent experiments.

Figure 5. Amino acid replenishment causes restoration of SQSTM1 in bafilomycinA₁-treated cells. (A) Wild-type MEFs were cultured in starvation medium containing 0.1 µM bafilomycinA₁ (Baf) for 1, 2, 4, 6, and 8 h. After 3 h of starvation treatment, the medium was replaced with regular DMEM containing amino acids with or without 250 nM Torin1. Densitometric quantification of SQSTM1 protein levels using ImageJ software is shown on the graph. Data represent the mean ± SEM of 3 independent experiments. (B) Wild-type MEFs were cultured in starvation medium containing 20 µM chloroquine (CQ) for 1, 2, 4, 6, and 8 h. After 3 h of starvation treatment, the medium was replaced with regular DMEM containing amino acids with or without 250 nM Torin1.

Figure 6. A model of SQSTM1 restoration during prolonged starvation. Under nutrient-rich conditions, SQSTM1 protein is maintained at basal levels. During short-term starvation (~2 h), SQSTM1 is rapidly degraded by autophagy. As starvation-induced transcriptional upregulation of SQSTM1 is only modest, overall SQSTM1 protein levels decrease. Following prolonged starvation, high levels of Sqstm1 mRNA and sufficient amounts of intracellular amino acids derived from autophagy promote de novo synthesis of SQSTM1, which restores SQSTM1 protein expression to basal levels.