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. 2018 May 22;9(6):604. doi: 10.1038/s41419-018-0642-6

Fig. 4. PPI-induced accumulation of exogenous SQSTM1 via ubiquitin-proteasome pathway.

Fig. 4

a AGS cells were transfected with HA-tagged SQSTM1 plasmid for 48 h, and then treated with PPI for another 48 h in both pH 6.5 and pH 7.4 conditions. The exogenous SQSTM1 protein was detected with antibody for HA. b AGS cells were treated with baf A1 (50 and 100 nM) and HCQ (10, 50, and 100 μM) for 24 h. c AGS cells were pretreated with baf A1 (100 nM) for 30 min, followed with 1 μM rapamycin or amino acid starvation by HBSS for another 24 h. d Different concentrations of two classical proteasome inhibitors Bortezomib and MG132 were added. e AGS cells were either untreated or treated with Bortezomib (25 nM) or MG132 (0.1 μM) for 24 h in the absence or presence of baf A1 (100 nM). f AGS cells transfected with HA-tagged SQSTM1 plasmid, were treated with 25 μg/ml cycloheximide (CHX) over a 240-min time period (left) or treated with 100 μg/ml PPI for 48 h in pH 7.4 conditions, and then followed by 25 μg/ml CHX over a 240-min time period (right). Cells were lysed at the indicated time points (0, 60, 120, and 240 min). Right panel showed the half-life of HA, which reflected the stability of exogenous SQSTM1 protein. g–i The accumulated SQSTM1 by either proteasome inhibitors or PPI was sensitive to autophagy mediated degradation. AGS cells were pretreated with 25 and 50 nM bortezomib for 1 h, and then incubated with 500 nM torin 1 for 24 h (g). After pretreatment with PPI for 24 h in pH 7.4 or pH 6.5 condition, rapamycin (1 μM) or torin 1 (500 nM) was added for another 24 h (h). The protein level of SQSTM1 was measured by western blot analysis (g, h), and the change pattern of SQSTM1 mRNA was confirmed by qRT-PCR (i) (**p < 0.01, identified by one-way ANOVA with Dunnett’s multiple comparison test)