Fig. 2. Nt-Arg of proteins generated by ATE1 propagates lipophagy.

(A) WB of HepG2 cells following treatment of OA/PA (1 mM/500 μM, 9 h). (B) WB of HepG2 cells following treatment of OA/PA (1 mM/500 μM) for indicated time. (C) WB of HepG2 cells under siRNA-mediated knockdown of ATE1 (80 nM, 48 h) with sequential treatment of OA (1 mM) and PA (500 μM) for indicated time. (D) HepG2 cells treated with OA/PA (1 mM/500 μM) for 9 h were fractionated. Whole cell lysates (Total) in comparison with microsome (Micro.), cytosol (Cyto.) and LD fractions were analyzed by WB. Coomassie brilliant blue (CBB) staining of the gel was used to confirm the protein loading. (E) ICC of p62, N-terminal arginylated BiP (R-BiP) and BODIPY signals in Hep3B cells exposed to OA/PA (1 mM/500 μM, 24 h). Scale bar, 10 μm. (F) ICC of p62 and BODIPY signals and (G) quantification of LD area (n = 50 cells) in WT and Ate1^−/− MEFs exposed to OA (2 mM, 24 h) and PA (1 mM, 24 h), with or without sequential treatment of Baf. A1 (200 nM, 4 h). Scale bar, 10 μm. (H) TG quantification of WT and Ate1^−/− MEFs exposed to conjugating vehicle, bovine serum albumin (BSA), alone or OA/PA (2 mM/1 mM) for 24 h (n = 4). (I) ICC of p62 and BODIPY signals (left panel) and TG quantification (right panel, n = 4) in MEFs treated with tannic acid (TA, 10 μM, 24 h), OA/PA (1 mM/500 μM), or both. Scale bar, 10 μm. (J) TG quantification of WT and Ate1^−/− MEFs exposed to OA (2mM, 24 h) and PA (1 mM, 24 h), with or without sequential treatment of Lalistat-1 (30 μM, 24 h). *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.