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. 2021 Jul 17;11(11):3567–3584. doi: 10.1016/j.apsb.2021.07.012

Figure 5.

Figure 5

HA-1141 inactivates mTORC1 to induce autophagy in an ATF4-dependent manner. (A, B) HA-1141 inactivates mTORC1. H358 cells were treated with HA-1141 or tunicamycin at the indicated concentrations for 24 h, or with HA-1141 (20 μmol/L) or tunicamycin (3 μmol/L) for the indicated periods of time, followed by Western blotting analysis. (C) Autophagy measured by appearance of punctate vesicle structure. H358 were treated with HA-1141 at the indicated concentration for 24 h before photography under a fluorescent microscope (left panels). Cells with punctate vesicle structures of LC3 were counted in five independent areas and data are plotted in a bar graph (right panel). Shown are the mean ± SEM (n = 3), size bar, 10 μm ∗∗∗P < 0.001 vs. control. (D) Detection of autophagosomes by TEM: H358 cells were treated with HA-1141 (20 μmol/L) for 24 h, along with a DMSO vehicle control, followed by TEM analysis. Autophagosomes are indicated by arrows. Direct magnification: × 30,000, size bar, 1 μm. (E) Time-dependent LC3-Ⅱ conversion: H358 cells were treated with HA-1141 (20 μmol/L) or tunicamycin (3 μmol/L) for the indicated time followed by Western blotting analysis. (F, G) ATF4 knockdown decreases punctate vesicle structure by HA-1141. H358 cells were transfected with the indicated siRNAs for 60 h and then treated with DMSO or HA-1141 (20 μmol/L) for 24 h before photography (F). Cells with punctate vesicle structures of LC3 were counted in five independent areas and the data are plotted in a bar graph (G). Shown are the mean ± SEM (n = 3), size bar, 3 μm ∗∗∗P < 0.001 (H) ATF4 knockdown partially rescues autophagy. H358 cells were transfected with the indicated siRNAs for 60 h and then treated with DMSO or HA-1141 (20 μmol/L) for 8 h, followed by Western blotting analysis.