FIGURE 5.

Phosphorylation-defective ATG4B mutants show alterations in regulation of LC3. A, knocking down ATG4B reduces autophagy as measured by the actin-LC3B-dNGLUC substrate. MCF7 cells expressing a scrambled non-targeting shRNA or two different ATG4B targeting shRNA (1 and 2) were co-transfected with plasmids encoding Actin-LC3B-dNGLUC or Actin-dNGLUC together with CMV-Luc2. After 48 h, the activity of Gaussia luciferase in the culture medium was measured, and normalized to Firefly luciferase activity in the cells, setting the activity for ATG4B WT as 100%. Error bars indicate the S.D. of three independent experiments. ***, p < 0.001. B, knocking down ATG4B increases levels of endogenous LC3-II. Cells in A were treated with lysosomal inhibitors for the indicated times. Cell lysates were analyzed by immunoblotting using anti-LC3, anti-ATG4B, and anti-actin antibodies. The relative amount of LC3-II was quantified by scanning densitometry, normalized to actin and further normalized to cells expressing non-targeting shRNA with lysosomal inhibitor at 4 h (values shown below lanes). C, atg4b^−/− MEF cells stably expressing GFP alone, ATG4B WT or ATG4B C74A mutant were cultured in nutrient-rich medium (DMEM), followed by treatment with HBSS starvation for the indicated times. Cell lysates were analyzed by immunoblotting using anti-LC3, anti-ATG4B, and anti-actin antibodies. D, C-terminal truncation or phosphorylation mutants of ATG4B significantly increase the level of endogenous LC3-PE (LC3-II). atg4b^−/− MEF cells stably expressing ATG4B WT or various indicated ATG4B mutants were cultured in nutrient-rich medium (DMEM), followed by treatment with or without rapamycin (25 μg/ml) or HBSS starvation, in the presence or absence of lysosomal inhibitors (±L.I., 20 mm NH₄Cl and 100 μm leupeptin) for 4 h. Cell lysates normalized for total protein were analyzed by immunoblotting using anti-LC3, anti-ATG4B and anti-actin antibodies. The relative amount of LC3-II in the presence of lysosomal inhibitor was quantified by scanning densitometry, normalized to actin and further normalized to cells expressing ATG4B WT in nutrient-rich, rapamycin treatment or HBSS starvation conditions, respectively, setting the level of LC3-II for ATG4B WT as 1. Error bars indicate the S.D. of three independent experiments. *, p < 0.05; **, p < 0.01.