Metacaspase lies upstream of autophagy in the vacuolar cell death pathway. (A) Proteolytic activity of mcII-Pa in control and RNAi lines measured using the FESR-AMC peptide. Data represent the means ± SEM from three independent experiments. The mean values were normalized to the control line. *, P < 0.05; **, P < 0.01 (vs. control, Dunnett’s test). (B) Localization of mRFP-Atg8 in dying cells from control and RNAi lines with and without ConA treatment. Bars, 50 µm. DIC, differential interference contrast microscopy. (C) Quantification of autophagosomes. Data represent the means ± SEM number of mRFP-Atg8–positive puncta counted for ≥10 cell volumes, 10 µm3 each, per line. **, P < 0.01; ***, P < 0.001 (vs. control, Dunnett’s test). (D) Western blot analysis of NBR1 degradation. Numbers shown below the blots correspond to the relative amount of NBR1. The integrated band intensities were first normalized to corresponding intensities of Coomassie staining (loading) and then to the control. (E) Schematic pathway showing the relationship between autophagy and metacaspase mcII-Pa in the regulation of vacuolar and necrotic modes of cell death.
