FIGURE 3.

RCAN1-1L-induced mitochondrial degradation involves activation of autophagy. A, levels of LC3 (I and II) are increased after RCAN1-1L overexpression. ST14A cells were transfected with vector carrying RCAN1-1L fragment or with null vector (C) for the indicated time periods. Control (C), in this particular case, represents cells transfected with null construct for 9 h. Signals were detected and quantified as described under “Experimental Procedures.” Probing with β-tubulin was used to control protein loadings. Results represent data from two separate experiments, in which two separate cell samples were used for each time point. Values shown are means ± S.E. (error bars) At 9 h, levels of LC3-II and LC3-I were markedly (∼2 times) increased in cells overexpressing RCAN1-1L as compared with controls as evaluated by Student's t test (p < 0.05). B, autophagic flux after RCAN1-1L overexpression. Cells were transfected, and at the 6 h time point after transfection, they were incubated for another 3 h (total 9 h after transfection) either with (Inhib+) or without (Inhib−) inhibitors of lysosomal proteolysis (20 mm NH₄Cl + 100 μm leupeptin) to prevent autophagosome clearance. Levels of LC3-II were measured as in A. Three separate cell samples were used for each time point. Values shown are means ± S.E. C, the number of autophagosomes is increased after RCAN1-1L overexpression. Dividing ST14A cells were collected at 24 h after RCAN1-1L overexpression and analyzed using TEM as shown in supplemental Fig. 2. Whole cell sections were reconstructed, and, to ensure comparison only of cells cut close to the middle, only sections that spread into at least three photomicrographs were analyzed. Ten reconstructed cell sections were analyzed for each sample. Autophagosomes were counted manually. Autophagosome number was significantly (∼2.2-fold) increased in cells overexpressing RCAN1-1L in comparison with control cells, as evaluated by Student's t test (p < 0.05). A.U., arbitrary units.