FIGURE 3.

3-MA does not affect lysosomal functions. A, effect of 3-MA on autolysosome maturation and lysosomal degradation. The L929 cells with stable expression of the tfLC3 construct (tfLC3-L929) were treated with CQ (20 μm), 3-MA (5 mm), 3-MA + CQ and rapamycin (10 nm), or rapamycin + CQ for 9 h in full medium. The cells were examined using a confocal microscope (×600, top). The RFP- and GFP-LC3 puncta/cell were counted and presented (bottom; **, p < 0.01). B, effect of CQ on GFP-LC3 punctuation/aggregation induced by 3-MA. WT MEFs with stable expression of GFP-LC3 (same as in Fig. 2B without DOX treatment) were treated with 3-MA (5 mm), CQ (20 μm), or 3-MA + CQ for 9 h in full medium. The cells were examined by confocal microscopy (×600, left). The GFP-LC3 puncta/cell were counted and presented (right; **, p < 0.01). C, MEFs were treated as described in B, and the cell lysate were subjected to immunoblotting. D, effect of 3-MA on intralysosomal pH. WT MEF cells were treated with 3-MA (5 mm) or CQ (20 μm) for 9 h in full medium, and the intralysosomal pH values were determined using a Lysosensor probe coupled with flow cytometry, as described under “Experimental Procedures.” E, effect of 3-MA on cathepsin B/L activity. WT MEFs were treated with 3-MA (5 mm), CQ (20 μm), or cathepsin inhibitors (E64d + pepstatin A, 20 μg/ml each) for 9 h, and the lysate was subjected to the cathepsin B/L activity assay, as described under “Experimental Procedures.” The data in E are presented as means ± S.D. from three independent experiments and analyzed using Student's t test (**, p < 0.01). Ctrl, control.