Figure 2.

Induction of autophagy by L-asparaginase treatment. (a) Western blot analysis of REH and 697 cells. Fold change of LC3B-II level (normalized to β-actin) relative to that of untreated cells is indicated in the graph in the lower panel. (b) Immunofluorescence analysis of LC3B protein. Square areas are enlarged and shown in the lower panel. Scale bars represent 10 μm. (c) Representative images of electron microscopic analysis. Arrow and arrowhead indicate an autolysosome and autophagosome, respectively. Numbers and areas of these autophagic vacuoles per cell were analyzed using ImageJ. Fifty cells were investigated per group. Scale bars represent 500 nm. Data represent as mean±s.d. (d) Mitochondrial membrane potential assay with TMRE. Fluorescence intensity was measured using flow cytometry. (e) Measurement of intracellular and mitochondrial ROS level. Treated REH cells were stained with 10 μM of DCFDA (intracellular ROS) or 2.5 μM of MitoSox (mitochondrial ROS). REH cells were treated with 1 U/ml of L-asp for 48 h and/or 10 μM of CQ for the last 3 h (a–d) or for 48 h (d, e). Data in a, c–e represent as mean±s.d. (n=3); *P<0.05, ***P<0.001. P-values were calculated using one-way analysis of variance (ANOVA).