Figure 6

Granta519 cells form vacuoles in response to WIN55,212-2 treatment. (a) Light microscopy pictures of 0.5 μm sections prepared as for TEM of Granta519 cells treated with 10 μM WIN55,212-2 or vehicle for 24 h. Note the large cytoplasmic vacuoles. Occasional mitotic figures were also detected (black arrow); (b) TEM images of Granta519 cells treated with 10 μM WIN55,212-2. Photographs showed cells at different time points after 0 (i and ii); 2 (iii, iv); 6 (v and vi) and 24 (vii and viii) h of incubation. Normal ultrastructure morphology was predominately found in the control cells (0 h), showing a well-defined plasma membrane and uniformly distributed chromatin in the nucleus (i) and well-defined organelles in the cytoplasm (ii). After 2 h of WIN55,212-2 treatment, the cell cytoplasm displayed vacuoles with well-defined membranes (arrows; iii). Ribosomes (arrowheads) are attached to some of these membranes, indicating that these vacuoles (^*) were derived from the rough ER (iv). After 6 h, the number and size of these vacuoles increased and some contained membrane structures (arrows; v). The nuclear membrane was also affected, but the nuclear pore complexes were not disrupted (arrows; vi). At 24 h, more necrosis were found and the cells contained more vacuoles (vii). The vacuoles were mostly empty (^*), and mitochondria were often found in the cytoplasm (viii). Bars: i, iii, v and vii=2 μm; ii, iv, vi and viii=1 μm. (c) Fluorescence images of Granta519 cells incubated with vehicle or 10 μM WIN55,212-2 for 24 h immunostained for calreticulin (green) and DAPI (blue). White arrows indicate vacuoles surrounded by calreticulin