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Fig. 5. Accumulation of acidic vesicular organelles (AVOs) induced by ceramide treatment is impaired in Beclin KD cells. (A) Control and Beclin KD cells were maintained for 24 hours in medium with or without 10 µM C2-ceramide, as indicated. Cells were then incubated with acridine orange (AO) and examined by fluorescence microscopy, using a filter combination to detect red fluorescence. All digital micrographs were taken at the same exposure setting. Bar, 50 µm. (B) Control and Beclin KD cells were treated with 0, 10 or 20 µM C2-ceramide for 24 hours and incubated with AO. The relative amount of AO trapped in vesicular compartments (red fluorescence; excitation at 488 nm, emission at 655 nm) was measured by fluorimetry and normalized to cellular DNA detected with ethidium bromide (EB). The data represent the mean ± s.e. of three determinations from parallel cultures.
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