FIG 7.

Increased fluorescence of the K⁺-sensitive dye APG3 in LEs. (A) Loading of endosomes with APG3. A549 cells were serum starved for 4 h prior to EGF-AF647 (200 ng/ml) binding on ice for 30 min. Cells were then washed with growth medium and loaded for 30 min with APG3 at 37°C (APG3 pulse). Live cells were imaged with a confocal microscope. EGF-AF647 signal was false colored to red for better visualization of the resulting yellow colocalization signal. (B) APG3 fluorescence upon internalization over time. Cells were loaded with APG3 as described in the legend to panel A. Following 30 min incubation with the dye, the cells were washed, transferred to the microscope, and imaged at the indicated time points. In the case of nocodazole treatment, cells were incubated in the presence of the drug together with APG3 for 30 min, followed by exchange to fresh nocodazole-containing medium for the imaging time. (Top) Representative images for both conditions at 60 min after the APG3 pulse; (bottom) mean fluorescence intensities at the indicated time points after APG3 loading were determined with ImageJ-based quantification. (A, B) Cell borders and nuclei were determined in transmission mode and are indicated as dashed lines in the images. Bars, 20 μm.