Quantification of cellular autophagy
To examine the effect of PLB on autophagy in PANC-1 and BxPC-3 cells, cellular autophagy was detected using flow cytometry as described previously.21 Briefly, PANC-1 and BxPC-3 cells were seeded in 60 mm Petri dishes. After cells were seeded for 24 hours, the cells reached ~75% confluence and then treated with fresh medium alone, control vehicle alone (0.05% DMSO, v/v), or PLB (0.1, 1, and 5 μM) for 24 hours. In separate experiments, PANC-1 and BxPC-3 cells were treated with 5 μM PLB for 4, 8, 12, 24, 48, and 72 hours. Following the PLB treatment, cells were resuspended in 250 μL of phenol red-free culture medium containing 5% FBS, and 250 μL of the diluted Cyto-ID®Green stain solution was added to each sample and mixed well. Cells were incubated for 30 minutes at 37°C in the dark and then collected by centrifugation at 250× g. The cell pellet was washed with 1× assay buffer in the Cyto-ID® autophagy detection kit and resuspended in 500 μL fresh 1× assay buffer. Cells were analyzed using the green (FL1) channel of a flow cytometer (Becton Dickinson Immunocytometry Systems).
Confocal fluorescence microscopy
In order to further detect the cellular autophagy level, the cellular autophagy level was examined using confocal fluorescence microscopy. Briefly, PANC-1 and BxPC-3 cells were seeded into an 8-well chamber slide. The cells were treated with PLB at 0.1, 1, and 5 μM for 24 hours. In separate experiments, cells were treated with 5 μM of PLB for 4, 8, 12, 24, 48, and 72 hours. After the PLB treatment, the cells were washed with 1× assay buffer in the Cyto-ID® autophagy detection kit, following by incubation with 100 μL of microscopy dual detection reagent for 30 minutes at 37°C in the dark. After the incubation, the cells were washed with 1× assay buffer to remove detection reagent, and the cells were then examined using a Leica TCS SP2 laser scanning confocal microscopy (Wetzlar, Germany) using a standard fluorescein isothiocyanate filter set for imaging the autophagic signal at wavelengths of 405/488 nm. Full paper
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