Fig. 9

The detection of autophagy by LC3 accumulation. a–o Fluorescent microscope was used to visualize the LC3-II punctate staining pattern (green); the cell nuclei were counterstained with DAPI (blue). a–l The A549 cells were treated with 10 µM fisetin (FIS) and/or 0.1 µM paclitaxel (PTX) for 24 h or left untreated (control, CTRL). m–o As negative control, bafilomycin A1 (Baf A1; 100 nM) was added to the cells for a period of 4 h, followed by washing with PBS and subsequent incubation with FIS and PTX for 24 h. Note the increase in LC3-II puncta following FIS and PTX treatment in the absence or presence of Baf A1 (j, m). Bar 50 µm. p Real-time PCR measurement of LC3-II mRNA expression in A549 cells. The expression was normalized to glucose 6-phosphate dehydrogenase (G6PD) and presented as a fold difference relative to a calibrator sample (untreated A549 cells; designated as 1). Error bars represent standard deviation from duplicate qRT-PCR assays