Fig. 5. RACK1 induces autophagy and enhances autophagic flux in colon cancer cells.

a Western blot analysis showing the expression levels of BECN1, ATG5, IC3-II, and SQSTM1 in the SW480-RACK1 OE cells, SW620-RACK1 KD cells, and their control cells. b Immunofluorescent staining showing the number of LC3 puncta in the SW480-RACK1 OE cells, SW620-RACK1 KD cells, and their control cells. Cells were stained by indirect immunofluorescence using anti-LC3 antibody, and the number of LC3 puncta per cell was quantified. Scale bars = 100 μm. c Electron microscopic examination showing autophagic vacuoles (arrows) in the cytoplasm in the SW480-RACK1 OE cells, SW620-RACK1 KD cells, and their control cells. d RACK1 knockdown enhances autophagic flux. (top) Western blot analysis showing the LC3-II levels in the SW480-RACK1 OE cells and control cells treated bafilomycin A1 (BAF). (bottom) The number of EGFP-LC3 puncta in the SW480-RACK1 OE cells and control cells treated BAF. Cells were transfected with 1 μg of EGFP-LC3 plasmid. 24 h after transfection, cells were treated with BAF. 24 h after treatment, EGFP-LC3 puncta was examined by fluorescence microscopy. The number of EGFP-LC3 puncta per cell was quantified. Scale bars = 50 μm. Three experiments were done; Means, SDs, and statistical significance are denoted; ***P < 0.001