Transfection of HCT-116 cells with PFKFB3 siRNA stimulates autophagy. LC3-II and p62 protein levels were determined using Western blotting 48 hours after transfection with either control (ctrl) or a siRNA directed against PFKFB3 (PFKFB3) (A). Treatment with 1 nM bafilomycin A1 (Baf A1) was used to determine if LC3-II levels were a result of increased autophagic flux or impaired degradation (A). Quantitative densitometry was performed to assess relative protein levels (B, C). LC3-II and p62 levels are expressed as the mean fold change ± SD from three experiments relative to LC3-I or β-actin and control. After 48 hours of transfection with either control (ctrl) or PFKFB3-specific siRNA, cells also were stained with acridine orange, observed by fluorescent microscopy and collected by flow cytometry to measure the relative content of acidic compartments (D). Examination of the cells by electron microscopy demonstrated that PFKFB3 siRNA transfection resulted in cells containing intracellular structures consistent with autophagosomes (E; arrow). Data are presented as the mean ± SD from three experiments (*P <0.05).