PFKFB3 inhibition with 3PO stimulates autophagy. HCT-116 cells were treated with either vehicle, or 7.5, 10, or 15 μM 3PO for 24 hours and LC3-II and p62 expression was measured by Western blot (A) and densitometry (B, C). Addition of bafilomycin A1 (Baf A1) was used to determine if the changes in LC3-II were the result of increased synthesis or impaired degradation. LC3-II quantitation is relative to control + bafilomycin due to the absence of a visible band in the control sample. HCT-116 cells were also stained with 1 μg/mL acridine orange for 15 minutes, viewed using a fluorescent microscope, harvested for flow cytometry and gating was used to quantitate the number of cells with a high AO fluorescence and expressed relative to vehicle (D, E). Using electron microscopy, autophagic structures were seen in cells exposed to 3PO (F; arrow).