CQ increased the formation of Acidic vesicular organelles (AVOs). (A) The formation of AVOs was quantified by flow-cytometry after acridine orange staining in HT-29 cells treated with 5-FU for 48 h after 12-h pretreatment with CQ. Treatment of HT-29 cells with 5-FU alone resulted in increased AVOs formation, but the effect was potentiated by CQ-pretreatment. The results are expressed as the mean fluorescein intensity of acridine orange stained cells. (B) The similar results were obtained with the fluorescence microscopic examination, confirming the induction of autophagy. HT-29 cells treated with CQ and 5-FU displayed great number of red fluorescence vesicles in the cytoplasm, which represent AVOs. Because CQ inhibits the late step of autophagy, the accumulation, and not inhibition, of AVOs was induced by the treatment with CQ.