Co-treatment with I3C and genistein does not induce maturation of autophagosomes. Quantification of acidic vesicular organelles by acridine orange staining using flow cytometry. HT-29 cells treated with DMSO (control), amino acid-deprived medium (AAS) in the presence or absence of 3-MA (10 mmol/L), or the combination of I3C (300 μmol/L) and genistein (40 μmol/L) for 12 h were stained with acridine orange, and then subjected to a flow cytometric analysis as described in Materials and Methods. Left, FL1-H indicates the intensity of green fluorescence that is endogenous to the nucleus. FL3-H indicates the intensity of red fluorescence. Top of the grid was considered as AVOs. GEN, genistein. Right, Columns, mean (n = 3); bars, SD. *, P < 0.05, significantly different compared with the control.