Figure 7

Effects of simultaneous inhibition of Akt activity and progression of autophagic process in HT-29 cells. A, HT-29 cells were exposed to DMSO (control), LY294002 (50 μmol/L) or Akt inhibitor IV (1.25 μmol/L) for 48 h and then subjected to immunofluorescent staining for LC3 as described in Materials and Methods. Bars, 20 μm. B, Quantification of acidic vesicular organelles by acridine orange staining using flow cytometry. HT-29 cells treated with DMSO (control), amino acid-deprived medium (AAS), LY294002 (50 μmol/L), or Akt inhibitor IV (1.25 μmol/L) for 48 h were stained with acridine orange and then the development of AVOs was analyzed 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. Right, Columns, mean (n = 3); bars, SD. *, P < 0.05, significantly different compared with the control. C, The sub-G1 population was quantified by flow cytometry after exposure to LY294002 (50 μmol/L) or Akt inhibitor IV (1.25 μmol/L) with or without bafilomycin A1 (10 nmol/L) or 3-MA (10 mmol/L) for 48 h. Columns, mean (n = 3); bars, SD. *, P < 0.05, significantly different compared with the control.