Fig. 7. Assessment of autophagy induction after PKM2 knockdown. (A) DU145 cells were transfected with the indicated siRNAs (100 nM for 72 h) and were stained with acridine orange. Formation of autophagic vacuoles was observed under a confocal microscope. Acridine orange staining resulted in the fluorescent green staining of both the cytoplasm and nucleus and fluorescent bright red or orange-red staining of the autophagic vacuoles. (B) MDC staining showing the induction of autophagy in DU145 cells after PKM2 knockdown as compared with normal and negative control. Cells was examined using confocal microscopy. Scale bars indicate 50 μm. (C) Western blotting of autophagic proteins in cells transfected with the indicated siRNAs. Western blotting was performed using the whole-cell lysates, with GAPDH as the internal loading control. Representative blots are shown. (D) The ratio of LC3-II/GAPDH, Beclin 1/GAPDH were measured and depicted in the bar graph. One-way ANOVA was used to compare the means of different groups. Differences between the means were considered to be significant at P<0.05 by using Tukey multiple comparison tests; ^*P<0.05, **P<0.01, and ***P<0.001 compared with the normal control cells.