Figure 5.

Evaluation of induction of autophagy after PKM2 expression knockdown. (A) 786-O cells were transfected with specified siRNAs and stained with acridine orange (1 μg/mL). Formation of acidic vesicular organelles (AVOs) was observed under a confocal microscope at 400× magnification. Fluorescent green staining in cytoplasm and nucleus and fluorescent bright red or orange-red staining of autophagic vacuoles were observed. (B) Analysis of autolysosome formation through MDC staining. Cells were transfected, stained with MDC (50 μM), and examined using confocal microscopy. Scale bars indicate 50 μm. (C,D) Western blot analysis of autophagic proteins in 786-O cells. Immunoblotting was performed using whole-cell lysates, and GAPDH was used as internal loading control. Representative blots are shown. Ratios of LC3-II/GAPDH and beclin 1/GAPDH were measured and depicted as bar graphs. One-way ANOVA was used to compare means of different groups. Differences between means were considered significant at p < 0.05 using Tukey’s multiple comparison test; ** p < 0.01, and *** p < 0.001 as compared with normal control cells. (E) Immunofluorescence analysis was performed to evaluate expression of light chain 3B (LC3B) after siRNA transfection and observed under a confocal microscope at 400× magnification. Higher LC3B expression (indicated by red fluorescence) was observed in si156-transfected cells than in siPK-transfected, normal control, and negative control cells. DAPI was used for nuclear staining. Alexa Fluor 488-conjugated goat anti-rabbit antibody and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody Rhodamine Red-X were used to detect PKM2 and LC3B, respectively.