Figure 5

RB knockdown impaired VP-16-induced autophagy. (a) Representative Beclin-1 and LC-3 immunoblots for U87 cells 24 h post-VP-16 treatment (1 μM); experimental groups as indicated. α-Tubulin was used as loading control. Starved cells were used as positive control of autophagy induction. Starvation was induced by incubating the cells for 3 h in Krebs-Ringer buffer. (b) Graphic shows changes in intensity of red fluorescence (indicating acid vesicles labeled with acridine orange) in U87 (black bars) and GBM95 (white bars) cells after 24 h of VP-16 treatment (1 μM). Changes in red fluorescence were represented as fold increase (mean red fluorescence/total cell number) over control (siRNA-Neg group); experimental groups as indicated. **P<0.01 by ANOVA and Tukey’s post test. Data are represented as mean and S.EM. of three independent experiments. (c) Representative images of three independent experiments of acridine orange vital staining of U87 cells after 24 h of 1 μM VP-16 treatment; experimental groups as indicated. Acid vesicles are stained in red and total cells are represented by the nuclear–cytoplasmic green staining. Scale bar=10 μm. (d) Transmission electron microscopy of VP-16-treated cells (1 μM for 24 h) shows the presence of content filled autophagic vacuoles (white arrows) in both non-silenced and silenced groups (siRNA-Neg plus VP-16 and siRNA-RB plus VP-16 groups, respectively). Although non-silenced cells also show several clear vacuoles of which most of the content has been degraded (black arrows), RB-silenced cells show mainly content-filled vacuoles (white arrows), possibly indicating autophagic flux blockage. Control cells (siRNA-Neg) show vacuoles with hardly any content. N, nucleus. Scale bar=6 μm