Figure 6

RB knockdown blocked VP-16-induced autophagic flux. (a) Representative confocal images of three independent experiments showing LAMP 1 – a lysosome marker (red) and LC3 – a autophagosome marker (green) colocalization in U87 cells after 24 h of 1 μM VP-16 treatment; experimental groups as indicated. Nuclei were stained with DAPI. Scale bar=20 μm. (a′) Higher magnifications of siRNA-Neg plus VP-16 and siRNA-RB plus VP-16 conditions images. Arrows indicate cells with LAMP 1/LC3 colocalization. Arrowheads indicate cells without LAMP 1/LC3 colocalization. Scale bar=20 μm. (b) Histogram showing the LAMP1/LC3 colocalization rate in U87 cells after 24 h of VP-16 treatment. Experimental groups as indicated. ***P<0.005 by ANOVA and Tukey’s post test. Data are represented as mean and S.E.M. of three independent experiments. (c) Histogram shows fold changes of p62 levels over siRNA-Neg control group; after 24 h of VP-16 treatment of U87 cells; experimental groups as indicated. ***P<0.005 by ANOVA and Tukey’s post test. Data are represented as mean and S.E.M. of three independent experiments. (d) Histogram shows fold changes VRK1 levels over siRNA-Neg control group; after 24 h of VP-16 treatment of U87 cells; experimental groups as indicated. *P<0.05 by ANOVA and Tukey’s post test. Data are represented as mean and S.E.M. of three independent experiments. (e) Representative p62 and VRK1 immunoblots for U87 cells 24 h post-VP-16 treatment; experimental groups as indicated. α-Tubulin was used as loadisng control