Figure 5.
Knockdown of VAMP8 and Vti1b disturbed the maturation of canonical autophagosomes. (A) HeLa cells stably expressing GFP-LC3 were treated with siRNA in the same manner as described in Figure 4A. The cells were further cultured in growth medium (Fed) or EBS solution (Starved) for the indicated periods and then fixed and observed with a confocal microscope. Cellular DNA was stained with DAPI (blue). Bars, 10 μm. (B) Quantitative analysis of the number of GFP-LC3 puncta per cell shown in A was performed using ImageJ software. More than 100 cells were examined. Data are shown as the mean ± SD (C) siRNA-treated HeLa cells were cultured in Fed and Starved conditions for 240 min. The cellular lysates were subjected to western blotting using anti-LC3 and actin antibodies. (D) Quantitative analysis of Western blot bands shown in C was performed using ImageJ software. Data show the ratios of LC3-II band intensities to the actin bands. Values are shown as the mean ± SD from three independent experiments.