Figure 6. Gene delivery and formation of tubulovesicular autophagosomes require endocytosis. (A) MEF cells were incubated with cationic lipoplex for 1 h in nutrient media (i), or media with 80 μM dynasore (ii). Cells were immunostained for endogenous LC3. Scale bar: 10 μm. (iii) MEF cells were incubated with lipoplex vector (black) or polyplex (white) complexed to a luciferase reporter plasmid in nutrient media (filled columns) or nutrient media containing 80 μM dynasore (hatched columns). Luciferase activity was assayed at 16 h and 24 h from three independent experiments, error bars (SE) are shown. (B) HEK 293 cells stably expressing GFP-LC3 (green) were incubated with lipoplex vector for 4 h. Cells were fixed and immunostained for clathrin (i, red) or early endosome antigen 1(ii, red). (iii) MEF cells expressing RAB5-RFP (red) were incubated with lipoplex vector for 4 h. Cells were fixed and immunostained for endogenous LC3 (green). MEF cells expressing dominant-negative RAB7^T22N (iv) were incubated with lipoplex vector for 4 h. Cells were fixed and immunostained for endogenous LC3 (pseudo-colored green). Regions of interest are indicated by the white square and high magnification images of green, red and merged channels are presented to the right of each figure.