Figure 1. Cationic lipoplex, polyplex and calcium phosphate precipitates induce formation of tubulovesicular autophagosomes. CHO cells expressing GFP-LC3 were used to monitor autophagosome formation. Nuclei were stained with DAPI. Scale bar: 10 μm. (A) Cells were incubated in nutrient media (i), or starved in HBSS (ii). When incubated in nutrient media containing cationic lipoplex (iii) calcium phosphate precipitate complexed with DNA (iv) or polyplex. (B) Different cell types were incubated with lipoplex (Transfast) and immunostained for endogenous LC3 after 4 h. HeLa cells (i), Vero cells (ii), murine embryonic fibroblasts (iii), primary culture of murine dendritic cells. (C) Primary cultures of murine skin fibroblasts were incubated in starved in HBSS for 4 h (i) or incubated with lipoplex for 4 h (ii) and immunostained for endogenous LC3 (green). Insets show region of interest at higher magnification. (iii) CHO cells expressing GFP-LC3 were electroporated in the presence of a plasmid expressing LAMP1-RFP and MEF cells were electroporated in the presence of a plasmid expressing GFP. LC3 distribution (gray) was analyzed in cells expressing the reporter proteins 7 h after electroporation. (D). MEFs were transfected with a plasmid expressing human GABARAPL2 tagged with GFP (green).¹⁴ Cells were incubated in nutrient media (i), starved in HBSS for 4 h (ii) or incubated with lipoplex (iii) and immunostained for endogenous LC3 (red) after 4 h. Insets show region of interest at higher magnification.