Figure 4.

Distribution of [³H]DNA/[¹⁴C]bPEI polyplexes after 4 h pulse in cells fractionated using a 5–20% continuous iodixanol density gradient. (a) Schematic of density-gradient centrifugation method used to separate organelle populations. HeLa cells (2 × 10⁷) were pulsed with [³H]DNA/[¹⁴C]bPEI polyplexes for 4 h, and then prepared for fractionation. A 5–20% continuous iodixanol gradient was used to separate vesicular organelles. (b) Density of control gradients. Data are presented as mean ± S.D., n = 3. (c) The percent radioactivity measured in fractions from the 5–20% gradient. 100% radioactivity is equal to the sum of the radioactivity found in all 24 fractions collected from the 5–20% gradient. (d) Total protein was measured in untreated and treated gradient fractions. (e) Hexosaminidase A (lysosome) activity was also measured in untreated and treated gradient fractions. (f) An equivolume (250 μL) of gradient fractions from untreated and treated samples was precipitated, concentrated, and probed for CD49b (plasma membrane), LAMP2 (lysosome), and Rab5 (endosome). (g) The optical density of each band was measured using ImageJ. Each time point presented is representative of duplicate experiments.