Fluid Phase Endocytosis Contributes to Transfection of DNA by PEI-25 Hansjörg Hufnagel, Parvez Hakim, Aline Lima, Florian Hollfelder  Molecular Therapy  Volume 17, Issue 8, Pages 1411-1417 (August 2009) DOI: 10.1038/mt.2009.121 Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Rottlerin affects the transfection rate. Rottlerin decreases the amount of cells successfully expressing GFP in (a) CHO-K1 and (b) HeLa cells after transfection with PEI-25/DNA complexes. Cells were preincubated with rottlerin (0–10 µmol/l) for 1 hour at 37 °C and subsequently treated with PEI-25/DNA complexes at N/P ratios 5 and 10 (0.4 µg DNA) for 4 hours at 37 °C. After further incubation for 36 hours at 37 °C, transfection rates were determined by measuring the amount of cells expressing GFP. Cells treated with polyplexes alone were set to 100%. Mean values ± SD were obtained from experiments performed at least in triplicate and in two independent experiments. All values shown to be smaller compared to sample values of 100% were tested to ensure statistical significance (P < 0.05). GFP, green fluorescent protein; PEI, polyethylene imine. Molecular Therapy 2009 17, 1411-1417DOI: (10.1038/mt.2009.121) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Rottlerin affects gene expression. Rottlerin decreases the amount of EGFP expressed by (a) CHO-K1 and (b) HeLa cells after transfection with PEI-25/DNA complexes at N/P 5 and N/P 10. Cells were preincubated with rottlerin (0–10 µmol/l) for 1 hour at 37 °C and subsequently treated with PEI-25/DNA complexes at N/P ratios 5 and 10 (0.4 µg DNA) for 4 hours at 37 °C. After further incubation of 36 hours at 37 °C cells were lysed and EGFP emission was measured at 520 nm. The EGFP expression was normalized versus the protein content. Cells treated with polyplexes alone were set to 100%. Mean values ± SD were obtained from experiments performed at least in triplicate and in two independent experiments. All values shown to be smaller compared to sample values of 100% were tested to ensure statistical significance (P < 0.05). EGFP, enhanced green fluorescent protein; PEI, polyethylene imine. Molecular Therapy 2009 17, 1411-1417DOI: (10.1038/mt.2009.121) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Flow cytometry of markers of uptake. (a–d) HeLa and (e–f) CHO-K1 cells take up markers of endocytosis and PEI/DNA polyplexes. a and e show the intrinsic fluorescence of a cell population. Fluorescence is enhanced by uptake of Oregon Green 488–labeled PEI-25/DNA complexes (0.4 µg DNA, N/P 5) (b and f). For comparison, cells were also incubated with fluorescently labeled molecules that are markers for endocytotic pathways (c: FITC-cholera toxin B subunit 1 µg/ml; d: FITC-transferrin, 0.5 mg/ml). Cells were incubated with markers of endocytosis for 4 hours at 37 °C. CTB, cholera toxin B subunit; FITC, fluorescein isothiocyanate. Molecular Therapy 2009 17, 1411-1417DOI: (10.1038/mt.2009.121) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Rottlerin reduces the internalization of PEI-25/DNA in HeLa and CHO-K1 cells. Cells were preincubated with rottlerin (0–10 µmol/l) for 1 hour at 37 °C and subsequently treated with Oregon 488–labeled PEI-25/DNA complexes (0.4 µg DNA, N/P ratio: 5) for 4 hours at 37 °C before measuring uptake using flow cytometry. Values for cells treated with polyplexes alone were set as 100%. Mean values ± SD were obtained from experiments performed at least in triplicate and in two independent experiments. All values shown to be smaller compared to sample values of 100% were tested to ensure statistical significance (P < 0.05). Molecular Therapy 2009 17, 1411-1417DOI: (10.1038/mt.2009.121) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 CHO-K1 and HeLa cells show complete uptake of PEI-25/DNA polyplexes. CHO-K1 and HeLa cells were seeded into 8-well chamber slides and preincubated with rottlerin (0–4 µmol/l) for 1 hours at 37 °C and subsequently treated with Oregon 488–labeled PEI-25/DNA polyplexes (0.4 µg DNA, N/P ratios 5 and 10) for 4 hours at 37 °C. Despite extensive washing (three times with PBS) cells showed intracellular distribution of fluorescence, ruling out nonspecific attachment of polyplexes to cell membranes after application of rottlerin. For visualization chamber slides were mounted onto the stage of a Leica 6000 CS inverted microscope. Images were acquired after excitation with a 488 nm argon laser by monitoring emission at 520 nm. Molecular Therapy 2009 17, 1411-1417DOI: (10.1038/mt.2009.121) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Rottlerin affects DNA uptake. Application of rottlerin reduces the internalization of fluorescently labeled DNA in PEI-25/DNA in (a) CHO-K1 and (b) HeLa cells. Cells were preincubated with rottlerin (0–4 µmol/l) for 1 hour at 37 °C and subsequently treated with PEI-25/DNA complexes (for 4 hours at 37 °C; 0.4 µg DNA, N/P ratio: 5 and 10) in which the DNA was fluorescently labeled. Cells were lysed and fluorescence was measured at 420 nm. The fluorescence was normalized against protein content in wells. Fluorescence values of cells treated with polyplexes alone were set as 100%. Mean values ± SD were obtained from experiments performed at least in triplicate and in two independent experiments. All values shown to be smaller compared to sample values of 100% were tested to ensure statistical significance (P < 0.05). Molecular Therapy 2009 17, 1411-1417DOI: (10.1038/mt.2009.121) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Impact of rottlerin on uptake of markers of endocytosis. Rottlerin does not inhibit the uptake of transferrin and cholera toxin subunit B (CTB) as marker of (a) clathrin- and (b) caveolin-mediated endocytosis, respectively, in HeLa and CHO-K1 cells up to a concentration of 6 µmol/l. Cells were preincubated with rottlerin (0–10 µmol/l) for 1 hour at 37 °C and subsequently treated with FITC-cholera-toxin subunit B (1.0 mg/ml) and FITC-transferrin (0.5 mg/ml) for 4 hours at 37 °C. Values of cells treated with markers alone were set to 100%. Mean values ± SD were obtained from experiments performed in triplicate and two independent experiments. CTB, cholera toxin B subunit; FITC, fluorescein isothiocyanate. Molecular Therapy 2009 17, 1411-1417DOI: (10.1038/mt.2009.121) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Cell viability of CHO-K1 and HeLa cells after treatment with rottlerin and PEI/DNA. Cells were preincubated with rottlerin (0–10 µmol/l) for 1 hour at 37 °C and subsequently treated with PEI-25/DNA complexes (0.4 µg DNA, N/P ratio: 5) for 4 hours at 37 °C. After further incubation (36 hours at 37 °C) cells were washed and their viability measured using crystal violet staining. Values of cell viability of cells neither treated with polyplexes nor inhibitor were set to 100%. Mean values ± SD were obtained from experiments performed in triplicate. Molecular Therapy 2009 17, 1411-1417DOI: (10.1038/mt.2009.121) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions