1. Volume 10, Issue 4, Pages 706-718 (October 2004)
Membrane-permeant, DNA-binding agents alter intracellular trafficking and increase the transfection efficiency of complexed plasmid DNA 
Sylvia Fong, Yong Liu, Timothy Heath, Paul Fong, Denny Liggitt, Robert J. Debs 
Molecular Therapy 
Volume 10, Issue 4, Pages (October 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Hoechst:CLDC complexes transfect DNA more efficiently than CLDC alone in a variety of cell types. Comparison of luciferase gene expression following cellular transfection with (A) Hoechst:DNA; (B) CLDC alone; (C) Hoechst:DNA, CLDC alone, or Hoechst:CLDC; (D) DNA:PEI or DNA:Hoechst:PEI. (E) Comparison of the percentage of X-GAL-positive cells following transfection with CLDC or Hoechst:CLDC. B16-F10 cells were transfected with the pHCMV-Luc plasmid complexed at a series of different charge ratios to either (A) Hoechst or (B) DOTAP MLV liposomes. Percentage cell death ± SEM is shown under the x axis for each ratio tested. In A, *P < 0.05 vs. 1:30. In B, *P < vs. 1:3.03, +P < < 0.001, #P < (C) B16-F10 (□), 4T1 (▪), CT26 ( ), and LM1 (□) cells were transfected with Hoechst:DNA, CLDC alone, or Hoechst:CLDC at previously optimized charge ratios. *P < vs. CLDC, **P < 0.01 vs. CLDC, +P < vs. CLDC, ++P < vs. CLDC, #P < vs. DNA alone, ##P < vs. DNA < vs. DNA alone, ###P < 0.05 vs. DNA alone. (D) B16-F10 (□), 4T1 (▪), CT26 ( ), and LM1 (□) cells were transfected with DNA:PEI or DNA:Hoechst:PEI complexes at previously optimized charge ratios. +P < 0.05 vs. PEI alone, ++P < vs. PEI alone. (E) B16-F10 cells were transfected with pHCMV-β-GAL using either CLDC alone or Hoechst:CLDC. *P < vs. CLDC. All cells were harvested 24 h posttransfection and analyzed for luciferase activity (A–D) or for β-GAL activity by X-GAL staining (E).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. FIG. 1
Hoechst:CLDC complexes transfect DNA more efficiently than CLDC alone in a variety of cell types. Comparison of luciferase gene expression following cellular transfection with (A) Hoechst:DNA; (B) CLDC alone; (C) Hoechst:DNA, CLDC alone, or Hoechst:CLDC; (D) DNA:PEI or DNA:Hoechst:PEI. (E) Comparison of the percentage of X-GAL-positive cells following transfection with CLDC or Hoechst:CLDC. B16-F10 cells were transfected with the pHCMV-Luc plasmid complexed at a series of different charge ratios to either (A) Hoechst or (B) DOTAP MLV liposomes. Percentage cell death ± SEM is shown under the x axis for each ratio tested. In A, *P < 0.05 vs. 1:30. In B, *P < vs. 1:3.03, +P < < 0.001, #P < (C) B16-F10 (□), 4T1 (▪), CT26 ( ), and LM1 (□) cells were transfected with Hoechst:DNA, CLDC alone, or Hoechst:CLDC at previously optimized charge ratios. *P < vs. CLDC, **P < 0.01 vs. CLDC, +P < vs. CLDC, ++P < vs. CLDC, #P < vs. DNA alone, ##P < vs. DNA < vs. DNA alone, ###P < 0.05 vs. DNA alone. (D) B16-F10 (□), 4T1 (▪), CT26 ( ), and LM1 (□) cells were transfected with DNA:PEI or DNA:Hoechst:PEI complexes at previously optimized charge ratios. +P < 0.05 vs. PEI alone, ++P < vs. PEI alone. (E) B16-F10 cells were transfected with pHCMV-β-GAL using either CLDC alone or Hoechst:CLDC. *P < vs. CLDC. All cells were harvested 24 h posttransfection and analyzed for luciferase activity (A–D) or for β-GAL activity by X-GAL staining (E).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. FIG. 2
Ability of Hoechst:DNA and Hoechst:CLDC to overcome fucoidan-mediated suppression of cellular transfection by CLDC alone. pHCMV-Luc complexed to Hoechst (□), to DOTAP MLV (▪), or to Hoechst:CLDC ( ) at previously optimized charge ratios was used to transfect (A) B16-F10 cells or (B) CT26 cells. Cells were pretreated with 0, 10, or 100 nM fucoidan for 30 min before transfection and were analyzed for luciferase activity 24 h after transfection. In A, *P < vs. no fucoidan added. In B, *P < 0.05 vs. no fucoidan added, +P < vs. no fucoidan added.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. FIG. 3
Increased ability of Hoechst:CLDC versus CLDC alone to transfect nondividing cells. Growth-arrested (96 h postconfluency) or dividing (80% confluent) CV1-P cells were transfected with either CLDC (□) or Hoechst:CLDC (▪) at previously optimized charge ratios and analyzed for luciferase activity 24 h after transfection. *P < 0.01 vs. 80% confluency, +P < 0.05 vs. 80% confluency.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. FIG. 4
Increased ability of Hoechst:CLDC versus CLDC alone to transfect mouse tissues following intravenous injection into mice. ICR mice received pHCMV-Luc as either CLDC (▪) alone or Hoechst:CLDC (□) at previously optimized charge ratios. (A) Lung and (B) heart, liver, and spleen were harvested 24 h later and analyzed for luciferase activity. In A, *P < 0.05 vs. CLDC. In B, *P < vs. CLDC, +P < 0.01 vs. CLDC.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. FIG. 5
Hoechst accelerates and preferentially targets plasmid DNA to nuclei. Cells were transfected with rhodamine-labeled plasmid DNA either as (A) CLDC or as (B) Hoechst:CLDC, harvested at 1 h posttransfection, and assessed by fluorescence microscopy. (a, d, and g) Staining of nuclei, (b, e, and h) localization of the rhodamine-labeled DNA, and (c, f, and i) overlapping images of a/b, d/e, and g/h. (C) Cells were transfected with rhodamine-labeled plasmid DNA either as CLDC alone or as Hoechst:CLDC, harvested at 1 h posttransfection, and assessed by fluorescence (a and b) and bright-field (c and d) microscopy. (a and c) The fluorescent and corresponding bright-field images for Hoechst:CLDC-transfected cells; (b and d) fluorescent and corresponding bright-field images for CLDC-alone-transfected cells.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

8. FIG. 5
Hoechst accelerates and preferentially targets plasmid DNA to nuclei. Cells were transfected with rhodamine-labeled plasmid DNA either as (A) CLDC or as (B) Hoechst:CLDC, harvested at 1 h posttransfection, and assessed by fluorescence microscopy. (a, d, and g) Staining of nuclei, (b, e, and h) localization of the rhodamine-labeled DNA, and (c, f, and i) overlapping images of a/b, d/e, and g/h. (C) Cells were transfected with rhodamine-labeled plasmid DNA either as CLDC alone or as Hoechst:CLDC, harvested at 1 h posttransfection, and assessed by fluorescence (a and b) and bright-field (c and d) microscopy. (a and c) The fluorescent and corresponding bright-field images for Hoechst:CLDC-transfected cells; (b and d) fluorescent and corresponding bright-field images for CLDC-alone-transfected cells.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions