Journal: Journal of Lipid Research

Article Title: Apo B100 similarities to viral proteins suggest basis for LDL-DNA binding and transfection capacity

doi: 10.1194/jlr.M003277

Figure Lengend Snippet: Synthetic peptide mediated transfection of HeLa cells. Top panel: Synthetic peptide mediated transfection of HeLa cells with BOBO-1-labeled phMGFP plasmid. Dye-labeled plasmid DNA complexed to apo B100-derived peptides was used to transfect HeLa cells grown in DMEM medium as described in Methods. All frames show a merged overlay image of the fluorescence micrograph and the corresponding phase contrast version. Frames A thru C show cells transfected using cocktails of synthetic peptides B1-1 and B1-2 (Table 2) derived from N-terminal region of Apo B100 mixed with 1.0 μg BOBO-1-labeled phMGFP plasmid DNA: A: 1.5 nmol of B1-1 and 1.9 nmol of B1-2 plus plasmid; B: 5.9 nmol of B1-1 and 7.5 nmol of B1-2 plus labeled plasmid; C: 15 nmol of B1-1 and 19 nmol of B1-2 plus labeled plasmid. All transfection experiments were conducted in 400 μl of DMEM. Enlarged cut-out images in inserts D–H show intracellular loci of label. All images were obtained using a Zeiss Axiovert 25 scope and the Optronics MicroFire CCD camera. Bottom panel: Cytotoxic effects of peptides B1-1 and B1-2 on HeLa cells are demonstrated using Trypan Blue dye. Cytotoxicity caused by mixtures of synthetic peptides B1-1 and B1-2, 4 µg each peptide (frame I) and 10 µg each peptide (frame J), added to preconditioned HeLa cells in DMEM. In contrast, no cytotoxicity is seen for HeLa cells treated similarly with mixtures containing the same quantities of synthetic peptides B2-1/B2-2, frames K and L, respectively.

Article Snippet: The LUMAM TM EPI-Fluorescence microscope (LOMO America, Inc.) equipped with an Optronics MacroFire® 2.0 CCD camera and the Zeiss Axiovert 25 microscope with the Optronics MicroFire CCD camera were used to obtain all other cell images.

Techniques: Transfection, Labeling, Plasmid Preparation, Derivative Assay, Fluorescence