Fig 1.
Structure of Amino-Prenols, the AP molecule contains an polyisoprenoid chain composed of n isoprene residues.
Fig 2.
Gel retardation analysis with different carrier:pDNA complexes at various N/P ratios (of amino-group of the carriers to the phosphate group of the nucleic acid).
(a) AP-11:pDNA, (b) AP-15:pDNA, (c) AP-7:pDNA, (d) AP-8:pDNA, (e) PEI:pDNA. M- molecular weight size marker 1kb+.
Fig 3.
Evaluation of transfection efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes.
(a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from Attractene treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P<0.05, **P<0.005. Transfections were performed using 4 μg of respective pDNA.
Fig 4.
(a) cell viability and (b) total cell number of B16-F10 cells transfected with AP:pGFP complexes at various N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15), or with AP-15/DOPE:pGFP and AP-15/DOPE/DMEM:pGFP lipoplexes at 2.5 μg of lipids/μg of pDNA dose, analysed by FACS.
Transfections were performed with the use of 4 μg of pDNA. *P<0.05, **P<0.001.
Fig 5.
TIMP2 protein expression in B16-F10 cells assessed by Western blot in.
(a) cell lysates, (b) media from cells. (c) Control Gapdh protein level in cell lysates. Lines: 1- B16-F10 cells, 2- B16-F10 cells transfected with AP-15/DOPE:pGFP, 3- B16-F10 cells transfected with AP-15/DOPE:pTIMP2.
Fig 6.
Number of viable cells in samples treated with the various APs / AP-based reagents alone (grey bars: AP-7, AP-8, AP-11, AP-15, AP-15/DOPE, AP-15/DOPE/DMEM) and AP:pGFP / AP-15 and DOPE-containing complexes (patterned bars: AP:pGFP: AP-7, AP-8, AP-11 r = 1.7, AP-15 r = 2.0; AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP 2.5 μg of lipids/μg of pDNA), analysed by FACS.
Transfections were performed with the use of 4 μg of pDNA. *P<0.05.
Table 1.
Effect of transfection of B16-F10 cells with AP-15/DOPE:pDNA complexes on expression of lipid metabolism-related gene.
Fig 7.
In vivo transfection study of tumor cells using AP-15-based complexes.
Analyses of PCR products using DNA templates from: (a) B16-F10 tumors, injected with: 2-4- H2O, 5-7- AP-15/DOPE, 8-10- AP-15, 11-13- pSec, 14-16- pTIMP2, 17-20- AP-15/DOPE:pTIMP2 complexes, 21-24- AP-15:pTIMP2 complexes; 25- positive control for PCR (pTIMP2), (b) L1 tumors, injected with: 2-3- H2O, 4-5- AP-15, 6-7- AP-15/DOPE/DMEM, 8-14- AP-15:pTIMP2 complexes, 15-21- AP-15/DOPE/DMEM:pTIMP2 complexes; 25- positive control for PCR (pTIMP2). M- molecular weight size marker 1kb+, 1- reagent control for PCR.