Fig. 2

Characterization of pDNA-lipoplexes (lipo-pDNA) and the functional validation of pDNA-lipoplexes (lipo-pDNA) in vitro. a Schematic diagram of pU6gRNA1Cas9EGFPU6gRNA2 plasmid and sgRNAs targeting CD47 gene. b The change of particle sizes and zeta potentials of pDNA-lipoplexes (lipo-pDNA) at various N/P ratio. c, Expressions of CD47 in GL261 cells transfected with different preparations. Lipo-SpDNA is short for liposome-scrambled pDNA. d, Western blot-based quantifications of CD47 expressions in GL261 cells transfected with different preparations. The results are expressed as mean ± SD (n = 3). e, CD47 expressions on cells were analysed by Firefly Luciferase Reporter Gene Assay Kit after GL261 cells were transfered with different preparations for 24 h. Data are expressed as mean ± SD (n = 3). ***P < 0.001. f Fluorescence picture GL261 cells after transfection of 48 h with (a) blank, (b) PEI-pDNA, (c) lipo2000-pDNA (2), (d) lipo-pDNA (N/P = 2) (2.5 μg/mL equivalent pDNA). Scale bar = 100 μm. g CD47 expressions on cells were analysed by flow cytometry after GL261 cells were transfered with different formations for 72 h. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01. h Evaluation of cell viability (CCK-8 assay) of GL261 cell lines after 24 h of treatment with different preparations, respectively. Values are expressed as mean ± SD (n = 6). i Evaluation of cell viability (CCK-8 assay) of GL261 cells after 24 h of treatment with different preparations, respectively. Values are expressed as mean ± SD (n = 6). j Confocal fluorescentmicroscopy of macrophages 8 h after phagocytosis of GL261 cells precultured with (A) blank, (B) naked pDNA, (C) lipo2000-pDNA (2) and (D) lipo-pDNA (N/P = 2) for 72 h. Scale bar = 60 μm. The green fluorescence is for GL261 tumor cells and the red fluorescence is for macrophages