Figure 4.

Lipoplex-mediated delivery of anti-miR-21 oligonucleotides in U87 human GBM cells and miR-21, PTEN and PDCD4 expression after oligonucleotide-mediated miR-21 silencing. For the assessment of lipoplex-cell association, U87 cells were incubated with lipoplexes for 4 h, rinsed with PBS and prepared for flow cytometry analysis (as described in Supplementary Materials and Methods). Viable cells were gated based on morphologic features, including cell volume (given by the forward scatter, FSC) and cell complexity (given by the side scatter, SSC) (left plots in [A and B]). Fluorescent intensity plots of U87 cells (A) untreated or (B) transfected with lipoplexes at a final concentration of 100 nm anti-miR-21 oligonucleotides are shown in the right plots in (A) and (B). Mean fluorescence values (geometric mean) are indicated for each plot. (C) miR-21 and (D) PTEN and PDCD4 mRNA expression levels in U87 cells 48 h after transfection with anti-miR-21 or scrambled oligonucleotides (n = 3), at a final oligonucleotide concentration of 50 or 100 nm. MiR-21 expression levels, normalized to the reference U6snRNA, as well as PTEN and PDCD4 expression levels, normalized to the reference HPRT1, are presented as relative expression values to control untreated cells. (E) Representative gel showing PTEN and PDCD4 protein levels in U87 cells 48 h after transfection with anti-miR-21 or scrambled oligonucleotides (n = 3) at a final oligonucleotide concentration of 100 nm. (F) Quantification of PTEN and PDCD4 bands observed in (E), corrected for individual α-tubulin signal intensity. Results are presented as PTEN and PDCD4 expression levels relative to control. *P < 0.05, **P < 0.01, ***P < 0.001 when compared with cells transfected with a similar amount of scrambled oligonucleotides.