Figure 1.
Morphology and fiber diameter of electrospun scaffolds.
SEM images of Control scaffolds without DNA (A, B) and those containing plasmid DNA encoding for Cdk2 (Cdk2i) or EGFP (EGFPi) shRNA (D, E and G, H, respectively). C, F, and I indicate the fiber diameter distribution between the three types of scaffolds (using corresponding images in A, C and E). Scale bar in A = 10 µm, B = 2 µm and in D and G = 35 µm, E and H = 5 µm.
Figure 2.
Plasmid DNA release from electrospun scaffolds.
Graphs indicates the amount of DNA release per time point (A) and cumulative release (B) over the 21 day study from the two plasmid DNA containing scaffolds, Cdk2i and EGFPi. (C) Agarose gel electrophoresis showing the integrity of the released DNA from both scaffolds. Lane 1, 100 bp MW marker; Lane 2 and 4, EGFP (pKD-EGFP-v1) and Cdk2 (pKD-Cdk2-v5) control (unincorporated) plasmid DNA; Lane 3 and 5, 21 day released EGPF and Cdk2 plasmid DNA, respectively.
Figure 3.
Electrospun scaffold degradation.
SEM images of Control scaffolds without DNA (A,C,E,G) and with scaffold containing plasmid DNA encoding for Cdk2 shRNA (Cdk2i, B, D, F, H) at the specified days (0, 8, 14 and 21). Scale bar in A, C, E, G = 2 µm, and in B, D, F, H = 5 µm.
Figure 4.
Silencing of Cdk2 and suppression of MCF-7 cell proliferation.
The effectiveness of the plasmid DNA encoding Cdk2 shRNA was tested on MCF-7 cells growing on tissue culture plates, both in terms of suppressing Cdk2 expression, as measured by Q-PCR on culture day 4 (A) and cellular proliferation, as measured by the MTS assay on culture day 1 (B). The effect on proliferation and cell death was also examined via microscopy in the three conditions (C). The plasmid DNA encoding for EGFP shRNA was used as a control in both experiments. Control indicates results from untransfected cells. (A) *p<.03, **p<.05. (B) *p<.001, **p<.001.
Figure 5.
Silencing of Cdk2 and suppression of cell proliferation by the Cdk2i scaffold.
The effectiveness of the plasmid DNA containing scaffolds was tested on MCF-7 cells growing directly on them. Control scaffold and those containing plasmid DNA encoding for Cdk2 (Cdk2i) or EGFP (EGFPi) shRNA, both in terms of suppressing Cdk2 mRNA, as measured by Q-PCR on culture day 4 (A) and cellular proliferation, as measured by the MTS assay on culture day 1 (B). The Control and EGFPi scaffolds served as controls in both experiments. (A) *p<.01, **p<.04. (B) *p<.02, **p<.001.
Figure 6.
Silencing of Cdk2 and cell death by the Cdk2i scaffold.
LIVE/DEAD assay of MCF-7 cells grown on each of the three scaffolds, Control (A, B, C), and those containing plasmid DNA encoding for Cdk2 (Cdk2i, D, E, F) or EGFP (EGFPi, G, H, I) shRNA. A, D, H show living cells (green); B, E, H show dead cells (red); C, F, I represent the overlay of the two corresponding red/green images. Scale bar = 50 µm.