Supplemental Figures -
(PDF) (334 kb).
Figure I. Optimization of knocking-down target of mouse PHD2 gene. (a) Schema of the tandem type shRNA structure. Two H1 promoters are used to drive the separate transcription of the sense and antisense strands. The two strands would then anneal to form a double-stranded siRNA complex within the cell. (b) Forty-eight hours after siRNA fragment transfection, comparison of knocking-down efficiency was tested by RT-PCR of mouse PHD2 gene. Data shown here indicate that the site-2 siRNA fragment had the best interference efficiency.
Figure II. Evaluation of cardiac function in infarcted mice following shRNA therapy. (a) Representative echocardiogram (M-mode) of mice with LAD ligation following injection of shScramble plasmid (upper panel) versus shPHD2 plasmid (lower panel) at pre-surgery, week 2, week 4 and week 8. (b) Quantitative analysis of left ventricular fractional shortening (LVFS) between the two groups. Animals injected with shPHD2 had significant improvement in LVFS at week 4 but not at week 8.
Figure III. Injection of shPHD2 plasmid improves myocardial neovascularization. (a) Representative histology of infarcted heart injected with shScramble, infarcted heart injected with shPHD2, and control non-infarcted heart injected with PBS at week 4. Trichrome stains of the peri-infarct area indicate the infarction size based on collagen staining. (b) Immunofluorescence staining of CD31 endothelial marker (green) indicate small vessels in the myocardium. Cardiomyocyte staining is identified by troponin (red; 400x magnification). Nuclear staining is identified by DAPI (blue; 400x magnification). (c) Quantitative analysis of capillary density was significantly higher in the shPHD2 group compared with the control shScramble group at week 4 (P<0.01) (20x magnification).
