Figure 5
Figure 5. Influence of Sp1 siRNA siRNA in vivo on cardiac ischemia. (A) Murine model of in situ siRNA repression. A surgically inserted carotid catheter was advanced into the left ventricle. Blood pressure recording confirmed correct placement of the catheter tip within the left ventricle. After placement of the catheter's infusion port within the left ventricle, an infusion of target (Sp1 siRNA) or control siRNA (SCR) together with transfection agent (siPORT Amine; Ambion) was given over indicated time periods (1.5 μg siRNA/g body weight over indicated time periods). Changes in cardiac Sp1 transcript (B) or protein (C) after in vivo siRNA repression. C57BL/6 mice were treated with a left ventricular infusion (as described for panel A) of siRNA specific for Sp1 or SCR siRNR. After indicated time periods, hearts were excised, total RNA isolated, and Sp1 transcript levels were determined by real-time RT-PCR. Data were calculated relative to an internal housekeeping gene (β-actin) and are expressed as fold change compared with control (0 hour of siRNA infusion ± SEM at each indicated time, n = 6). (C) Western blot. (D) CD39 transcript of myocardial biopsies from the area at risk after Sp1 repression. C57BL/6 mice were treated with scrambled control siRNA (SCR siRNA) or Sp1-specific siRNA (Sp1 siRNA) via intraventricular infusion over 2 hours before exposure to in situ myocardial ischemia (0-60 minutes). Cardiac tissue from the area at risk was excised at indicated time points, flash-frozen, and CD39 transcript was determined by real-time RT-PCR. Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as fold change compared with control (0 minutes of myocardial ± SEM at each indicated time, n = 6). *P < .05 compared to 0 hours (B,D). (E) C57BL/6 mice were treated with control (SCR siRNA) or Sp1-specific siRNA (Sp1 siRNA) via intraventricular infusion over 2 hours before exposure to in situ myocardial ischemia (45 minutes). Cardiac tissue from the area at risk was harvested after 120 minutes of reperfusion, sectioned, and stained using CD39 antibody. Cardiac tissue from sham-operated mice served as control (original magnification ×400). To control for nonspecific staining, IgG was used at identical concentrations and staining conditions as the target primary antibodies for CD39.

Influence of Sp1 siRNA siRNA in vivo on cardiac ischemia. (A) Murine model of in situ siRNA repression. A surgically inserted carotid catheter was advanced into the left ventricle. Blood pressure recording confirmed correct placement of the catheter tip within the left ventricle. After placement of the catheter's infusion port within the left ventricle, an infusion of target (Sp1 siRNA) or control siRNA (SCR) together with transfection agent (siPORT Amine; Ambion) was given over indicated time periods (1.5 μg siRNA/g body weight over indicated time periods). Changes in cardiac Sp1 transcript (B) or protein (C) after in vivo siRNA repression. C57BL/6 mice were treated with a left ventricular infusion (as described for panel A) of siRNA specific for Sp1 or SCR siRNR. After indicated time periods, hearts were excised, total RNA isolated, and Sp1 transcript levels were determined by real-time RT-PCR. Data were calculated relative to an internal housekeeping gene (β-actin) and are expressed as fold change compared with control (0 hour of siRNA infusion ± SEM at each indicated time, n = 6). (C) Western blot. (D) CD39 transcript of myocardial biopsies from the area at risk after Sp1 repression. C57BL/6 mice were treated with scrambled control siRNA (SCR siRNA) or Sp1-specific siRNA (Sp1 siRNA) via intraventricular infusion over 2 hours before exposure to in situ myocardial ischemia (0-60 minutes). Cardiac tissue from the area at risk was excised at indicated time points, flash-frozen, and CD39 transcript was determined by real-time RT-PCR. Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as fold change compared with control (0 minutes of myocardial ± SEM at each indicated time, n = 6). *P < .05 compared to 0 hours (B,D). (E) C57BL/6 mice were treated with control (SCR siRNA) or Sp1-specific siRNA (Sp1 siRNA) via intraventricular infusion over 2 hours before exposure to in situ myocardial ischemia (45 minutes). Cardiac tissue from the area at risk was harvested after 120 minutes of reperfusion, sectioned, and stained using CD39 antibody. Cardiac tissue from sham-operated mice served as control (original magnification ×400). To control for nonspecific staining, IgG was used at identical concentrations and staining conditions as the target primary antibodies for CD39.

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