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1.
Figure 1

Figure 1. Hoechst-IGF1 synthesis and bioactivity.. From: Targeting Extracellular DNA to Deliver IGF-1 to the Injured Heart.

(A) Hoechst-amine was functionalized by the addition of a PEG-Biotin to allow for conjugation with IGF-1. (B) Using the biotin-streptavidin interaction, Hoechst-biotin was incubated with biotinylated-IGF-1 and tetravalent streptavidin to create the Hoechst-IGF1 (H-IGF1) complex. A control compound, streptavidin-IGF1 (S-IGF1) containing only streptavidin, biotin and biotinylated-IGF-1 was also assembled. (C) Immunoblot for IGF-1 demonstrating the size of H-IGF1 species at 70 and 80 kDa (right lane) compared to biotinylated-IGF-1 at 10 kDa (left lane). (D) Live and methanol fixed RAW 264.7 macrophages were treated with H-IGF1 or Hoechst-biotin for 15 minutes. Hoechst compounds remained membrane impermeable to live cells as demonstrated by low levels of blue fluorescence. In contrast, permeabilized cells had high levels of blue, nuclear fluorescence demonstrating the DNA binding capability of Hoechst. White scale bar represents 100 μm. (E) Cardiomyoblasts (H9c2 cells) were treated with control media, streptavidin, IGF-1, biotinylated-IGF-1 or H-IGF1 for 15 minutes. Cells were homogenized and immunoblotted for Akt phosphorylation. H-IGF1 treatment activated Akt to a similar extent as IGF-1 and biotinylated-IGF-1.

Raffay S. Khan, et al. Sci Rep. 2014;4:4257.
2.
Figure 3

Figure 3. Hoechst-IGF1 preserves cardiac function and attenuates cardiac fibrosis following MI.. From: Targeting Extracellular DNA to Deliver IGF-1 to the Injured Heart.

(A) Representative echocardiographic M-mode images and bar graph depicting cardiac contractility at 28 days following IR. As shown, H-IGF1 treated mice demonstrated greater percent fractional shortening than PBS and S-IGF1 treated mice (%FS, mean ± SEM: 41.55 ± 2.49 vs. 29.18 ± 3.51 vs. 27.83 ± 1.06). * p < 0.01 vs. S-IGF1, ** p < 0.001 vs. PBS, n = 5–9 per group, one-way ANOVA followed by Tukey's post-test. (B–D) Invasive cardiac hemodynamic measurements determined at 28 days following IR. H-IGF1 treatment led to significantly improved (B) Ejection fraction and (C) End-diastolic volumes compared to PBS and S-IGF1 treated mice. (D) End-systolic volumes were not different between sham and H-IGF1 treated mice (n = 5 per group, *p < 0.05 vs. sham mice, # p < 0.05 vs. S-IGF1 treated mice, one-way ANOVA followed by Tukey's post-test). (E) Representative Picrosirius red images from histologic sections of hearts from PBS, S-IGF1 and H-IGF1 treated mice. Black scale bar represents 1 mm. H-IGF1 treatment reduced collagen content in the left ventricle compared to PBS treated mice (n = 5–6 per group, *, p < 0.05 vs. PBS, one-way ANOVA followed by Tukey's post-test).

Raffay S. Khan, et al. Sci Rep. 2014;4:4257.
3.
Figure 2

Figure 2. Hoechst increases IGF-1 localization to infarcted myocardium.. From: Targeting Extracellular DNA to Deliver IGF-1 to the Injured Heart.

(A) Rats were subjected to ischemia-reperfusion (IR) surgery. One day later Hoechst-biotin was injected and allowed to circulate for 1 h. Frozen sections were made and tissue was examined via red autofluorescence of the myocardium. Sham operated animals showed cell membrane integrity in the left ventricle (LV) and little blue fluorescence. IR-operated animals showed necrosis in the LV and diffuse blue staining suggesting release of DNA (Scale bar = 200 μm). (B) Imaging of hearts harvested from mice at the indicated time-point. Hoechst increased IGF-1 accumulation at 18 hours compared to S-IGF1 (n = 4–7 per group; * p < 0.05 vs. S-IGF1, ** p < 0.01 vs. SIGF1, two-way ANOVA followed by Bonferroni post-test). (C) Imaging was used to quantify IGF-1 as %ID per gram of tissue at 18 h. While other organs were similar, there was a significant increase in the heart of H-IGF1 treated mice compared to S-IGF1 treatment (n ≥ 5 per group, *p < 0.05; t-test). (D) Western blots showing phosphorylation of Akt in the LV of mice treated with S-IGF1 or H-IGF1. By 18 hours, Akt phosphorylation in H-IGF1 treated mice was significantly greater than S-IGF1 treated mice. Line represents PBS treated IR mice. (* p < 0.05 vs. S-IGF1 at 18 hours, n = 3–7 per group, two-way ANOVA followed by Bonferroni post-test). (E) Mice were treated with S-IGF1, H-IGF1, or H-IGF1 pre-incubated with 1000× excess of double-stranded DNA. Pre-incubation with DNA significantly decreased IGF-1 levels. (*p < 0.05 vs. S-IGF1, # p < 0.05 vs. H-IGF1, n = 5–7 per group, one-way ANOVA followed by Bonferroni post-test).

Raffay S. Khan, et al. Sci Rep. 2014;4:4257.

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