Recombinant human bfgf

Growth factor reduced Matrigel (BD) with 400 ng ml⁻¹ recombinant human bFGF (Millipore) was injected subcutaneously in Balb/C mice. Mice were injected i.v with 10 μg control or anti-miRs 3 days after plugs were implanted. Four days later mice were irradiated with the indicated doses on a Shepherd ¹³⁷cesium irradiator at a rate of ∼166 cGy min⁻¹. 3 days after irradiation mice were injected with 10 μg FITC-conjugated Griffonia simplificola lectin, the plugs were harvested, lysed in RIPA-buffer and the FITC content was measured on a spectrophotometer (Glomax, Promega) with a standard FITC filter set.

All animal work was approved by the OHSU Institutional Animal Use and Care Committee. WT male and female C57Bl/6 N and Ddx58 (−/−) 8–10-week-old mice were purchased from Jackson Labs and injected subcutaneously with Growth factor reduced (Matrigel BD) with 400 ng mL⁻¹ recombinant human bFGF (Millipore). One-week later Matrigel plugs, as well as lung, liver, and heart, were harvested from mice and RNA was isolated using the Eurx RNA purification kit according to manufacturer’s instructions. Matrigel plugs were homogenized and analyzed for hemoglobin content using a colorimetric assay kit (Sigma). In addition, RNA from tissue was used to analyze endothelial activity using the Qiagen endothelial cell activity qRT-PCR array according to manufacturer’s instructions.

All animal work was approved by the OHSU Institutional Animal Use and Care Committee. Immune-compromised 8- to 10-week-old female nu/nu mice were purchased from Jackson Labs. Growth factor-reduced Matrigel (BD) with 400 ng/ml recombinant human bFGF (Millipore) was injected subcutaneously in nu/nu mice. Mice were injected intravenously (i.v.) with 7C1 nanoparticles containing miR-494 or control miR (~1 mg/kg, i.v) 3 or 4 days after plugs were implanted. At day 7 mouse tissues were harvested and processed to obtain RNA or frozen in OCT for tissue staining. 4T1 cells (1 × 10⁴) were implanted into the mammary fat pad #4 of 6- to 8-week-old female Balb/C mice in 100 μl Matrigel. Mice were randomized into groups once the average tumor volume reached 150 mm³, approximately 10 days after implantation. Mice were treated with 7C1 nanoparticles containing miR-494 or control miR (0.7 mg/kg, i.v.). Mice were euthanized ~day 18–20 for analysis of metastatic burden in lungs. Additional experiments were carried out in postpubertal female FVB/n mice (10–12 weeks), which received injections of 50 000 PyMT-derived tumor cells into the right lower mammary fat pad for induction of orthotopic tumors as described in ref. ^{47 (link)}.