Deletion of both <i>Ets1</i> and <i>Ets2</i> ablates <i>Hras^G12V</i> transformation of MEFs.

<p><i>E1+E2+</i> and <i>E1−E2−</i> MEFs were infected with <i>HRasv^G12V</i> or empty vector control retrovirus, then selected with Hygromycin for at least 5 days before functional examination of the four generated cellular genotypes, as shown. A) Western blotting analysis of 20 µg protein lysates probed with antibodies against proteins shown, with α-tubulin as loading control. B) Representative images and C) Quantification of the cellular colonies that grew in soft agar from the four different genetic groups Asterisk indicates P<0.05 as determined by the Student <i>t</i> test. D) Cells were injected subcutaneously into nude mice (10⁶ cells per injection site) and after 3 weeks the tumors were harvested. The ratios represent the percentage of tumors that grew from the total number of injections for each of the different genotypes.</p

<i>MiR-17-92</i> overexpression in <i>Ets1/Ets2</i>-null MEFs rescue <i>Hras^G12V</i> transformation.

<p><i>E1−E2−/pBabe</i> control and <i>E1−E2−/H-Rasv12</i> were infected with either MSCV-puro empty control or MSCV-puro-<i>miR-17- 92</i> vector and cells were selected by Puromycin before further functional analysis. A) pre-<i>mir17-92</i> cluster expression in the indicated genotypes relative to control vector. Asterisk indicated P<0.05. B) Graph demonstrating the percentage of tumors formed over the total number of injections for the different cellular groups. N/A indicates that there were no tumors observed for the specified group. (C) Representative images showing the total <i>E1+E2+</i>/<i>H-Rasv12</i> and <i>E1−E2−/H-Rasv12/MSCV-puro-miR-17-92</i> derived tumors. D) Graph indicating individual and average volume of <i>E1+E2+</i>/<i>H-Rasv12</i> and <i>E1−E2−/H-Rasv12/MSCV-puro-miR-17-92</i> tumors.</p

<i>mir17-92</i> expression depends on <i>Ets1</i> and <i>Ets2</i> in <i>Hras^G12V</i> transformed MEFs.

<p>A) Fold change of pre-<i>mir17-92</i> and B) individual <i>microRNAs</i> of the <i>mir17-92</i> cluster: in the indicated genetic groups relative to control cells. Asterisk indicates P<0.05. C) Schematic illustration of the <i>mir17-92</i> promoter showing conserved ETS(box) and MYC (triangle) binding sites, as well as the ChIP primers used (arrows) relative to the start RNA start site. D) ChIP performed on the indicated MEFs genotypes using IgG control and specific antibodies as indicated. Asterisk indicates P<0.05.</p

Overexpression of <i>c-Myc</i> in <i>Ets1/Ets2</i>-null MEFs rescued <i>Hras^G12V</i> transformation.

<p><i>E1−E2−</i> control and <i>E1−E2−/H-Rasv^G12V</i> cells were infected with either MSCV-<i>GFP</i> empty control or MSCV-<i>GFP-c-Myc</i> vector, and cells were sorted for GFP expression by FACS. A) 20 µg of protein lysates from <i>E1−E2−/H-Rasv12/MSCV-GFP control</i> and <i>E1−E2−/H-Rasv12/MSCV-GFP-c-Myc</i> cells analyzed by western blot against MYC and β-Actin (protein loading control) antibodies. B) Graph representing the percentage of tumors formed over the total number of injections for the indicated cellular genotypes. N/A indicates that there were no tumors observed for the specified group. C) Representative pictures of all tumors derived from <i>E1+E2+</i>/<i>H-Rasv12</i> and <i>E1−E2−/H-Rasv12/MSCV-GFP-c-Myc</i> cells. D) Graph showing individual and average volumes of <i>E1+E2+</i>/<i>H-Rasv12</i> and <i>E1−E2−/H-Rasv12/MSCV-GFP-c-Myc</i> tumors. Asterisk indicates P<0.05.</p

<i>Ets1</i> and <i>Ets2</i> activate <i>c-Myc</i> expression in <i>Hras^G12V</i> transformed MEFs.

<p>A) Fold change of <i>c-Myc</i> gene expression by real time rt-PCR in the 4 different genetic groups examined. B) Western blotting analysis of 20 µg protein lysates probed with antibodies against MYC and α-tubulin. C) Schematic illustration of the <i>c-Myc</i> promoter showing conserved Ets binding sites (box) and the ChIP primers used (arrows) relative to the P2 promoter. D) ChIP performed on MEFs with indicated genotypes using anti-ETS1 and ETS2 antibodies with IgG as control. The threshold value for the promoter being studied was normalized to that of input values and represented as relative enrichment. Asterisk indicates P<0.05.</p

<i>Ets1</i>, <i>Ets2</i> and <i>c-Myc</i> activate miR17-92 transcription.

<p><i>C-Myc^f/f</i> MEFs were infected with <i>pBabe-Cre</i> retroviral vector or control <i>pBabe-empty</i> vector, selected with puromycin before functional examination. A) Protein analysis of 20 µg lysates from MEFs by western blot using antibodies as indicated B) Relative expression of <i>Ets1</i>, <i>Ets2</i> and <i>c-Myc</i> mRNAs after 48 hrs of transfection of expression vector. Asterisk represents P<0.05. C and D) Fold change relative real time PCR gene expression of pre-<i>mir17-92</i> (C) and individual miRs of the cluster (D) after 48 hrs transient transfection of the indicated vectors in <i>c-Myc^−/−</i> MEFs relative to control empty vector. Asterisk indicates P<0.05.</p

Cry Protein Crystals: A Novel Platform for Protein Delivery

<div><p>Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium <i>Bacillus thuringiensis</i>. We demonstrate that fusion of the <i>cry3Aa </i>gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines <i>in vitro</i>, and can be delivered to mice <i>in vivo</i> via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.</p></div

Fluorescence and TEM images of Cry3Aa-GFP and Cry3Aa-mCherry crystals uptaken into cells.

<p>Macrophage cells (RAW264.7) were incubated with Cry3Aa-GFP crystals for different time periods. Fluorescence images at (a) 15-min, demonstrating fast initial uptake and at (b) 1-h, where the green fluorescence intensity reached its maximum value. Note the punctate fluorescence pattern, which supports uptake of the whole crystal. (c) Red fluorescence image of primary mouse fibroblasts after 1.5 h incubation with Cry3Aa-mCherry protein crystals is not as intense as seen with macrophages. TEM images of (d) untreated RAW264.7 cells, and (e) cells treated with Cry3Aa-GFP. The Cry3Aa-GFP treated cells exhibit a distinct cytoplasmic particulate not observed in the control cells. (f) Enlarged TEM image of the cytoplasmic particulate observed in the Cry3Aa-GFP treated macrophages highlighting the likely crystal degradation in the cytoplasm.</p

Bioluminescence of Cry3Aa-luciferase crystals delivered to mice.

<p>(a) No Cry3Aa-luciferase crystal control (b) nasal uptake, and (c) intraperitoneal injection. The luminescence measurements were taken 10 min after crystal and D-luciferin delivery. (d) Luciferase activity following oral gavage of Cry3Aa-luciferase crystals after 5, 10, 20, 30, 40, and 50 min. The D-luciferin substrate was replenished at 20 min intervals. Activity and lifetime of luciferase activity of Cry3Aa-luciferase crystals (0.5 mg or 0.25 mg as indicated in the figure) and luciferase protein (2.5 mg or 1.25 mg as indicated in the figure) delivered to C57BL/6 mice via (e) nasal spray, (f) intraperitoneal injection and (g) oral gavage. Measurements were made by selecting a region of interest (ROI) in the area where the maximal intensity was obtained from the crystal and the protein. Lifetime of luminescence was measured in the selected ROI over the indicated times. D-luciferin was replenished every 20 min. The lifetime of the luciferase activity of the Cry3Aa-luciferase crystals was found to be higher that of the recombinant luciferase protein in each route of delivery, supporting the ability of the Cry3Aa crystal framework to protect the luciferase protein from degradation.</p

TNF regulates Notch-1 expression in vivo.

<p><b>A.</b> Muscle homogenates were prepared from 7–8 week old <i>mdx</i> mice that either contained or lacked IKKβ and western analysis was performed probing for Notch-1. The blot was stripped and reprobed for α-tubulin used as a loading control. <b>B.</b> Nude mice were implanted with vector control CHO cells or CHO cells expressing TNF. Once tumors were established, muscle injury was induced to tibialis anterior muscles with cardiotoxin injections. At indicated days post-toxin injections, muscles were harvested and homogenates prepared for western analysis probing for Notch-1 and α-tubulin as a loading control.</p