Characterization of Ran transgenic mice.

<p><i>A</i>, Tail DNA was extracted from transgenic mouse lines expressing Ran-WT, Ran T24N or Ran G19V in pancreatic β cells under RIP control, and PCR products were amplified using RIP-HA (<i>top</i>) or RIP-Ran (<i>bottom</i>) primers. (+) or (−) refers to Ran-positive or –negative genotype, or positive or negative control primers for the amplification reaction. M, molecular weight markers. <i>B</i>, Pancreas tissues extracted from the various mouse cohorts genotyped for the presence (+) or absence (−) of Ran transgenes were analyzed by Western blotting. 3T3, extracts from NIH3T3 cells transiently transfected with HA-Ran cDNA used as control. a-HA, antibody to HA. <i>C</i>: Pancreas or liver tissues were isolated from PCR-confirmed Ran transgenic mice, and analyzed by Western blotting. β-actin was used as a loading control.</p

Defective islet cell proliferation in Ran transgenic mice.

<p><i>A</i>, Pancreas section from non-TG or Ran-WT transgenic mice were harvested at the indicated postnatal (P) age and analyzed for Ki-67 reactivity, by immunohistochemistry. Sections from normal mouse spleen were used as control. <i>B</i>, The number of Ki-67⁺ cells/islet surface area (0.02 mm²) was quantified by morphometry. **, p = 0.0081. <i>C</i>, Pancreas section from non-TG or Ran-WT transgenic mice (P50) were analyzed for TUNEL reactivity and the numbers of positive cells was quantified. Mean±SD.</p

Postnatal defect in islet development in Ran transgenic mice.

<p><i>A</i>, E18 non-TG or Ran-WT embryos were analyzed by H&E or immunohistochemical staining with an antibody to insulin. <i>Arrows</i>, insulin-stained islets. <i>B</i>, Islet number (<i>left</i>), or islet surface area (<i>right</i>), was quantified in E18 non-TG or Ran-WT transgenic embryos by morphometry of insulin-stained sections. The differences among groups are not statistically significant. <i>C</i>, Littermates of Ran-WT diabetic mice crossed with normal mice were analyzed for changes in blood glucose levels at the indicated time intervals. E, embryonic; P, postnatal. Glucose levels in PCR-confirmed non-TG (<i>open circles</i>) or Ran-WT transgenic (<i>closed circles</i>) mice are shown. Each point corresponds to an individual mouse. Glucose concentrations (mg/dl) in each group (number of mice in parentheses), and statistical analyses <i>versus</i> E18 values are as follows: E10 (n = 15), 108.7±4.93; E18 (n = 15), 120.7±2.7; P30 Ran-WT (n = 7), 196.3±17.8, p<0.0001; P30 non-TG (n = 4), 134±5.4; P45 Ran-WT (n = 7), 187.1±13.5, p<0.0001; P45 non-TG (n = 4), 134.8±3.4; P60 Ran-WT (n = 7), 193.1±15.5, p<0.0001; P60 non-TG (n = 4), 132.5±13.9. Statistical data re-analysis using Mann-Whitney test was as follows: P30 Ran-WT, p = 0.013; P45 Ran-WT, p = 0.0006; P60 Ran-WT, p = 0.0009.</p

Defective islet development in Ran transgenic mice.

<p><i>A</i>, Pancreas tissues from non-TG, Ran-WT, Ran-G19V or Ran-T24N transgenic mice were analyzed by H&E or immunohistochemical staining with an antibody to insulin. <i>B</i>, Pancreas sections from non-TG, asymptomatic Ran-WT or diabetic Ran-WT transgenic mice were analyzed for islet number or islet surface area by morphometry of insulin-stained areas. Islet number, non-TG (n = 2), 29.5±1.5; Ran-WT (n = 3), 23±1.73, n.s., not significant; Ran-WT diabetic (n = 3), 9.33±0.88, **, p = 0.001; islet surface area, non-TG (n = 2), 0.51±0.02; Ran-WT (n = 3), 0.35±0.021, *, p = 0.017; Ran-WT diabetic (n = 3), 0.057±0.004, **, p<0.0001. <i>C</i>, Pancreas tissues from Ran-WT, Ran-G19V or Ran-T24N transgenic mice were analyzed by immunohistochemical staining with antibodies to somatostatin or glucagon.</p

PDGF-AA, PDGF-BB and survivin levels are increased in eNOS (−/−) mice in response to remodeling stimulus.

<p>(A), Immunohistochemistry of PDGF-BB protein shows that it is present in adventitia of RC but is increased in all layers of LC post-ligation. Scale bar, 10 µm. (B), Quantitative RT-PCR of PDGF-AA and BB shows there was no change of PDGF mRNA level in wild type mice after LEC ligation. However, PDGF-AA and BB mRNA are all elevated in ligated LC compare to contralateral RC in eNOS (−/−) mice, 18 s was used as internal control. (C), Survivin immunohistochemistry shows strong staining throughout whole vessel wall of LC, and there is little or no staining in RC. Scale bar equals to 10 µm. (D), RT-PCR shows equal expression of survivin mRNA of contralateral RC and ligated LC in wild type mice. However, survivin was upregulated in ligated LC of eNOS (−/−) mice. 18 s was used as internal control.</p

NO donor dose dependently decreases cell number induced by PDGF-BB and blocks survivin expression and <i>in vitro</i>.

<p>(A), Rat SMC were treated or not with PDGF (10 ng/ml), in the absence or presence of the NO donor, DETA/NO (10, 30, and 100 µM) and cell number quantified after 48 h. (B), NO reduces PDGF induced survivin levels. VSM were treated with PDGF (10 ng/ml) with or withoutt DETA/NONO (10, 30 and 100 µM) and the levels of survivin assessed by Western blotting. DETA/NO dose dependently decreased survivin levels relative to Hsp90 (a protein loading control). Densitometric values of the ratio of survivin to Hsp90 are shown below the blots. * P<0.05, ** P<0.01 with one way ANOVA with Bonferroni posttest.</p

Transgenic expression of Ran impairs glucose metabolism.

<p><i>A and B</i>, The indicated PCR-confirmed transgenic mice were analyzed for blood glucose content at 2 mo of age under <u>ad libitum</u> feeding (<i>A</i>) or fasting (<i>B</i>) conditions. Non-TG, non-transgenic mice. Glucose concentrations (mg/dl) in each group in <i>A</i> (number of mice in parentheses), and statistical analyses (unpaired <i>t</i> test) are as follows: Non-TG (n = 24), 125±2; Ran-WT (n = 26), 189.4±17.8, p = 0.013; Ran-G19V (n = 24), 153.7±6.5, p = 0.003; Ran-T24N (n = 21), 172.4±21.1, p = 0.02. Statistical data re-analysis of the groups in <i>A</i> using ANOVA and post-hoc multiple tests with Bonferroni procedure was as follows: Ran-WT, p<0.0001; Ran-G19V, 0.017; Ran-T24N, p = 0.029. <i>C</i>, The indicated non-TG or Ran transgenic mice were analyzed for blood insulin concentrations. Insulin levels (ng/ml) in each group (number of mice in parentheses), and statistical analyses (unpaired <i>t</i> test) are as follows: Non-TG (n = 8), 1.26±0.1; Ran-WT (n = 11), 0.7±0.16, p = 0.019; Ran-G19V (n = 5), 0.61±0.03, p = 0.0008; Ran-T24N (n = 7), 0.49±0.14, p = 0.0009. Statistical data re-analysis using ANOVA and post-hoc multiple tests with Bonferroni procedure was as follows: Ran-WT, p = 0.023; Ran-G19V, p = 0.031; Ran-T24N, p = 0.004. One outlier mouse in the Ran-G19V group with aberrantly high insulin level (2.35 ng/ml) was excluded from the analysis. For panels <i>A–C</i>, each point corresponds to an individual mouse. <i>D</i>, Islets (20/well) isolated from non-TG or Ran-WT transgenic mice were incubated with 5 mM D-glucose, and analyzed for insulin release in the supernatant at the indicated time intervals. Mean±SD of replicates. <i>E</i>, Islets (20/well) isolated from non-TG (<i>black</i>) or Ran-WT (<i>purple</i>), Ran-G19V (<i>grey</i>) or Ran-T24N (<i>blue</i>) transgenic mice were incubated with 16.7 mM glucose, and analyzed for insulin release in the supernatant at the indicated time intervals. Mean±SD of replicates.</p

Deregulated PDX-1 expression in Ran-targeted cells.

<p><i>A</i>, Pancreas sections from non-TG, Ran-WT, Ran-G19V or Ran-T24N transgenic mice were stained with an antibody to insulin or PDX-1, by immunohistochemistry. <i>B</i>, The number of insulin- or PDX-1-stained cells was quantified by morphometry in the indicated surface area. Insulin⁺ cells, non-TG (n = 2), 28.2±0.4; Ran-WT (asymptomatic, n = 3), 29.8±1.2; Ran-WT (diabetic, n = 3), 6.2±0.6, ***, p<0.0001; PDX-1⁺ cells, Non-TG (n = 2), 34.2±4.1; Ran-WT (asymptomatic, n = 3), 37.5±2.3; Ran-WT (diabetic, n = 3), 22.8±1.8, **, p = 0.007. <i>C</i>, Islets from 2 mo-old non-TG or a Ran-WT transgenic mouse were analyzed by Western blotting. <i>D</i>, Pancreatic islets isolated from non-TG mice were transduced <u>ex vivo</u> with control pAd-GFP or pAd-GFP-Ran-WT, pAd-GFP-Ran-G19V or pAd-GFP-T24N and analyzed after 48 h by fluorescence microscopy for GFP expression (<i>left</i>), or Western blotting (<i>right</i>). *, non-specific. <i>E</i>, INS-1 cells were left untreated (INS-1) or transduced with control lentivirus (pLKO) or lentivirus encoding Ran-directed shRNA (Ran, 74V1), and analyzed by Western blotting. <i>F</i>, Parental INS-1 cells or four independent clones of INS-1 cells stably transduced with Ran-directed shRNA (77V-2, 74V-1, 75V-1, 75V-2) were analyzed for changes in insulin release in the supernatant. Representative experiment out of at least two independent determinations.</p

SU9518 selectively inhibits PDGFR.

<p>SU9518 selectively inhibits PDGFR.</p

Supplementary Data from Global Targeting of Subcellular Heat Shock Protein-90 Networks for Therapy of Glioblastoma

Supplementary Data from Global Targeting of Subcellular Heat Shock Protein-90 Networks for Therapy of Glioblastom