J. Biol. Chem., Vol. 282, Issue 52, 37429-37435

Luciferase Assay—Dual luciferase assay was performed in triplicate according to the instructions of the manufacturer (Promega). The pGL2-p21A luciferase reporter under the control of the two p53-responsive elements in the p21 promoter was used (29). Briefly, 100 ng of pGL2-p21A luciferase reporter, 100 ng of pcDNA3 or pcDNA3-HA-p53, and 5 ng of Renilla luciferase assay vector pRL-CMV (Promega) were co-transfected into H1299 or MCF7 cells. The -fold increase in relative luciferase activity is a product of the luciferase activity induced by p53 divided by that induced by an empty pcDNA3 vector.

Mol Cell Biol. 2005 April; 25(7): 2808–2818.

Fluorometric assay of caspase activity. Caspase activity was determined as described previously (11). Briefly, cell lysates were incubated with 50 μM fluorogenic caspase-3 substrate DEVD-AMC or the caspase-8 substrate IETD-AMC in 200 μl of buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 10% sucrose, 0.1% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, and 10 mM dithiothreitol. The release of aminomethylcoumarin was measured by fluorometry using an excitation wavelength of 360 nm and an emission wavelength of 475 nm.

Heart Rhythm. 2007 July; 4(7): 916–924

Signal-to-Noise Ratio Evaluation The endoscopic mapping system was compared with the direct mapping system by evaluating their respective signal-to-noise ratios (SNRs) during SR and AF. The direct mapping system consisted of the same camera and light source used for the endoscope but arranged in a conventional epifluorescence setting to image the PLA through a minimal LAA incision.10 In both approaches, signal levels for the respective SR and AF movies were determined as the peak-to-peak amplitude minus twice the noise level. On the other hand, noise levels were calculated as the standard deviation of the peak-to-peak amplitude during quiescent episodes of background subtracted movies for SR, and of background and sequentially subtracted movies for AF.9 Pixel-by-pixel SNRs were combined in maps generated for both unfiltered and filtered (see above) background subtracted data. The SNR of the direct system was determined for movies during both SR and cholinergic AF (0.5 μM ACh) and analyzed as described above. SNR histograms for the pixels in the maps were generated and average SNR values were calculated for the full width half height (FWHH) range.

Heart Rhythm. 2007 July; 4(7): 916–924

Endoscopic Fluorescence Mapping Set-up The cardio-endoscopic mapping system is schematically described in Figure 1. Its principal component is a dual-channel flexible and steerable endoscope. To achieve fluorescence mapping of cardiac impulses the endoscope is coupled to an excitation 532 nm Laser (1–5 W, CW Diode-pumped, Millenia Pro 5sJ, Spectra Physics, Inc.) at the proximal end of the illuminating channel (green arrow) and to a 14 bit CCD camera (SciMeasure, Inc) with a 2×2 mm2 chip size. The camera is C-coupled with a 12 mm, 1:1.4 maximal N.A. and 2/3” diagonal field focusing lens to a 645±50 nm band-pass filter and to the eyepiece of the imaging channel (red arrow). The following endoscopes were chosen for mapping the different regions of the LA: (i) a sigmoidoscope (Pentax, Inc., FS-34P2) of 11.5 mm diameter, 120° field of view and 63 cm working length. This endoscope features a deflectable direct view tip (Figure 1, upper right panel) with angulations of 180°/180° (up/down) and 160°/160° (right/left) or (ii) a therapeutic duodenoscope (Olympus, Inc. JF1T) of 11.0 mm diameter, 80° field of view and 103 cm working length. This endoscope features a deflectable side view tip (Figure 1, lower right panel) with angulations of 130°/130° up/down and 90°/90° right/left. Endoscopes showed transmittance of about 13% and 11%, respectively, as assessed by a 532 nm laser input in the range of 0.2–5W with a digital power meter (FieldMaster-GS, Coherent, Inc.). Figure 1 Left, Experimental set-up showing the dual-channel cardio-endoscope inserted in the LA though a minimal left ventricular opening and across the mitral valve (MV). Right, Deflectable direct-view and side-view tips and the corresponding working (W), light (more ...)

EMBO J. 2008 January 23; 27(2): 373–383.

Luciferase assay NF-κB activation was determined using 0.5 × 105 HEK293T cells transfected with expression plasmids in the presence of reporter plasmids, NF-κB-dependent pBxIV-luc and control pEF1BOS-β-gal, as described (Inohara et al, 2000; Kobayashi et al, 2002).

hepatology. 2008 june 47(6): 1983–1993

Fluorescence Microscopy HSCs were cultured on glass slides and exposed to experimental conditions. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X for 5 minutes. Blocking was performed with 3% milk and 10% goat serum. Primary antibody (3-nitrotyrosine, Cayman) was incubated at room temperature for 2 hours. Alexafluor 488-tagged secondary antibody was incubated for 2 hours at room temperature. Cells were analyzed with fluorescent microscopy using a Zeiss LSM confocal microscope.

J. Clin. Invest. 118(1): 173-182 (2007).

Immunofluorescence. Frozen 10-μm-thick cryosections were used. The sections were allowed to thaw at room temperature for at least 2 h and then fixed in cold acetone for 2 min. Primary anti-ZO-1 (1:50), anti-CD31 (1:500), or anti-occludin (1:10) antibodies were used. After 3 5-min washings, sections were incubated with a mixture of FITC goat anti-rabbit antibody (1:50) and streptavidin (1:500) for 1 h in 1% blocking buffer. After incubation for 15 min in Hoechst stain, the sections were washed and examined under the microscope.

J. Clin. Invest. 118(1): 133-148 (2007).

Laser-scanning confocal microscopy for double-immunofluorescence analysis. To identify the coexpression of cell type–specific markers in SN-immunoreactive GFP+ and BrdU+ cells, immunofluorescent colocalization study with 3D images was performed to test for the expression of GFAP, α-SMA, vWF, MAP-2, Musashi-1, and Neu-N. The double-immunofluorescence technique with specific antibodies against BrdU (1:400; Mannheim), GFAP (1:400; Sigma-Aldrich), MAP-2 (1:200, Boehringer Mannheim), Nestin (1:400, Sigma-Aldrich), Neu-N (1:200, Chemicon), vWF (1:400, Sigma-Aldrich), Musashi-1 (1:100, Serotec) and SMA (1:100, BD Pharmingen) conjugated with Cy3, Cy5, or FITC (1:500, Jackson Immunoresearch) has been described previously (14). The tissue sections were analyzed with a Carl Zeiss LSM510 laser-scanning confocal microscope.

J. Clin. Invest. 118(1): 40-50 (2007)

Adrenomedullin signaling is necessary for murine lymphatic vascular developmentJ. Clin. Invest. Kimberly L. Fritz-Six, et al. 118:40 doi:10.1172/JCI33302 [Go to this article.] Figure 8AM signaling preferentially mediates enhanced ERK activation in HMVEC-dLys compared with HUVECs. (A) Cultured HMVEC-dLys and HUVECs are morphologically and genetically distinct cell lines based on histology and expression pattern of Prox1. Original magnification, ×400. (B) Stimulation of HUVECs and HMVEC-dLys with the potent growth factor VEFGA resulted in a dose-dependant increase in cell proliferation that was not significantly different between the 2 cell lines. Data represent averages of 4 independent experiments, each performed in duplicate. (C) Stimulation of HUVECs and HMVEC-dLys with AM peptide resulted in a dose-dependant increase in cell proliferation that was significantly greater in HMVEC-dLys compared with HUVECs. *P < 0.05. Data represent averages of 4 independent experiments, each performed in duplicate. (D) Stimulation of HUVECs and HMVEC-dLys with 10 nM AM peptide resulted in a significantly greater induction of ERK phosphorylation in HMVEC-dLys compared with HUVECs over a 30-minute time course. *P < 0.03 at 15- and 20-minute time points. Data represent averages of 3 independent experiments, each performed in duplicate. (E) Induction of ERK activation by AM stimulation in HMVEC-dLys was significantly reduced by the RAMP2-specific peptide inhibitor AM22-52 and completely blocked by the MAPK inhibitor PD98057 (PD). *P < 0.05 compared with untreated; #P < 0.05 compared with AM-treated. Data represent averages of 3 independent experiments, each performed in duplicate.

PLoS Med. 2006 October; 3(10): e420.

Luciferase AssayH838 cells stably expressing NQO1-ARE luciferase were seeded onto a 24-well dish at a density of 0.2 × 106 cells/ml for 12 h before transfection. WT-KEAP1 cDNA constructs along with the mutant cDNA constructs (G333C and L413R) were transfected into the cells along with pRL-TK plasmid expressing Renilla luciferase as a transfection control. Twenty-four hours after transfection, cells were lysed and both firefly and Renilla luciferase activities were measured with a Dual-Luciferase Reporter Assay System (Promega).

The Journal of Neuroscience, July 4, 2007, 27(27):7083-7093

Double-label immunohistochemistryFor identification of the cell types producing ROS, mice treated with hydroethidine were anesthetized with sodium pentobarbital (120 mg/kg) and perfused transcardially with 4% paraformaldehyde. Brains were removed, frozen, and sectioned through the parietal cortex. Brain sections were incubated with antibodies against the neuronal marker NeuN (1:100; Millipore), the endothelial cell marker CD31 (1:100; BD Biosciences, San Diego, CA), or the astrocytic marker glial fibrillary acidic protein (GFAP) (1:1000; Sigma-Aldrich). Sections were then incubated with cyanine dye (Cy5)-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA). For identification of the cell types expressing 3-NT, frozen sections were first processed for 3-NT immunocytochemistry, followed by incubation with anti-NeuN, anti-CD31, or anti-GFAP antibodies, and, subsequently, Cy5-conjugated secondary antibodies. The specificity of the immunolabel was assessed by omitting the primary antibodies or by preadsorption with the antigen (Forster et al., 1999). Images of double-labeled neocortex were sequentially acquired using a Leica (Mannheim, Germany) TCS SP5 confocal laser-scanning microscope. ROS and 3-NT signals were pseudocolored red, whereas Cy5 signals were pseudocolored green.

Reproductive Biology and Endocrinology 2008, 6:49

Immunofluorescence For PBX1 and MEIS1/2, cells were grown on glass coverslips, fixed in freshly prepared 4% paraformaldehyde in PBS for 10 min at 4°C, and permeabilized with 0.1% Triton X-100 in PBS for 10 min at 25°C. For PBX2, cells on the coverslips were fixed in cold methanol and postfixed in cold methanol/acetone (1:1). The cells were incubated with primary antibodies, anti-PBX1 (1:20), anti-PBX2 (1:50) and anti-MEIS1/2 antibody (1:50). The binding of primary antibodies was followed by goat anti-mouse antibody (Alexa Fluor 594, Molecular Probes, Eugene, Oregon) or rabbit anti-goat antibody (Alexa Fluor 488, Molecular Probes). Control experiments were done in the absence of primary antibodies and verified their no or little background. For Hoechst staining, after incubation of the secondary antibodies, cells were incubated for 1 min with 5 μg/ml Hoechst 33258.

Am J Pathol. 2008 July; 173(1): 68–76.

Figure 3 Egr-1 regulates genes involved in gliosis. A: Egr-1 loss-of-function effects on human astrocytes transfected with siRNAs against Egr-1. RT-PCR analysis shows that knockdown of Egr-1 leads to reduction in the expression levels of genes encoding ECM components of the glial scar. c, control mock-transfected cells; −, Egr-1 knockdown. DNA size markers are shown on the left. Gene name abbreviations are as follows: CSPG2, 3, 4, chondroitin sulfate proteoglycan 2, 3, 4, respectively; Lamα1, laminin α1; Lamα2: laminin α2; Lamβ1: laminin β1. Real-time PCR quantification of the effects of the Egr-1 knockdown on the expression of putative gene targets shows a 60% drop in phosphacan RNA levels in Egr-1 siRNA-treated astrocytes. Adjusted for aldolase, relative phosphacan expression was 12.96 ± 1.21 U in controls versus 5.05 ± 0.89 U after Egr-1 knockdown (n = 5, P < 0.001). B–E: Immunofluorescence analysis after Egr-1 overexpression in astrocytes transfected with the CMV-Egr-1-IRES-EGFP construct. Transfected, EGFP-positive cells (green, marked by arrows), stain more intensely with antibodies recognizing laminin α1 (B, red) and phosphacan (C, red, anti-RPTPβ) than nontransfected neighboring cells. No difference in expression levels of GFAP (D, red) or β-tubulin (E, red) between Egr-1-overexpressing cells (green, marked by arrows) and nontransfected cells. Superimposed images (far right) confirm that transfected cells express higher levels of putative Egr-1 targets, but similar levels of other proteins. F: Western blotting of proteins isolated from astrocytes transfected with siRNAs against Egr-1 (Egr-1 siRNA), control siRNA (scrambled siRNA), or mock-transfected cells (c). siRNAs against Egr-1 diminish effectively Egr-1 protein levels and lead to down-regulation of phosphacan (Pcan, detected with the KAF13 antibody). β-Tubulin (β-Tub) levels remain unaffected serving as control. Quantification of blot images shows that Egr-1 protein levels are reduced 3.0-fold versus control (mock-transfected cells; SD, 0.4) and 2.8-fold (SD, 0.2) versus scrambled siRNA-transfected cells; phosphacan protein levels are down-regulated 2.18-fold versus control (mock; SD 0.1) and 2.23 times (SD 0.1) versus scrambled siRNA. G: Western blotting of proteins isolated from astrocytes transfected with the CMV-Egr-1-IRES-EGFP (Egr-1 cDNA) construct or the empty vector (c). Egr-1 protein levels increase 2.08-fold (SD, 0.4) leading to 1.42-fold up-regulation of phosphacan (Pcan; SD, 0.14). β-Tubulin (β-Tub) serves as control. Am J Pathol. 2008 July; 173(1): 77–92.

Am J Pathol. 2008 July; 173(1): 68–76.

Figure 2 Egr-1 expression in adult mouse brain after cerebral ischemia. A: Cerebral ischemia was induced by permanent occlusion of the MCA. The infarcted tissue appears white in the TTC-stained brain slice (inset). Immunohistochemistry using anti-Egr-1 antibody 4 days after MCAO detects Egr-1 expression in cells within the infarct area (arrows) and around the infarct border zone (star). B–D: Immunofluorescence analysis of brain tissue sections stained with anti-Egr-1 and GFAP antibodies 4 days after MCAO. Egr-1-expressing cells in the border zone (B, green color) stain positive for GFAP (C, red color). D: Double-labeled astrocytic cells appear yellow/orange in the superimposed images. E–G: High Egr-1 expression persists in cells around the injury site 6 weeks after infarction. Low-magnification images reveal strong Egr-1 expression (green) in cells accumulating around the infarct region (E) compared with the corresponding area of the contralateral hemisphere (F), or the ipsilateral area of sham-operated animals (G). H–J: Confocal microscopy images of brain tissue sections stained with anti-Egr-1 and GFAP antibodies 6 weeks after MCAO. Egr-1-positive cells (H, green) around and within the glial scar stain positive for GFAP (I, red). Double-labeled astrocytes appear yellow/orange (J). K and L: Confocal microscopy images of brain tissue sections stained with anti-Egr-1 antibody. K: The number of Egr-1-positive cells (in green) around the ventricles of infarcted hemispheres increases after MCAO (L, arrows) compared with contralateral controls. M: Western blotting detects higher levels of Egr-1 protein in scar tissue isolated from infarcted (left, l) hemisphere as compared with corresponding noninfarcted area of the contralateral side (right, r). β-Actin protein levels serve as control. Sham-operated animals have comparable Egr-1 amounts on both brain sides. Quantitative image analysis shows that Egr-1 protein levels are 2.3-fold higher in the peri-infarct region compared with control tissue (SD, 0.3), whereas sham-operated animals show equivalent amounts of Egr-1 protein in the corresponding areas of both hemispheres (0.98-fold difference; SD, 0.12). i: infarct; dotted lines demarcate infarct and peri-infarct areas. Scale bars: 100 μm (A, E–G, K, L); 25 μm (B–D, H–J). Am J Pathol. 2008 July; 173(1): 77–92.

Am J Pathol. 2008 July; 173(1): 30–41.

Figure 7 HGF does not block p65 NF-κB nuclear translocation but abolishes its binding to the cognate cis-acting element in RANTES promoter. A and B: HGF did not block the p65 NF-κB nuclear translocation induced by TNF-α. HKC-8 cells were treated with TNF-α or/and HGF as indicated. A: Representative micrographs showed the staining for p65 NF-κB after various treatments. B: The percentages of cell population with p65 nuclear translocation after various treatments are given. *P < 0.05 versus control. C: ChIP assay demonstrated that HGF signaling abolished the binding of p65 to its cis-acting element in the RANTES promoter. Marker, DNA size markers; IgG, precipitated with control IgG.