Journal: Antioxidants

Article Title: A Preliminary Study on the Effect of Hydrogen Gas on Alleviating Early CCl 4 -Induced Chronic Liver Injury in Rats

doi: 10.3390/antiox10121933

Figure Lengend Snippet: Changes in the expression of uncoupling protein 2 (UCP2) were observed by immunohistochemistry. ( a , b ) The expression of UCP2 in liver tissue of control group rats. A small number of UCP2-positive cells were brownish yellow in the cytoplasm. ( c , d ) The expression of UCP2 in liver tissue of CCl 4 group rats. A small number of UCP2 positive cells were brownish yellow in the cytoplasm. ( e , f ) The expression of UCP2 in liver tissue of CCl 4 + H 2 group rats. UCP2 was diffusely expressed in liver cells, and the staining sites were mostly located on the cell membrane. ( g , h ) The expression of UCP2 in liver tissue of H 2 group rats. The number of UCP2 positive cells with brownish-yellow staining in the cytoplasm increased significantly. ( a , c , e , g ) Fields of vision at 40× magnification. ( b , d , f , h ) Higher magnifications of the area outlined in ( a , c , e , g ). The long scale bar refers to 100 μm. The short scale bar refers to 50 μm.

Article Snippet: The membrane was incubated with antibodies of goat anti-UCP2 (bs-20750R, Bioss, Beijing, China) and mouse anti-β actin (A2228, Sigma, St. Louis, MO, USA) overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Antioxidants

Article Title: A Preliminary Study on the Effect of Hydrogen Gas on Alleviating Early CCl 4 -Induced Chronic Liver Injury in Rats

doi: 10.3390/antiox10121933

Figure Lengend Snippet: UCP2-positive area rate. N = 8, * p < 0.05.

Article Snippet: The membrane was incubated with antibodies of goat anti-UCP2 (bs-20750R, Bioss, Beijing, China) and mouse anti-β actin (A2228, Sigma, St. Louis, MO, USA) overnight at 4 °C.

Techniques:

Journal: Antioxidants

Article Title: A Preliminary Study on the Effect of Hydrogen Gas on Alleviating Early CCl 4 -Induced Chronic Liver Injury in Rats

doi: 10.3390/antiox10121933

Figure Lengend Snippet: Expression changes of UCP2 (Western blotting). ( a ) Representative western blotting image of UCP2. ( b ) Statistical results of western blotting. N = 8, * p < 0.05.

Article Snippet: The membrane was incubated with antibodies of goat anti-UCP2 (bs-20750R, Bioss, Beijing, China) and mouse anti-β actin (A2228, Sigma, St. Louis, MO, USA) overnight at 4 °C.

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

doi: 10.1371/journal.pone.0032349

Figure Lengend Snippet: A , PCs and NPCs were isolated from the liver tissues of C57 and AKO mice. The protein (left and middle panel) and mRNA (right panel) expression of UCP2 was measured by Western blotting and QPCR, respectively. SSBP-1 was used as the protein loading control and β-actin as the internal control for quantifying gene expressions. QPCR results were plotted as fold changes against the C57 PC samples. *, P <0.05 vs C57 NPC samples, n = 3. B , AKO mice were treated with adenoviruses encoding luciferase (Luci) or adiponectin (ADN). UCP2 protein (left and middle panel) and mRNA (right panel) levels in PCs and NPCs were analyzed as above. QPCR results were presented as fold changes against the Luci PC controls. *, P <0.05 vs corresponding controls, n = 3.

Article Snippet: Protein lysates were heated at 95°C for 5 min, separated by SDS-PAGE, and transferred to PVDF membrane for immunoblotting with the specific antibodies against UCP2 (R&D Systems, #AF4739), single-strand binding protein-1 (SSBP-1, Santa Cruz, #sc-34727), liver sinusoidal endothelial cell marker SE-1 (Novus Biologicals, NB110-68095), F4/80 (Abcam, #ab6640), hnRNP K (Santa Cruz, #sc-25373) or β-actin (Sigma, #A5316).

Techniques: Isolation, Expressing, Western Blot, Control, Luciferase

Journal: PLoS ONE

Article Title: Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

doi: 10.1371/journal.pone.0032349

Figure Lengend Snippet: PBS or 50 µg of murine adiponectin protein was injected into the portal vein of AKO mice livers. Liver tissues were collected for evaluation of UCP2 expression. The protein abundance in mitochondria (A), mRNA expression in total tissue lysates (B), and the protein content in PC and NPC fractions (C) were analyzed as in . *, P <0.05 vs vehicle treated samples, n = 3.

Article Snippet: Protein lysates were heated at 95°C for 5 min, separated by SDS-PAGE, and transferred to PVDF membrane for immunoblotting with the specific antibodies against UCP2 (R&D Systems, #AF4739), single-strand binding protein-1 (SSBP-1, Santa Cruz, #sc-34727), liver sinusoidal endothelial cell marker SE-1 (Novus Biologicals, NB110-68095), F4/80 (Abcam, #ab6640), hnRNP K (Santa Cruz, #sc-25373) or β-actin (Sigma, #A5316).

Techniques: Injection, Expressing, Quantitative Proteomics

Journal: PLoS ONE

Article Title: Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

doi: 10.1371/journal.pone.0032349

Figure Lengend Snippet: AKO mice were treated as in . The NPCs were used for further fractionation to collect those enriched with Kupffer (K)- and sinusoidal endothelial (E) cells. The enrichment of the two cell types were confirmed by Western blotting using macrophage marker F4/80 and sinusoidal endothelial marker SE-1, respectively (A). UCP2 expression was monitored as in . After densitometry analysis, the protein ratio of UCP2/β-actin was calculated and presented as fold changes against Luci Kupffer samples (B). UCP2 gene expression was also quantified in four types of cells treated with or without adiponectin (10 µg/ml) (C). *, P <0.05 and **, P <0.01 vs corresponding controls, n = 3.

Article Snippet: Protein lysates were heated at 95°C for 5 min, separated by SDS-PAGE, and transferred to PVDF membrane for immunoblotting with the specific antibodies against UCP2 (R&D Systems, #AF4739), single-strand binding protein-1 (SSBP-1, Santa Cruz, #sc-34727), liver sinusoidal endothelial cell marker SE-1 (Novus Biologicals, NB110-68095), F4/80 (Abcam, #ab6640), hnRNP K (Santa Cruz, #sc-25373) or β-actin (Sigma, #A5316).

Techniques: Fractionation, Western Blot, Marker, Expressing, Gene Expression

Journal: PLoS ONE

Article Title: Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

doi: 10.1371/journal.pone.0032349

Figure Lengend Snippet: Two inhibitors, actinomycin D (ActD, A, B and C) or cycloheximide (CHX, A, B and D), were administered together with or without adiponectin protein into liver tissues of AKO mice livers. The protein (A) and mRNA (B) abundance of UCP2 was monitored by Western blotting and QPCR, respectively. The relative protein expression was also monitored in PCs/NPCs lysates (C and D). *, P <0.05 vs corresponding controls, n = 3.

Article Snippet: Protein lysates were heated at 95°C for 5 min, separated by SDS-PAGE, and transferred to PVDF membrane for immunoblotting with the specific antibodies against UCP2 (R&D Systems, #AF4739), single-strand binding protein-1 (SSBP-1, Santa Cruz, #sc-34727), liver sinusoidal endothelial cell marker SE-1 (Novus Biologicals, NB110-68095), F4/80 (Abcam, #ab6640), hnRNP K (Santa Cruz, #sc-25373) or β-actin (Sigma, #A5316).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

doi: 10.1371/journal.pone.0032349

Figure Lengend Snippet: Mitochondrial respiration chain inhibitors, including rotenone (Rot), sodium azide (NaN 3 ) and antimycin A (AntA) were administered into liver tissues of AKO mice as described in . The effects of each inhibitor on adiponectin-induced UCP2 protein expression in mitochondria of liver tissues (A), UCP2 mRNA expression in total tissue lysates (B) were evaluated. The mitochondria protein content of UCP2 (C) and hnRNPK (D) in PCs and NPCs were compared by Western blotting. *, P <0.05 vs corresponding controls, n = 3.

Article Snippet: Protein lysates were heated at 95°C for 5 min, separated by SDS-PAGE, and transferred to PVDF membrane for immunoblotting with the specific antibodies against UCP2 (R&D Systems, #AF4739), single-strand binding protein-1 (SSBP-1, Santa Cruz, #sc-34727), liver sinusoidal endothelial cell marker SE-1 (Novus Biologicals, NB110-68095), F4/80 (Abcam, #ab6640), hnRNP K (Santa Cruz, #sc-25373) or β-actin (Sigma, #A5316).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

doi: 10.1371/journal.pone.0032349

Figure Lengend Snippet: A schematic summary: Adiponectin-evoked transient elevation of mitochondrial O 2• − serves as a trigger for the translocation hnRNP K, which subsequently promotes the stabilization of UCP2 mRNA and its protein synthesis in mitochondria of hepatic endothelial cells.

Article Snippet: Protein lysates were heated at 95°C for 5 min, separated by SDS-PAGE, and transferred to PVDF membrane for immunoblotting with the specific antibodies against UCP2 (R&D Systems, #AF4739), single-strand binding protein-1 (SSBP-1, Santa Cruz, #sc-34727), liver sinusoidal endothelial cell marker SE-1 (Novus Biologicals, NB110-68095), F4/80 (Abcam, #ab6640), hnRNP K (Santa Cruz, #sc-25373) or β-actin (Sigma, #A5316).

Techniques: Translocation Assay

Journal: Molecular Medicine Reports

Article Title: Modulation of fatty acid metabolism is involved in the alleviation of isoproterenol-induced rat heart failure by fenofibrate

doi: 10.3892/mmr.2015.4466

Figure Lengend Snippet: Expression of PPARα and UCP2. (A) Neonatal rat cardiomyocytes; (B) rat myocardial tissue. Samples from the CON, FF+ISO and ISO groups were analyzed (n=10/group). CON, control; FF, fenofibrate; ISO, isoproterenol; PPAR, peroxisome proliferator-activated receptor; UCP2, uncoupling protein 2.

Article Snippet: Horseradish peroxidase (HRP)-conjugated rabbit anti-rat PPARα (cat. no. sc-130640) and goat anti-rat UCP2 (cat. no. sc-390189) primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Control

Journal: Critical Care Medicine

Article Title: Renal Tubular Cell Mitochondrial Dysfunction Occurs Despite Preserved Renal Oxygen Delivery in Experimental Septic Acute Kidney Injury

doi: 10.1097/CCM.0000000000002937

Figure Lengend Snippet: Renal uncoupling protein by Western blot and urine isoprostane levels. Circles and squares represent individual data points in the scatter plot. A , Uncoupling protein-2 (UCP-2) in kidney homogenate from sham-operated and 24-hour septic animal. The Western blot demonstrates a clear increase in renal UCP-2 expression in septic animals (1.56-fold increase) compared with sham-operated animals ( p = 0.05). B , UCP-2 in kidney slice exposed to sham and septic serum. The Western blot demonstrates a modest increase in renal UCP-2 expression in live kidney slices exposed to septic serum (1.11-fold increase) compared with sham serum ( p = 0.10). C , Urine isoprostane levels from sham-operated and septic animals at 24 hours. There is an increase in urine isoprostane levels in septic animals compared with sham-operated animals at 24 hours, which approaches statistical significance ( p = 0.06).

Article Snippet: This membrane was then incubated with goat anti-mouse immunoglobulin-G polyclonal uncoupling protein (UCP-2) antibody (Santa Cruz, Dallas, TX) at 1:250 in 5% bovine serum albumin (BSA; Sigma) in phosphate-buffered saline overnight at 4 o C. Following incubation with the primary antibody, horseradish peroxidase rabbit anti-goat IgG (Sigma) was added at 1:3000 in 5% milk for 1 hour.

Techniques: Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: Negative regulation of UCP2 by TGF β signaling characterizes low and intermediate-grade primary breast cancer

doi: 10.1038/cddis.2010.30

Figure Lengend Snippet: UCP2 immunohistochemistry of primary invasive breast cancer

Article Snippet: Tissue arrays comprised of formalin-fixed primary breast cancer samples obtained from the Department of Pathology, California Pacific Medical Center under IRB-approved guidelines, were deparaffinized and used for immunoperoxidase localization of a goat anti-human UCP2 antibody (cat. no. C20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the supplier-recommended protocol, and counterstained with nuclear fast red.

Techniques: Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Negative regulation of UCP2 by TGF β signaling characterizes low and intermediate-grade primary breast cancer

doi: 10.1038/cddis.2010.30

Figure Lengend Snippet: UCP2 is differentially expressed by primary breast cancer of varying differentiation status. ( a ) Distinctive UCP2 expression patterns in four representative cases each of grade 3 and grade 1 primary breast cancer. Immunoperoxidase signal (blue) in sections of tissue cores (top 3 panels) and full-sized blocks (bottom panels at higher magnification, Bar=25 μ m). Red, nuclear counter stain. ( b ) Top – UCP2 transcript levels in primary tumor cell lines of all grades analyzed with QPCR probes labeled with SYBR Green (open bars), or FAM (solid bars). Bottom – western blot analysis reflects variation in UCP2 transcript levels shown above in bar graph. Tubulin, protein-loading control. ( c ) UCP2 immunofluorescence (green) in primary breast cancer cell lines evaluated in panel b . Nuclei counterstained with propidium iodide (red). Bar=25 μ m. ( d ) Cell polarity in Matrigel examined by immunostaining for the basal membrane marker, α 6-integrin (blue signal). Actin, a polarity-independent control, was localized with phalloidin (green). Nuclei were counterstained with propidium iodide. UCP2 knockdown of grade 3 tumor cells (CCdl672) propagated in Matrigel restored polarized growth as shown in right hand panels. Bar=25 μ m. ( e ) Quantitation of polarity induction by UCP2 knockdown. Apolar structures were defined by random nuclear orientation and individual α 6-integrin encircled cells. Polarized structures were characterized by redistribution of α 6-integrin around the periphery of an entire colony. Bar graph represents the average of polarized and apolar grade 3 (CCdl672) colonies of control versus UCP2 knockdown cells in Matrigel per × 10 field in four independent experiments. Asterisks indicate statistical significance ( P <0.05)

Article Snippet: Tissue arrays comprised of formalin-fixed primary breast cancer samples obtained from the Department of Pathology, California Pacific Medical Center under IRB-approved guidelines, were deparaffinized and used for immunoperoxidase localization of a goat anti-human UCP2 antibody (cat. no. C20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the supplier-recommended protocol, and counterstained with nuclear fast red.

Techniques: Expressing, Staining, Labeling, SYBR Green Assay, Western Blot, Control, Immunofluorescence, Immunostaining, Membrane, Marker, Knockdown, Quantitation Assay

Journal: Cell Death & Disease

Article Title: Negative regulation of UCP2 by TGF β signaling characterizes low and intermediate-grade primary breast cancer

doi: 10.1038/cddis.2010.30

Figure Lengend Snippet: UCP2 expression in breast cancer cells is suppressed by TGF β in a histological grade-dependent manner. ( a ) TGF β inhibits cell proliferation in grade 1 and 2 but not in grade 3-derived breast cancer cell lines. Left panel – cell proliferation measured by BrdU incorporation and FACS analysis. Averages taken across three independent experiments. Asterisks indicate statistical significance ( P <0.05). Right panel – morphological changes induced by TGF β exposure. Dramatic effects are exemplified in a grade 1 cell line (CCdl68). Such effects were not observed in grade 3 cell lines (CCdl54 shown). ( b ) Transcript levels of UCP2 and other TGF β target genes were induced or repressed in TGF β -treated grade 1 and 2 tumor cells only. Data for each test gene were normalized to the reference gene, ACTB . Expression of additional housekeeping genes, GAPDH , G6PD and ALDOA is also shown. Each data point is the average of triplicate values; error bars denote standard deviation; and asterisks denote P <0.05. Open bars, no treatment control; filled bars, TGF β treatment. Sky blue, grade 1 cell line (CCdl68); navy blue, grade 2 cell line (CCdl78); pink, grade 3 cell line (CCdl54); purple, grade 3 cell line (CCdl672). ( c ) Exogenously induced UCP2 inhibits effects of TGF β on grade 1 and 2 tumor cell lines. UCP2 -transfected cells (CCdl66) were analyzed after 5 days of exposure to 4 ng/ml TGF β . Representative phase contrast images show UCP2-mediated inhibition of TGF β -induced morphological changes. Bar=50 μ m. ( d ) Top panel – diagrammatic representation of the human UCP2 promoter showing five potential RSBE-containing regions and their respective coordinates with respect to the transcriptional start site. The figures in parentheses represent the number of potential RSBE sites in each region. Bottom panel – ChIP assay shows SMAD4 binding to multiple regions in the UCP2 promoter in a grade 1 (CCdl68) cell line. Maximum binding was observed in region 5. M, 100 base pair ladder; I, input DNA; C, control IgG immunoprecipitation; IP, anti-SMAD4 immunoprecipitation. ( e ) Left panel – TGF β -mediated UCP2 repression is compromised by SMAD4 knockdown. QPCR analysis shows significant alleviation of UCP2 repression in SMAD4 siRNA-transfected cell lines. Open bars – grade 1 cell line (CCdl68); solid bars – grade 2 cell line (CCdl78). Each data point is the average of triplicate values; error bars denote standard deviation. Asterisks indicate that fold change in expression between control and treated samples is statistically significant ( P <0.05). Right panel – Effect of SMAD4 knockdown on SMAD4 and UCP2 protein levels. Cells transfected with control or smart pool SMAD4 siRNA and protein extracts analyzed 48 h later by western blotting. Partial (50%) reduction in SMAD4 protein resulted in enhanced UCP2 levels. Actin, loading control

Article Snippet: Tissue arrays comprised of formalin-fixed primary breast cancer samples obtained from the Department of Pathology, California Pacific Medical Center under IRB-approved guidelines, were deparaffinized and used for immunoperoxidase localization of a goat anti-human UCP2 antibody (cat. no. C20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the supplier-recommended protocol, and counterstained with nuclear fast red.

Techniques: Expressing, Derivative Assay, BrdU Incorporation Assay, Standard Deviation, Control, Transfection, Inhibition, Binding Assay, Immunoprecipitation, Knockdown, Western Blot

Journal: Cell Death & Disease

Article Title: Negative regulation of UCP2 by TGF β signaling characterizes low and intermediate-grade primary breast cancer

doi: 10.1038/cddis.2010.30

Figure Lengend Snippet: Enhancement of mitochondrial membrane potential (Δ ψ m) by UCP2 is an underlying factor for apoptosis inhibition in tumor cells. ( a ) UCP2 inhibits apoptosis by blocking CCCP-mediated uncoupling and mitochondrial depolarization. siRNA-transfected grade 3 cell lines 20 h after recovery from 250 μ M CCCP treatment display Annexin V–FITC staining by FACS analysis. Apoptotic index represents ratio of CCCP-induced apoptosis: baseline apoptosis. Each experiment was carried out in triplicate; asterisks represent significance between control and UCP2 knockdown cultures ( P <0.05). Reduction in UCP2 protein levels are shown in the bottom panel. ( b ) UCP2 knockdown reduces Δ ψ m and enhances apoptosis. Bivariate JC-1 FACS analysis of Δ ψ m and apoptosis in grade 3 (CCdl675) cells, 24 h after UCP2 knockdown. The y axis represents Δ ψ m, calculated as the ratio of JC-1 red fluorescence: JC-1 green fluorescence. Mitochondrial depolarization and subsequent apoptosis induction are indicated by decreased red: green fluorescence intensity ratios. Average of triplicate values shown; asterisk represents significance ( P <0.05) when compared with NS siRNA control. ( c ) Microscopic analysis of reduction in Δ ψ m by JC-1 staining of UCP2 siRNA-transfected grade 3 tumor cell lines. Tumor cells in glass-bottomed plates analyzed by live imaging 24 h after siRNA transfection. Cells with depolarized mitochondria exhibit reduced orange/red fluorescence. Bar=25 μ m. ( d ) UCP2 -RNA interference promotes both spontaneous and staurosporine-induced apoptosis in grade 3 (CCdl672) tumor cells propagated in Matrigel for 6 days after transfection. Cleaved caspase-3-associated fluorescence in cells treated with vehicle or 250 μ M CCCP. Apoptotic cells stained green, nuclei counterstained with propidium iodide (red). Bar=50 μ m. Bottom panel – cell counts derived from four × 20 microscopic fields of each treatment combination indicated. Asterisks represent significance ( P <0.05) when compared with untreated control. Differences between bar 4 versus bars 2 and 3 are also significant ( P <0.05). ( e ) Exogenous UCP2 induction in grade 1 and 2 tumor cell lines protects from CCCP-mediated mitochondrial depolarization. Top left – elevation of Δ ψ m in tumor cells stably transduced with exogenous UCP2 determined by bivariate JC-1 and FACS analysis. Bottom left – neutralization of CCCP-induced mitochondrial depolarization in cells shown in top panel. Data represents MFI of JC-1. Average of triplicate data shown; asterisks indicate significant differences ( P <0.05) when compared with vector-alone samples. Right panel – representative FACS dot plots of JC-1 Red ( y axis): JC-1 Green ( x axis) of data summarized in left panels

Article Snippet: Tissue arrays comprised of formalin-fixed primary breast cancer samples obtained from the Department of Pathology, California Pacific Medical Center under IRB-approved guidelines, were deparaffinized and used for immunoperoxidase localization of a goat anti-human UCP2 antibody (cat. no. C20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the supplier-recommended protocol, and counterstained with nuclear fast red.

Techniques: Membrane, Inhibition, Blocking Assay, Transfection, Staining, Control, Knockdown, Fluorescence, Imaging, Derivative Assay, Stable Transfection, Transduction, Neutralization, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Negative regulation of UCP2 by TGF β signaling characterizes low and intermediate-grade primary breast cancer

doi: 10.1038/cddis.2010.30

Figure Lengend Snippet: UCP2 promotes growth of tumor cells by regulating endogenous ROS. ( a ) Intrinsic total ROS levels of breast cancer cell lines of varying histological grade measured as MFI of C400 by FACS analysis. Error bars represent the standard deviation of triplicate samples, and asterisks indicate significance ( P <0.05) of comparisons between control and UCP2-silenced cells. Representative overlay histograms are shown for each cell line. ( b ) Enhancement of mitochondrial superoxide production in grade 3 UCP2 -KD tumor cells shown by live confocal imaging of dual staining with MitoSOX Red and MitoFluor Green. Note colocalization of both probes at sites of mitochondrial superoxide production. Bar=12.5 μ M. ( c ) Flow cytometric analysis of UCP2-regulated mitochondrial superoxide production. MitoSOX Red-stained live grade 3 tumor cells (CCdl54) were analyzed by FACS at 15, 20, 25 and 30 min after trypsinization (corresponding to 0, 5, 10 and 15 min measurements by time-lapse microscopic analysis). Significant differences in MitoSOX Red MFI were observed between control and UCP2 siRNA-transfected cells at 5, 10 and 15 min of antimycin A treatment ( * P <0.02, ** P <0.001). ( d ) UCP2 knockdown enhances oxidative stress-induced growth suppression, reversed by antioxidant treatment. Data represents UCP2 siRNA-transfected grade 3 cell line (CCdl675) treated with 500 μ M tert-butyl hydroperoxide (H 2 O 2 ) alone or in combination with 1 mM reduced glutathione (GSH) followed by the MTT cell growth assay. Each experiment was performed in six replicates. Asterisks indicate significant differences ( P <0.05) in comparison with peroxide alone treatment

Article Snippet: Tissue arrays comprised of formalin-fixed primary breast cancer samples obtained from the Department of Pathology, California Pacific Medical Center under IRB-approved guidelines, were deparaffinized and used for immunoperoxidase localization of a goat anti-human UCP2 antibody (cat. no. C20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the supplier-recommended protocol, and counterstained with nuclear fast red.

Techniques: Standard Deviation, Control, Imaging, Staining, Transfection, Knockdown, Growth Assay, Comparison

Journal: Cell Death & Disease

Article Title: Negative regulation of UCP2 by TGF β signaling characterizes low and intermediate-grade primary breast cancer

doi: 10.1038/cddis.2010.30

Figure Lengend Snippet: UCP2 promotes proliferation of tumor cells by regulating [Ca 2+ ]m (mitochondrial calcium). ( a ) Live cell confocal images showing steady state mitochondrial Ca 2+ levels in breast cancer lines of varying histological grade determined by dual staining with the [Ca 2+ ]m probe, Rhod-2 AM (red), and the mitochondrial dye, MitoFluor Green (green). Bar=12.5 μ m (upper panel) and 25 μ m (lower panel). Mitochondria displayed as green punctate stain. Mitochondrial calcium imaged as red punctate stain. ( b ) UCP2 induction results in a striking decline in [Ca 2+ ]m retention within grade 1 and 2 tumor cell lines. Top panel – [Ca 2+ ]m detected as described in panel c . Bar=25 μ m. Bottom panel – UCP2 induction in tumor cells confirmed by western blot. Tubulin, protein-loading control. ( c ) Time-lapse video microscopy of UCP2 -induced inhibition of [Ca 2+ ]m overload. Top panel – quantitative data acquired in real-time in vector versus UCP2 stably transduced grade 1 tumor cells (CCdl68). Bottom panel – microscopic images exemplifying real-time changes in Rhod-2 AM fluorescence within a single representative cell with (lower series) and without (upper series) UCP2 transduction at progressively increasing time points between 0 and 1 h. ( d ) UCP2 overexpression enhances proliferation. Comparison of cell cycle profiles of vector-transfected versus exogenous UCP2 -induced grade 2 cells with grade 3 cells. Cells were pulse labeled with BrdU to visualize DNA replicating fraction by FACS analysis. Representative data in pie charts show increased percentage of S-phase cells after UCP2 induction. Baseline cell cycle distribution of moderately and poorly differentiated tumor cells is distinctly unique reflecting their proliferation patterns. UCP2 transduction of grade 2 cells induces a striking resemblance to the proliferation pattern of grade 3 cells. Each experiment was run in triplicate. Difference in percentage of S-phase cells was significant ( P <0.05) between grade 1 and grade 3, and also between control versus UCP2 -transduced grade 2

Article Snippet: Tissue arrays comprised of formalin-fixed primary breast cancer samples obtained from the Department of Pathology, California Pacific Medical Center under IRB-approved guidelines, were deparaffinized and used for immunoperoxidase localization of a goat anti-human UCP2 antibody (cat. no. C20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the supplier-recommended protocol, and counterstained with nuclear fast red.

Techniques: Staining, Western Blot, Control, Microscopy, Inhibition, Plasmid Preparation, Stable Transfection, Fluorescence, Transduction, Over Expression, Comparison, Transfection, Labeling

Journal: Cell Death & Disease

Article Title: Negative regulation of UCP2 by TGF β signaling characterizes low and intermediate-grade primary breast cancer

doi: 10.1038/cddis.2010.30

Figure Lengend Snippet: Schematic representation of UCP2-mediated inhibition of tumor cell death and differentiation. Loss of TGF β signaling in histological grade 3 breast tumor cells promotes constitutive expression of UCP2, which facilitates maintenance of Δ Ψ m and restricts [Ca 2+ ]m accumulation leading to reduced mitochondrial ROS production, thereby augmenting tumor cell survival and proliferation. Conversely, in grade 1 tumors under microenvironmental stress, UCP2 transcriptional repression is maintained through SMAD-mediated TGF β signaling, which forces [Ca 2+ ]m overload and higher ROS levels, culminating in differentiation cues and/or cell death

Article Snippet: Tissue arrays comprised of formalin-fixed primary breast cancer samples obtained from the Department of Pathology, California Pacific Medical Center under IRB-approved guidelines, were deparaffinized and used for immunoperoxidase localization of a goat anti-human UCP2 antibody (cat. no. C20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the supplier-recommended protocol, and counterstained with nuclear fast red.

Techniques: Inhibition, Expressing