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  • 85
    Thermo Fisher leucine aminopeptidase lap
    Leucine Aminopeptidase Lap, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore acp2 acid phosphatase
    Figure 4. Pharmacological inhibition of BAX channel activity in MPP + -treated cells. ( A and B ) Enzymatic activities of ( A ) <t>ACP2</t> and ( B ) β-hexosaminidase in lysosomal-free cytosolic fractions from MPP + -treated SH-SY5Y cells, in the presence
    Acp2 Acid Phosphatase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioneer Corporation acp2
    <t>ACP2</t> is necessary for the replication of various wild-type influenza A and B viruses. A549 cells were transfected with siScr, siACP2, or siCSE1L. After 48 hours, cells were infected with the indicated viral strains at an MOI of 1 for 10 hours. The cells were stained with an anti-NP antibody and analyzed by confocal microscopy. Virus replication was determined by calculating the percentage of NP-expressing cells and normalized to that of the siScr-transfected cells. Bars show means ± SD from quadruplicated experiments. Statistical significance between the indicated groups was tested using the Student’s t-test with a threshold of *** p
    Acp2, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime acp2
    Autophagy impairment couples with lysosomal and mitochondrial dysfunction. NIH3T3 cells were treated with or without H 2 O 2 . (A) In vitro <t>ACP2/acid</t> phosphatase activity assay was performed at d 3 after H 2 O 2 treatment. (B) Lysosomes in cells were probed with LysoTracker Red DND-99 at day 3 and images were taken by confocal microscopy. The images in rectangles are 1.5-fold enlarged. Relative lysosome size in control and H 2 O 2 -treated cells was quantified. (C) CTSB in control and H 2 O 2 -treated cells at the indicated time points were examined by western blot. (D) Cellular CTSB activity was visualized by using the Magic Red Cathepsin B detection kit at the indicated days. Cells treated with bafilomycin A 1 (50 nM) for 12 h are shown as a negative control. Scale bars: 50 μm. (E) Mitochondrial membrane potential in cells was measured by JC-1 staining at d 3. Scale bars: 20 μm. (F) Cellular ATP level of control (5 d) and H 2 O 2 -treated (1 d, 3 d, 5 d) cells was measured. (G) Mitochondrial COX activity of control (5 d) or H 2 O 2 -treated (1 d, 3 d, 5 d) cells was assayed. (H) Cells pretreated with DMSO or 10 μM CCCP for 6 h followed by H 2 O 2 treatment as described in the Materials and Methods. Images of DCFH-DA fluorescence was taken at the indicated days. Scale bars: 50 μm. (I) SA-GLB1 staining was performed at day 5 on CCCP-pretreated cells. Scale bars: 50 μm. (J) Relative Il6 mRNA expression of CCCP-pretreated cells at d 5. The data are presented as means ± SD from 3 independent experiments. *p
    Acp2, supplied by Beyotime, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    LAP&P Consultants em enzymatic activity
    Autophagy impairment couples with lysosomal and mitochondrial dysfunction. NIH3T3 cells were treated with or without H 2 O 2 . (A) In vitro <t>ACP2/acid</t> phosphatase activity assay was performed at d 3 after H 2 O 2 treatment. (B) Lysosomes in cells were probed with LysoTracker Red DND-99 at day 3 and images were taken by confocal microscopy. The images in rectangles are 1.5-fold enlarged. Relative lysosome size in control and H 2 O 2 -treated cells was quantified. (C) CTSB in control and H 2 O 2 -treated cells at the indicated time points were examined by western blot. (D) Cellular CTSB activity was visualized by using the Magic Red Cathepsin B detection kit at the indicated days. Cells treated with bafilomycin A 1 (50 nM) for 12 h are shown as a negative control. Scale bars: 50 μm. (E) Mitochondrial membrane potential in cells was measured by JC-1 staining at d 3. Scale bars: 20 μm. (F) Cellular ATP level of control (5 d) and H 2 O 2 -treated (1 d, 3 d, 5 d) cells was measured. (G) Mitochondrial COX activity of control (5 d) or H 2 O 2 -treated (1 d, 3 d, 5 d) cells was assayed. (H) Cells pretreated with DMSO or 10 μM CCCP for 6 h followed by H 2 O 2 treatment as described in the Materials and Methods. Images of DCFH-DA fluorescence was taken at the indicated days. Scale bars: 50 μm. (I) SA-GLB1 staining was performed at day 5 on CCCP-pretreated cells. Scale bars: 50 μm. (J) Relative Il6 mRNA expression of CCCP-pretreated cells at d 5. The data are presented as means ± SD from 3 independent experiments. *p
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    GBF fusion protein restored promoter activity
    Autophagy impairment couples with lysosomal and mitochondrial dysfunction. NIH3T3 cells were treated with or without H 2 O 2 . (A) In vitro <t>ACP2/acid</t> phosphatase activity assay was performed at d 3 after H 2 O 2 treatment. (B) Lysosomes in cells were probed with LysoTracker Red DND-99 at day 3 and images were taken by confocal microscopy. The images in rectangles are 1.5-fold enlarged. Relative lysosome size in control and H 2 O 2 -treated cells was quantified. (C) CTSB in control and H 2 O 2 -treated cells at the indicated time points were examined by western blot. (D) Cellular CTSB activity was visualized by using the Magic Red Cathepsin B detection kit at the indicated days. Cells treated with bafilomycin A 1 (50 nM) for 12 h are shown as a negative control. Scale bars: 50 μm. (E) Mitochondrial membrane potential in cells was measured by JC-1 staining at d 3. Scale bars: 20 μm. (F) Cellular ATP level of control (5 d) and H 2 O 2 -treated (1 d, 3 d, 5 d) cells was measured. (G) Mitochondrial COX activity of control (5 d) or H 2 O 2 -treated (1 d, 3 d, 5 d) cells was assayed. (H) Cells pretreated with DMSO or 10 μM CCCP for 6 h followed by H 2 O 2 treatment as described in the Materials and Methods. Images of DCFH-DA fluorescence was taken at the indicated days. Scale bars: 50 μm. (I) SA-GLB1 staining was performed at day 5 on CCCP-pretreated cells. Scale bars: 50 μm. (J) Relative Il6 mRNA expression of CCCP-pretreated cells at d 5. The data are presented as means ± SD from 3 independent experiments. *p
    Fusion Protein Restored Promoter Activity, supplied by GBF, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore alkaline phosphatase activity
    Autophagy impairment couples with lysosomal and mitochondrial dysfunction. NIH3T3 cells were treated with or without H 2 O 2 . (A) In vitro <t>ACP2/acid</t> phosphatase activity assay was performed at d 3 after H 2 O 2 treatment. (B) Lysosomes in cells were probed with LysoTracker Red DND-99 at day 3 and images were taken by confocal microscopy. The images in rectangles are 1.5-fold enlarged. Relative lysosome size in control and H 2 O 2 -treated cells was quantified. (C) CTSB in control and H 2 O 2 -treated cells at the indicated time points were examined by western blot. (D) Cellular CTSB activity was visualized by using the Magic Red Cathepsin B detection kit at the indicated days. Cells treated with bafilomycin A 1 (50 nM) for 12 h are shown as a negative control. Scale bars: 50 μm. (E) Mitochondrial membrane potential in cells was measured by JC-1 staining at d 3. Scale bars: 20 μm. (F) Cellular ATP level of control (5 d) and H 2 O 2 -treated (1 d, 3 d, 5 d) cells was measured. (G) Mitochondrial COX activity of control (5 d) or H 2 O 2 -treated (1 d, 3 d, 5 d) cells was assayed. (H) Cells pretreated with DMSO or 10 μM CCCP for 6 h followed by H 2 O 2 treatment as described in the Materials and Methods. Images of DCFH-DA fluorescence was taken at the indicated days. Scale bars: 50 μm. (I) SA-GLB1 staining was performed at day 5 on CCCP-pretreated cells. Scale bars: 50 μm. (J) Relative Il6 mRNA expression of CCCP-pretreated cells at d 5. The data are presented as means ± SD from 3 independent experiments. *p
    Alkaline Phosphatase Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2774 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc c ebpβ liver enriched activating protein lap
    Autophagy impairment couples with lysosomal and mitochondrial dysfunction. NIH3T3 cells were treated with or without H 2 O 2 . (A) In vitro <t>ACP2/acid</t> phosphatase activity assay was performed at d 3 after H 2 O 2 treatment. (B) Lysosomes in cells were probed with LysoTracker Red DND-99 at day 3 and images were taken by confocal microscopy. The images in rectangles are 1.5-fold enlarged. Relative lysosome size in control and H 2 O 2 -treated cells was quantified. (C) CTSB in control and H 2 O 2 -treated cells at the indicated time points were examined by western blot. (D) Cellular CTSB activity was visualized by using the Magic Red Cathepsin B detection kit at the indicated days. Cells treated with bafilomycin A 1 (50 nM) for 12 h are shown as a negative control. Scale bars: 50 μm. (E) Mitochondrial membrane potential in cells was measured by JC-1 staining at d 3. Scale bars: 20 μm. (F) Cellular ATP level of control (5 d) and H 2 O 2 -treated (1 d, 3 d, 5 d) cells was measured. (G) Mitochondrial COX activity of control (5 d) or H 2 O 2 -treated (1 d, 3 d, 5 d) cells was assayed. (H) Cells pretreated with DMSO or 10 μM CCCP for 6 h followed by H 2 O 2 treatment as described in the Materials and Methods. Images of DCFH-DA fluorescence was taken at the indicated days. Scale bars: 50 μm. (I) SA-GLB1 staining was performed at day 5 on CCCP-pretreated cells. Scale bars: 50 μm. (J) Relative Il6 mRNA expression of CCCP-pretreated cells at d 5. The data are presented as means ± SD from 3 independent experiments. *p
    C Ebpβ Liver Enriched Activating Protein Lap, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fuji Pharma lap
    Autophagy impairment couples with lysosomal and mitochondrial dysfunction. NIH3T3 cells were treated with or without H 2 O 2 . (A) In vitro <t>ACP2/acid</t> phosphatase activity assay was performed at d 3 after H 2 O 2 treatment. (B) Lysosomes in cells were probed with LysoTracker Red DND-99 at day 3 and images were taken by confocal microscopy. The images in rectangles are 1.5-fold enlarged. Relative lysosome size in control and H 2 O 2 -treated cells was quantified. (C) CTSB in control and H 2 O 2 -treated cells at the indicated time points were examined by western blot. (D) Cellular CTSB activity was visualized by using the Magic Red Cathepsin B detection kit at the indicated days. Cells treated with bafilomycin A 1 (50 nM) for 12 h are shown as a negative control. Scale bars: 50 μm. (E) Mitochondrial membrane potential in cells was measured by JC-1 staining at d 3. Scale bars: 20 μm. (F) Cellular ATP level of control (5 d) and H 2 O 2 -treated (1 d, 3 d, 5 d) cells was measured. (G) Mitochondrial COX activity of control (5 d) or H 2 O 2 -treated (1 d, 3 d, 5 d) cells was assayed. (H) Cells pretreated with DMSO or 10 μM CCCP for 6 h followed by H 2 O 2 treatment as described in the Materials and Methods. Images of DCFH-DA fluorescence was taken at the indicated days. Scale bars: 50 μm. (I) SA-GLB1 staining was performed at day 5 on CCCP-pretreated cells. Scale bars: 50 μm. (J) Relative Il6 mRNA expression of CCCP-pretreated cells at d 5. The data are presented as means ± SD from 3 independent experiments. *p
    Lap, supplied by Fuji Pharma, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lap
    Autophagy impairment couples with lysosomal and mitochondrial dysfunction. NIH3T3 cells were treated with or without H 2 O 2 . (A) In vitro <t>ACP2/acid</t> phosphatase activity assay was performed at d 3 after H 2 O 2 treatment. (B) Lysosomes in cells were probed with LysoTracker Red DND-99 at day 3 and images were taken by confocal microscopy. The images in rectangles are 1.5-fold enlarged. Relative lysosome size in control and H 2 O 2 -treated cells was quantified. (C) CTSB in control and H 2 O 2 -treated cells at the indicated time points were examined by western blot. (D) Cellular CTSB activity was visualized by using the Magic Red Cathepsin B detection kit at the indicated days. Cells treated with bafilomycin A 1 (50 nM) for 12 h are shown as a negative control. Scale bars: 50 μm. (E) Mitochondrial membrane potential in cells was measured by JC-1 staining at d 3. Scale bars: 20 μm. (F) Cellular ATP level of control (5 d) and H 2 O 2 -treated (1 d, 3 d, 5 d) cells was measured. (G) Mitochondrial COX activity of control (5 d) or H 2 O 2 -treated (1 d, 3 d, 5 d) cells was assayed. (H) Cells pretreated with DMSO or 10 μM CCCP for 6 h followed by H 2 O 2 treatment as described in the Materials and Methods. Images of DCFH-DA fluorescence was taken at the indicated days. Scale bars: 50 μm. (I) SA-GLB1 staining was performed at day 5 on CCCP-pretreated cells. Scale bars: 50 μm. (J) Relative Il6 mRNA expression of CCCP-pretreated cells at d 5. The data are presented as means ± SD from 3 independent experiments. *p
    Lap, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Selleck Chemicals lap
    N 6 -(1-iminoethyl)-L-lysine (L-NIL)-mediated inhibition of inducible nitric oxide synthase (iNOS) in <t>hPSC-derived</t> cardiomyocytes decreases <t>LAP-plus-DOX-induced</t> toxicity. (A) Cytoplasmic iNOS protein expression as determined in hiPSC-CMs treated with vehicle (Control), LAP alone (2.5 μM), DOX alone (1.0 μM), or both LAP-plus-DOX. Cells were labeled with anti-iNOS, or an isotype-matched antibody. Percentage of iNOS-expressing cells are displayed as representative flow cytometry histogram plots (left) and quantitative analysis (right). (B) iNOS activity, as a function of total nitric oxide product formation (nitrate + nitrite), was measured using a colorimetric assay. (C) Proportion of viable, apoptotic and necrotic cells, based on flow cytometry analysis of 7-AAD and Annexin V co-labeling. (D) Quantification of fold changes in apoptosis upon L-NIL co-administration. (E) Quantification of L-NIL-mediated iNOS inhibition based on total nitric oxide product formation. (F) Mitochondrial function of hiPSC-CMs cultured in each experimental group. Oxygen consumption rate (OCR) of hiPSC-CMs demonstrating basal OCR and spare OCR respiratory capacity measured after consecutive addition of oligomycin, FCCP, and antimycin. (G) Flow cytometry analysis of HER2 expression in hiPSC-CMs. (H) Effects of L-NIL on LAP-plus-DOX-induced HER2 activity, PI3K/Akt activation, IκBα phosphorylation, and nuclear translocation of NF-κB p65 subunit. All data are presented as mean ± SD (n≥3). * P
    Lap, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend lap tgf β1 pe
    ADAP enhances <t>TGF-β1/TβRI</t> signaling via TRAF6-TAK1-SMAD3. (A) Wild type controls, ADAP-overexpressing Jurkat cells or ADAP deficient Jurkat cells (JDAP) were transfected with the (CAGA) 12 -luciferase and (SBE) 4 -luciferase reporters respectively. After exogenous TGF-β1 treatment (5ng/mL), the luciferase activity was assessed. Data are representative of two independent experiments. (B) Wild type or ADAP -/- CD8 + OT1 CTLs were generated with 10nM OVA 257-264 peptide stimulation, then treated with 5ng/mL exogenous TGF-β1 as the indicated time points. The levels of pSMAD3 (ser 423/425), total SMAD3 and β-actin were examined. The relative ratio between p-SMAD3 and β-actin was shown. Data are representative of two independent experiments. (C) WT and ADAP KO CD8 + CTLs were treated with 5ng/mL TGF-β1 for 30min, followed by preparation of cytoplasmic and nuclear extracts. Equal amounts of cytoplasmic and nuclear extracts were loaded for western blotting assay with antibodies against SMAD3. SP1 expression in nuclear extracts and tubulin expression in cytoplasmic extracts were used as controls. Data are representative of two independent experiments. (D) Wild type or ADAP -/- CD8 + OT1 Tg cells were stimulated with 10nM OVA 257-264 peptide and 5ng/mL exogenous TGF-β1 as the indicated time points. The levels of p-p38, p-JNK, p-p85 (Tyr458), p-AKT (Ser473) and β-actin were examined. Data are representative of two independent experiments. (E) Cell lysates from OT-I CD8 + CTLs were immunoprecipitated with anti-TRAF6 antibody, followed by immunoblotting with antibodies against ADAP, TAK1 and TRAF6. Data are representative of two independent experiments. (F)The mRNA levels of TGF-β1 were examined in CD8 + CTLs from wild type or ADAP -/- OT1 Tg mice that were generated with 10nM OVA peptide and 5ng/mL exogenous TGF-β1 in the presence or absence of the 2uM TAK1 inhibitor 5Z-7-oxozeaenol. Data are representative of three independent experiments.
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    Huntingdon Life Sciences β lap
    <t>β-LAP</t> inhibited the phosphorylation of MAPKs and AKT and DNA binding of NF-κB and AP-1 in LPS-stimulated BV2 cells. a Western blots for MAPKs and AKT activities. Cell extracts were prepared from BV2 cells pretreated with β-LAP (0.5, 1, and 2 μM, for 1 h), followed by LPS (100 ng/ml, for 1 h), and then subjected to immunoblot analysis using antibodies against the phospho- or total forms of JNK, ERK, p38 MAPK, and Akt. The autoradiograms are representative of three independent experiments. b Quantification of Western blot data. Levels of the phosphorylated forms of MAPKs and AKT were normalized with respect to the level of each total form and expressed as relative fold changes vs. the control group. Data are the means ± SEM for three independent experiments. * P
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    BioLegend anti mouse lap tgf β1
    GARP expression on human ASCs. (A): Human ASCs and human platelets were stained for GARP expression and analyzed on a flow cytometer. In order to compare the relative GARP levels on hASCs and platelets we calculated the GARP MFI/Isotype MFI ratio (upper panel). GARP (gray histograms) and isotype control (white histograms) stainings of hASCs and platelets are shown (lower panel). (B–D): Human ASCs were transduced with lentiviral vectors expressing a nonspecific shRNA (LV-CTRL) or human GARP-specific shRNAs (LV#18 and LV#19). The general vector copy number/hASC for the LVs used was one to two copies per cell. (B): Representative dot plots of <t>LAP/TGF-β1/GARP</t> costaining on LV-CTRL (left) and LV#19 (right) hASCs are shown. (C, D): A quantification of GARP (C) and LAP/TGF-β1 (D) expression levels on NT, LV-CTRL, LV#18, and LV#19 hASCs are shown as mean (SEM) of three independent experiments. *, p
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    The Jackson Laboratory lap tta c57bl6j mice
    Effects of acute leptin suppression on activity in Tet-off hyperleptinemic transgenic obob mice. ( A ) Strategy followed to obtain transgenic mice chronically over-expressing hleptin on an obob background. <t>LAP-tTa/TRE-hleptin/</t> obob mice are skinny since hleptin is over-produced until doxycycline (DOX) is administered. ( B ) In Tg obob mice, plasma hleptin levels were suppressed 1, 3, 6, and 13 days after beginning chronic administration DOX in the food at concentrations of 0.1, 0.5, and 2 g/kg. After DOX suppression, hleptin can be turned on again by switching back to regular chow. The “recovery” time necessary to document detectable hleptin in the plasma (1M = 1 month, 2M = 2 months) is a function of the DOX concentration in the food and the duration of the administration as shown after a 13 days of DOX. ( C ) Administration of DOX to LAP-tTa/Tre-hleptin/ obob at 8 weeks of age was accompanied by an increase in body weight. ( D ) The top panel compares an 8 week old LAP-tTa/Tre-hleptin/ obob mouse ( left ) and a littermate obob control ( center ) before DOX. The bottom panel shows the same mice 5 weeks after beginning DOX. ( E–F ) Effect of acute leptin suppression on activity in Tg obob mice. ( E ) After beginning DOX, a steady decrease in HCA is observed in Tg obob mice (n = 5) as shown by linear regression analysis, becoming significant (p≤0.05) after 7 days of DOX and continuing through the end of the experiment (p
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    R&D Systems lap
    (a,b) Western blot detection of PEDF and thrombospondin‐1 in cell conditioned medium. 30 μg proteome were used with a 12.5% reducing SDS‐PAGE. (c) Western blot detection of <t>LAP/TGFβ‐1</t> in cell conditioned medium. 8 μg proteome were used with a 12.5% reducing SDS‐PAGE. The primary antibody was directed against the LAP domain (prodomain). In line with previous reports (Dubois et al., 1995), the ∼55 kDa band is thought to represent proTGFβ‐1 whereas the predominant ∼40 kDa band is thought to represent LAP. The elevated size in comparison to the primary sequence is thought to stem from glycosylation (Brunner et al., 1992; Dubois et al., 1995). (d) Western blot detection of mature TGFβ‐1 (disulfide linked dimer) in cell conditioned medium. 30 μg proteome were used with a 12.5% non‐reducing SDS‐PAGE. The primary antibody was directed against the actual TGFβ‐1 domain. In addition, N‐terminal degradomics showed TGFβ‐1 activation (see corresponding section). (e) Western blot detection of <t>L1CAM</t> in cell conditioned medium (CCM) and total cell lysate, including cell surface proteins. 8 μg (CCM) and 12 μg proteome (total cell lysate), respectively, were separated with a 7.5% reducing SDS‐PAGE. The molecular weight of 220 kDa for L1CAM is higher than expected but in line with previous reports (Li and Galileo, 2010).
    Lap, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rdi research diagnostics lap tgfβ1
    αvβ8 integrin and Band 4.1B localize to adhesion sites when astrocytes are plated on the αvβ8 ligand, <t>LAP-TGFβ1.</t> ( A-C ) Primary astrocytes express endogenous αvβ8 and Band 4.1B. ( A ) Astrocytes express
    Lap Tgfβ1, supplied by rdi research diagnostics, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc c ebpβ lap
    Inhibition of hMADS cells adipogenesis by activin A. A and B : hMADS3 cells were induced to undergo adipocyte differentiation in the absence or presence of the indicated concentrations of activin A. Twelve days later, adipogenesis was assessed by Oil red O for lipid droplets staining ( 34 ) and by GPDH activity ( 35 ). C : hMADS3 cells were induced to undergo adipocyte differentiation and treated with 100 ng/ml activin A for the indicated time intervals. GPDH activity was determined at day 12. Results are means of three culture wells (24-well plates). Values are means ± SEM ( N = 3). *Significant differences in GPDH activities in treated cells vs. controls. Similar results were obtained with hMADS2 cells and with hMADS7–B7 and -B9 clones. D : Effects of activin A on the expression of adipogenic genes. hMADS3 cells were induced to undergo differentiation in the absence or presence of 100 ng/ml activin A. RNAs were prepared 6 days after induction of differentiation and expression was investigated by semiquantitative PCR. E : Effects of activin A on the expression of <t>C/EBPb-LAP</t> and -LIP isoforms. hMADS3 cells were induced to undergo differentiation in the absence or presence of 100 ng/ml activin A. Proteins were prepared 6 days after induction of differentiation. LAP and LIP isoforms were examined by Western blot analysis using 25 μg total proteins per lane and <t>anti-C/EBPβ</t> antibodies. Similar results were obtained when proteins were prepared 3 days after induction of differentiation (supplementary Fig. S6). The approximate molecular weight of C/EBPβ isoforms is indicated. The 14 kDa C/EBPβ proteolytic degradation product was not detected. Tubulin was used as a loading control. Samples were run on the same gel. (A high-quality digital representation of this figure is available in the online issue.)
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    R&D Systems lap tgfβ1
    Inhibition of hMADS cells adipogenesis by activin A. A and B : hMADS3 cells were induced to undergo adipocyte differentiation in the absence or presence of the indicated concentrations of activin A. Twelve days later, adipogenesis was assessed by Oil red O for lipid droplets staining ( 34 ) and by GPDH activity ( 35 ). C : hMADS3 cells were induced to undergo adipocyte differentiation and treated with 100 ng/ml activin A for the indicated time intervals. GPDH activity was determined at day 12. Results are means of three culture wells (24-well plates). Values are means ± SEM ( N = 3). *Significant differences in GPDH activities in treated cells vs. controls. Similar results were obtained with hMADS2 cells and with hMADS7–B7 and -B9 clones. D : Effects of activin A on the expression of adipogenic genes. hMADS3 cells were induced to undergo differentiation in the absence or presence of 100 ng/ml activin A. RNAs were prepared 6 days after induction of differentiation and expression was investigated by semiquantitative PCR. E : Effects of activin A on the expression of <t>C/EBPb-LAP</t> and -LIP isoforms. hMADS3 cells were induced to undergo differentiation in the absence or presence of 100 ng/ml activin A. Proteins were prepared 6 days after induction of differentiation. LAP and LIP isoforms were examined by Western blot analysis using 25 μg total proteins per lane and <t>anti-C/EBPβ</t> antibodies. Similar results were obtained when proteins were prepared 3 days after induction of differentiation (supplementary Fig. S6). The approximate molecular weight of C/EBPβ isoforms is indicated. The 14 kDa C/EBPβ proteolytic degradation product was not detected. Tubulin was used as a loading control. Samples were run on the same gel. (A high-quality digital representation of this figure is available in the online issue.)
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    LAP&P Consultants leucine aminopeptidase lap
    Inhibition of hMADS cells adipogenesis by activin A. A and B : hMADS3 cells were induced to undergo adipocyte differentiation in the absence or presence of the indicated concentrations of activin A. Twelve days later, adipogenesis was assessed by Oil red O for lipid droplets staining ( 34 ) and by GPDH activity ( 35 ). C : hMADS3 cells were induced to undergo adipocyte differentiation and treated with 100 ng/ml activin A for the indicated time intervals. GPDH activity was determined at day 12. Results are means of three culture wells (24-well plates). Values are means ± SEM ( N = 3). *Significant differences in GPDH activities in treated cells vs. controls. Similar results were obtained with hMADS2 cells and with hMADS7–B7 and -B9 clones. D : Effects of activin A on the expression of adipogenic genes. hMADS3 cells were induced to undergo differentiation in the absence or presence of 100 ng/ml activin A. RNAs were prepared 6 days after induction of differentiation and expression was investigated by semiquantitative PCR. E : Effects of activin A on the expression of <t>C/EBPb-LAP</t> and -LIP isoforms. hMADS3 cells were induced to undergo differentiation in the absence or presence of 100 ng/ml activin A. Proteins were prepared 6 days after induction of differentiation. LAP and LIP isoforms were examined by Western blot analysis using 25 μg total proteins per lane and <t>anti-C/EBPβ</t> antibodies. Similar results were obtained when proteins were prepared 3 days after induction of differentiation (supplementary Fig. S6). The approximate molecular weight of C/EBPβ isoforms is indicated. The 14 kDa C/EBPβ proteolytic degradation product was not detected. Tubulin was used as a loading control. Samples were run on the same gel. (A high-quality digital representation of this figure is available in the online issue.)
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    Arvs Equipments long acting parenteral lap antiretroviral drugs
    NMCAB protects MDM from HIV-1 infection over time MDM were treated with NMCAB, CAB <t>LAP</t> or NCAB containing 100 µM drug for 8h. At days 0, 2, 5,10, and 15 post drug loading, MDM were challenged with HIV-1 ADA at 0.1 MOI for 4 h. Uninfected cells without treatment served as negative controls (Control); HIV-1 infected cells without any treatment served as positive controls (HIV-1). Samples were collected for <t>antiretroviral</t> activity test seven days after viral challenge. ( A ) HIV RT activities were measured in culture media. Results are shown as percentage of RT activities as compared to HIV-1 infected MDM. Data are expressed as mean ± SD for n = 6 samples per group. ( B ) Cells were fixed in paraformaldehyde and stained for HIV-1p24 antigen (brown). Scale bar: 100 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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    The Jackson Laboratory lap tta mice
    Myc and YAP cooperate in inducing liver growth and tumorigenesis. ( A ) Liver weight assessed at 5 wk of induction. Data are reported as percentage relative to total body weight. ( B ) Kaplan-Meier disease-free survival analysis. ( C ) Western blotting analysis of YAP and Myc levels in <t>LAP-tTA</t> tet-YAP mice at the pretumoral stage (4 wk of YAP activation) and in tumors. Vinculin (vin) was used as aninternal control for equal loading. ( D ) Box plot of the expression level of MDSR genes up-regulated in the liver upon YAP and/or Myc induction. ( Inset at the right ) Ranked heat map. ( E , top panel) Heat map of Myc and YAP/TAZ gene signatures based on the expression data of breast cancers (TCGA_BRCA). The heat map was clustered by breast cancer subtypes (basal-like, normal-like, and Luminal/Her2 + ). ( Bottom panel) The statistical track shows the logarithmic plot of P -values for each gene. (Red bars) Genes up in basal-like; (green bars) genes up in Luminal/Her + .
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    R&D Systems human lap tgf beta 1 biotinylated antibody
    Myc and YAP cooperate in inducing liver growth and tumorigenesis. ( A ) Liver weight assessed at 5 wk of induction. Data are reported as percentage relative to total body weight. ( B ) Kaplan-Meier disease-free survival analysis. ( C ) Western blotting analysis of YAP and Myc levels in <t>LAP-tTA</t> tet-YAP mice at the pretumoral stage (4 wk of YAP activation) and in tumors. Vinculin (vin) was used as aninternal control for equal loading. ( D ) Box plot of the expression level of MDSR genes up-regulated in the liver upon YAP and/or Myc induction. ( Inset at the right ) Ranked heat map. ( E , top panel) Heat map of Myc and YAP/TAZ gene signatures based on the expression data of breast cancers (TCGA_BRCA). The heat map was clustered by breast cancer subtypes (basal-like, normal-like, and Luminal/Her2 + ). ( Bottom panel) The statistical track shows the logarithmic plot of P -values for each gene. (Red bars) Genes up in basal-like; (green bars) genes up in Luminal/Her + .
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    Image Search Results


    Figure 4. Pharmacological inhibition of BAX channel activity in MPP + -treated cells. ( A and B ) Enzymatic activities of ( A ) ACP2 and ( B ) β-hexosaminidase in lysosomal-free cytosolic fractions from MPP + -treated SH-SY5Y cells, in the presence

    Journal: Autophagy

    Article Title: BAX channel activity mediates lysosomal disruption linked to Parkinson disease

    doi: 10.4161/auto.28286

    Figure Lengend Snippet: Figure 4. Pharmacological inhibition of BAX channel activity in MPP + -treated cells. ( A and B ) Enzymatic activities of ( A ) ACP2 and ( B ) β-hexosaminidase in lysosomal-free cytosolic fractions from MPP + -treated SH-SY5Y cells, in the presence

    Article Snippet: ACP2/acid phosphatase and CTSD activities of the cytosolic and lysosomal fraction were determined with the Acid Phosphatase Assay Kit from Sigma (CS0740) and the Cathepsin D Activity Assay Kit from Abcam (ab65302), respectively, according to the manufacturer’s instructions.

    Techniques: Inhibition, Activity Assay

    Figure 3. BAX mediates LMP in vivo and in purified brain lysosomes. ( A ) Enzymatic activities of lysosomal enzymes ACP2 (left panel) and CTSD (right panel) in cytosolic, lysosomal-free fractions from the ventral midbrain of MPTP-treated wild-type

    Journal: Autophagy

    Article Title: BAX channel activity mediates lysosomal disruption linked to Parkinson disease

    doi: 10.4161/auto.28286

    Figure Lengend Snippet: Figure 3. BAX mediates LMP in vivo and in purified brain lysosomes. ( A ) Enzymatic activities of lysosomal enzymes ACP2 (left panel) and CTSD (right panel) in cytosolic, lysosomal-free fractions from the ventral midbrain of MPTP-treated wild-type

    Article Snippet: ACP2/acid phosphatase and CTSD activities of the cytosolic and lysosomal fraction were determined with the Acid Phosphatase Assay Kit from Sigma (CS0740) and the Cathepsin D Activity Assay Kit from Abcam (ab65302), respectively, according to the manufacturer’s instructions.

    Techniques: In Vivo, Purification

    ACP2 is necessary for the replication of various wild-type influenza A and B viruses. A549 cells were transfected with siScr, siACP2, or siCSE1L. After 48 hours, cells were infected with the indicated viral strains at an MOI of 1 for 10 hours. The cells were stained with an anti-NP antibody and analyzed by confocal microscopy. Virus replication was determined by calculating the percentage of NP-expressing cells and normalized to that of the siScr-transfected cells. Bars show means ± SD from quadruplicated experiments. Statistical significance between the indicated groups was tested using the Student’s t-test with a threshold of *** p

    Journal: Scientific Reports

    Article Title: Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry

    doi: 10.1038/srep43893

    Figure Lengend Snippet: ACP2 is necessary for the replication of various wild-type influenza A and B viruses. A549 cells were transfected with siScr, siACP2, or siCSE1L. After 48 hours, cells were infected with the indicated viral strains at an MOI of 1 for 10 hours. The cells were stained with an anti-NP antibody and analyzed by confocal microscopy. Virus replication was determined by calculating the percentage of NP-expressing cells and normalized to that of the siScr-transfected cells. Bars show means ± SD from quadruplicated experiments. Statistical significance between the indicated groups was tested using the Student’s t-test with a threshold of *** p

    Article Snippet: Primers for ACP2 were purchased from Bioneer.

    Techniques: Transfection, Infection, Staining, Confocal Microscopy, Expressing

    ACP2 depletion inhibits IAV infection. ( A ) Reduction of ACP2 protein expression by siACP2 in both non-infected and infected cells. A549 cells were transfected with indicated siRNAs. After 48 hours, cells were infected with rPR8-GFP virus or not (Mock) for 10 hours and then subject to western blot analyses using the anti-ACP2 and anti-β-actin antibodies. ( B ) Reduced ACP2 mRNA levels in siACP2-transfected cells. ( C ) Viral protein expression in ACP2-depleted cells. A549 cells were transfected with indicated siRNAs for 48 hours. Cells were then infected with the rPR8-GFP virus at an MOI of 1. After 10 hours, cells were lysed and subject to western blot analyses using the anti-ACP2, anti-NP, anti-M2, anti-M1, and anti-β-actin antibodies. The band density was quantified by Image J software. All values are relative to those of cells transfected with siScr. ( D ) Reduced viral mRNA levels upon ACP2 depletion. A549 cells were transfected with siACP2, siScr, or siSCE1L as indicated prior to infection with rPR8-GFP virus at an MOI of 1. After 10 hours, total RNA was extracted and subject to qRT-PCR. Mean mRNA expression was normalized to that of siScr-transfected cells using the 2ΔCt method. Means from triplicate experiments are given. Error bars represent ± SD. ( E ) ACP2 depletion reduced GFP expression in cells infected with rPR8-GFP virus. A549 cells were transfected for 48 hours with the indicated siRNAs, before infection with the rPR8-GFP virus at an MOI of 1. After 10 hours, cells were fixed and observed with a microplate imaging reader. Quantification of GFP-positive cells was normalized to that of the siScr-transfected cells. Bars show means ± SD from quadruplicated experiments. ( F ) Viral particle production was impeded by ACP2 depletion. A549 cells were transfected with indicated siRNAs for 48 hours before infection with the rPR8-GFP virus at an MOI of 0.001. Supernatant from the infected cells was collected at 12, 24, 48, and 72 hours post-infection. Viral titer was determined by TCID 50 assays on MDCK cells. Data are represented as the mean ± SD of three independent experiments. Statistical significance between the indicated groups was tested using the Student’s t -test; * p

    Journal: Scientific Reports

    Article Title: Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry

    doi: 10.1038/srep43893

    Figure Lengend Snippet: ACP2 depletion inhibits IAV infection. ( A ) Reduction of ACP2 protein expression by siACP2 in both non-infected and infected cells. A549 cells were transfected with indicated siRNAs. After 48 hours, cells were infected with rPR8-GFP virus or not (Mock) for 10 hours and then subject to western blot analyses using the anti-ACP2 and anti-β-actin antibodies. ( B ) Reduced ACP2 mRNA levels in siACP2-transfected cells. ( C ) Viral protein expression in ACP2-depleted cells. A549 cells were transfected with indicated siRNAs for 48 hours. Cells were then infected with the rPR8-GFP virus at an MOI of 1. After 10 hours, cells were lysed and subject to western blot analyses using the anti-ACP2, anti-NP, anti-M2, anti-M1, and anti-β-actin antibodies. The band density was quantified by Image J software. All values are relative to those of cells transfected with siScr. ( D ) Reduced viral mRNA levels upon ACP2 depletion. A549 cells were transfected with siACP2, siScr, or siSCE1L as indicated prior to infection with rPR8-GFP virus at an MOI of 1. After 10 hours, total RNA was extracted and subject to qRT-PCR. Mean mRNA expression was normalized to that of siScr-transfected cells using the 2ΔCt method. Means from triplicate experiments are given. Error bars represent ± SD. ( E ) ACP2 depletion reduced GFP expression in cells infected with rPR8-GFP virus. A549 cells were transfected for 48 hours with the indicated siRNAs, before infection with the rPR8-GFP virus at an MOI of 1. After 10 hours, cells were fixed and observed with a microplate imaging reader. Quantification of GFP-positive cells was normalized to that of the siScr-transfected cells. Bars show means ± SD from quadruplicated experiments. ( F ) Viral particle production was impeded by ACP2 depletion. A549 cells were transfected with indicated siRNAs for 48 hours before infection with the rPR8-GFP virus at an MOI of 0.001. Supernatant from the infected cells was collected at 12, 24, 48, and 72 hours post-infection. Viral titer was determined by TCID 50 assays on MDCK cells. Data are represented as the mean ± SD of three independent experiments. Statistical significance between the indicated groups was tested using the Student’s t -test; * p

    Article Snippet: Primers for ACP2 were purchased from Bioneer.

    Techniques: Infection, Expressing, Transfection, Western Blot, Software, Quantitative RT-PCR, Imaging

    ACP2 is crucial for membrane fusion during entry of influenza A and B viruses. ( A and B ) ACP2 knockdown impaired the fusion step between viral and endosomal membranes. A549 cells were transfected with the indicated siRNAs at 37 °C for 48 hours prior to infection with viruses rPR8-GFP ( A ) or B/Florida/04/2006 ( B ) that had been labeled with R18 and DiOC18 at an MOI of 100 at 4 °C for 1 hour. Cells were then washed with PBS and incubated at 37 °C for an additional 1.5 hours with or without 0.1 μM of bafilomycin A1 (BafA1) prior to fixation. The number of the cells with red (R18) or green (DiOC18) speckles was quantified using in-house-developed image-mining (IM) software and normalized to siScr control cells (right panel). ( C ) Cytoplasmic M1 expression was decreased in ACP2-depleted cells. A549 cells were transfected with the indicated siRNAs at 37 °C for 48 hours prior to rPR8-GFP virus infection at an MOI of 100 at 4 °C. Cells were then further treated with 0.5% DMSO or 1 mM amantadine for 3 hours at 37 °C. Cells were then fixed and stained with the anti-M1 antibody. The number of the cells with dispersed M1 in cytoplasm was quantified and normalized as described in the legend to panel A and B (right panel). ( D ) vRNP import into the nucleus is impeded by knockdown of ACP2. A549 cells were transfected with the indicated siRNAs at 37 °C for 48 hours prior to rPR8-GFP infection at an MOI of 100 at 4 °C. Cells were then incubated at 37 °C for 3.5 hours before staining with the anti-NP antibody. The number of the nuclear NP-positive cells was quantified and normalized as described in the legend to panel A and B. Bar graphs show means ± SD from three independent experiments. Statistical significance between the indicated groups was tested using the Student’s t-test with a threshold of: *** p

    Journal: Scientific Reports

    Article Title: Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry

    doi: 10.1038/srep43893

    Figure Lengend Snippet: ACP2 is crucial for membrane fusion during entry of influenza A and B viruses. ( A and B ) ACP2 knockdown impaired the fusion step between viral and endosomal membranes. A549 cells were transfected with the indicated siRNAs at 37 °C for 48 hours prior to infection with viruses rPR8-GFP ( A ) or B/Florida/04/2006 ( B ) that had been labeled with R18 and DiOC18 at an MOI of 100 at 4 °C for 1 hour. Cells were then washed with PBS and incubated at 37 °C for an additional 1.5 hours with or without 0.1 μM of bafilomycin A1 (BafA1) prior to fixation. The number of the cells with red (R18) or green (DiOC18) speckles was quantified using in-house-developed image-mining (IM) software and normalized to siScr control cells (right panel). ( C ) Cytoplasmic M1 expression was decreased in ACP2-depleted cells. A549 cells were transfected with the indicated siRNAs at 37 °C for 48 hours prior to rPR8-GFP virus infection at an MOI of 100 at 4 °C. Cells were then further treated with 0.5% DMSO or 1 mM amantadine for 3 hours at 37 °C. Cells were then fixed and stained with the anti-M1 antibody. The number of the cells with dispersed M1 in cytoplasm was quantified and normalized as described in the legend to panel A and B (right panel). ( D ) vRNP import into the nucleus is impeded by knockdown of ACP2. A549 cells were transfected with the indicated siRNAs at 37 °C for 48 hours prior to rPR8-GFP infection at an MOI of 100 at 4 °C. Cells were then incubated at 37 °C for 3.5 hours before staining with the anti-NP antibody. The number of the nuclear NP-positive cells was quantified and normalized as described in the legend to panel A and B. Bar graphs show means ± SD from three independent experiments. Statistical significance between the indicated groups was tested using the Student’s t-test with a threshold of: *** p

    Article Snippet: Primers for ACP2 were purchased from Bioneer.

    Techniques: Transfection, Infection, Labeling, Incubation, Software, Expressing, Staining

    ACP2 knockdown does not affect attachment of the virus or acidification of endosomes. ( A ) ACP2 does not affect attachment of the viral particles to cells. A549 cells transfected with the indicated siRNAs were incubated with or without 0.25 U/ml neuraminidase for 4 hours then inoculated with an R18-labeled virus at an MOI of 100 for 1 hour at 4 °C. Cells were then fixed for observation. ( B ) A549 cells were transfected with the indicated siRNAs at 37 °C for 48 hours prior to infection with A/Udorn/307/1972 (H3N2) at an MOI of 100 at 4 °C for 1 hour. After inoculation, cells were incubated at 37 °C for an additional hour in the presence or absence of 0.1 μM bafilomycin A1 (BafA1). Cells were then fixed and stained with an anti-HA antibody that specifically recognizes the acid-induced conformation of the HA protein of H3.

    Journal: Scientific Reports

    Article Title: Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry

    doi: 10.1038/srep43893

    Figure Lengend Snippet: ACP2 knockdown does not affect attachment of the virus or acidification of endosomes. ( A ) ACP2 does not affect attachment of the viral particles to cells. A549 cells transfected with the indicated siRNAs were incubated with or without 0.25 U/ml neuraminidase for 4 hours then inoculated with an R18-labeled virus at an MOI of 100 for 1 hour at 4 °C. Cells were then fixed for observation. ( B ) A549 cells were transfected with the indicated siRNAs at 37 °C for 48 hours prior to infection with A/Udorn/307/1972 (H3N2) at an MOI of 100 at 4 °C for 1 hour. After inoculation, cells were incubated at 37 °C for an additional hour in the presence or absence of 0.1 μM bafilomycin A1 (BafA1). Cells were then fixed and stained with an anti-HA antibody that specifically recognizes the acid-induced conformation of the HA protein of H3.

    Article Snippet: Primers for ACP2 were purchased from Bioneer.

    Techniques: Transfection, Incubation, Labeling, Infection, Staining

    ACP2 is not required for the replication of Ebola or hepatitis C viruses. ACP2 depletion has no effect on the replication of Ebola virus ( A ) or hepatitis C virus ( B ). 293 T cells or Huh7.5 cells were transfected with the corresponding siRNAs and then infected with Ebola-trVLPs or HCV- luc , respectively, after 48 hours. At 72 hours post-infection, replication of each virus was quantified by measuring Renilla luciferase activity. Data are represented as the mean ± SD of three independent experiments.

    Journal: Scientific Reports

    Article Title: Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry

    doi: 10.1038/srep43893

    Figure Lengend Snippet: ACP2 is not required for the replication of Ebola or hepatitis C viruses. ACP2 depletion has no effect on the replication of Ebola virus ( A ) or hepatitis C virus ( B ). 293 T cells or Huh7.5 cells were transfected with the corresponding siRNAs and then infected with Ebola-trVLPs or HCV- luc , respectively, after 48 hours. At 72 hours post-infection, replication of each virus was quantified by measuring Renilla luciferase activity. Data are represented as the mean ± SD of three independent experiments.

    Article Snippet: Primers for ACP2 were purchased from Bioneer.

    Techniques: Transfection, Infection, Luciferase, Activity Assay

    Autophagy impairment couples with lysosomal and mitochondrial dysfunction. NIH3T3 cells were treated with or without H 2 O 2 . (A) In vitro ACP2/acid phosphatase activity assay was performed at d 3 after H 2 O 2 treatment. (B) Lysosomes in cells were probed with LysoTracker Red DND-99 at day 3 and images were taken by confocal microscopy. The images in rectangles are 1.5-fold enlarged. Relative lysosome size in control and H 2 O 2 -treated cells was quantified. (C) CTSB in control and H 2 O 2 -treated cells at the indicated time points were examined by western blot. (D) Cellular CTSB activity was visualized by using the Magic Red Cathepsin B detection kit at the indicated days. Cells treated with bafilomycin A 1 (50 nM) for 12 h are shown as a negative control. Scale bars: 50 μm. (E) Mitochondrial membrane potential in cells was measured by JC-1 staining at d 3. Scale bars: 20 μm. (F) Cellular ATP level of control (5 d) and H 2 O 2 -treated (1 d, 3 d, 5 d) cells was measured. (G) Mitochondrial COX activity of control (5 d) or H 2 O 2 -treated (1 d, 3 d, 5 d) cells was assayed. (H) Cells pretreated with DMSO or 10 μM CCCP for 6 h followed by H 2 O 2 treatment as described in the Materials and Methods. Images of DCFH-DA fluorescence was taken at the indicated days. Scale bars: 50 μm. (I) SA-GLB1 staining was performed at day 5 on CCCP-pretreated cells. Scale bars: 50 μm. (J) Relative Il6 mRNA expression of CCCP-pretreated cells at d 5. The data are presented as means ± SD from 3 independent experiments. *p

    Journal: Autophagy

    Article Title: Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

    doi: 10.1080/15548627.2016.1247143

    Figure Lengend Snippet: Autophagy impairment couples with lysosomal and mitochondrial dysfunction. NIH3T3 cells were treated with or without H 2 O 2 . (A) In vitro ACP2/acid phosphatase activity assay was performed at d 3 after H 2 O 2 treatment. (B) Lysosomes in cells were probed with LysoTracker Red DND-99 at day 3 and images were taken by confocal microscopy. The images in rectangles are 1.5-fold enlarged. Relative lysosome size in control and H 2 O 2 -treated cells was quantified. (C) CTSB in control and H 2 O 2 -treated cells at the indicated time points were examined by western blot. (D) Cellular CTSB activity was visualized by using the Magic Red Cathepsin B detection kit at the indicated days. Cells treated with bafilomycin A 1 (50 nM) for 12 h are shown as a negative control. Scale bars: 50 μm. (E) Mitochondrial membrane potential in cells was measured by JC-1 staining at d 3. Scale bars: 20 μm. (F) Cellular ATP level of control (5 d) and H 2 O 2 -treated (1 d, 3 d, 5 d) cells was measured. (G) Mitochondrial COX activity of control (5 d) or H 2 O 2 -treated (1 d, 3 d, 5 d) cells was assayed. (H) Cells pretreated with DMSO or 10 μM CCCP for 6 h followed by H 2 O 2 treatment as described in the Materials and Methods. Images of DCFH-DA fluorescence was taken at the indicated days. Scale bars: 50 μm. (I) SA-GLB1 staining was performed at day 5 on CCCP-pretreated cells. Scale bars: 50 μm. (J) Relative Il6 mRNA expression of CCCP-pretreated cells at d 5. The data are presented as means ± SD from 3 independent experiments. *p

    Article Snippet: ACP2 (acid phosphatase 2, lysosomal) activity was assayed by a commercially available kit (Beyotime, P0326) according to the manufacturer's instructions.

    Techniques: In Vitro, Phosphatase Assay, Confocal Microscopy, Western Blot, Activity Assay, Negative Control, Staining, Fluorescence, Expressing

    N 6 -(1-iminoethyl)-L-lysine (L-NIL)-mediated inhibition of inducible nitric oxide synthase (iNOS) in hPSC-derived cardiomyocytes decreases LAP-plus-DOX-induced toxicity. (A) Cytoplasmic iNOS protein expression as determined in hiPSC-CMs treated with vehicle (Control), LAP alone (2.5 μM), DOX alone (1.0 μM), or both LAP-plus-DOX. Cells were labeled with anti-iNOS, or an isotype-matched antibody. Percentage of iNOS-expressing cells are displayed as representative flow cytometry histogram plots (left) and quantitative analysis (right). (B) iNOS activity, as a function of total nitric oxide product formation (nitrate + nitrite), was measured using a colorimetric assay. (C) Proportion of viable, apoptotic and necrotic cells, based on flow cytometry analysis of 7-AAD and Annexin V co-labeling. (D) Quantification of fold changes in apoptosis upon L-NIL co-administration. (E) Quantification of L-NIL-mediated iNOS inhibition based on total nitric oxide product formation. (F) Mitochondrial function of hiPSC-CMs cultured in each experimental group. Oxygen consumption rate (OCR) of hiPSC-CMs demonstrating basal OCR and spare OCR respiratory capacity measured after consecutive addition of oligomycin, FCCP, and antimycin. (G) Flow cytometry analysis of HER2 expression in hiPSC-CMs. (H) Effects of L-NIL on LAP-plus-DOX-induced HER2 activity, PI3K/Akt activation, IκBα phosphorylation, and nuclear translocation of NF-κB p65 subunit. All data are presented as mean ± SD (n≥3). * P

    Journal: Theranostics

    Article Title: The HER2 inhibitor lapatinib potentiates doxorubicin-induced cardiotoxicity through iNOS signaling

    doi: 10.7150/thno.23207

    Figure Lengend Snippet: N 6 -(1-iminoethyl)-L-lysine (L-NIL)-mediated inhibition of inducible nitric oxide synthase (iNOS) in hPSC-derived cardiomyocytes decreases LAP-plus-DOX-induced toxicity. (A) Cytoplasmic iNOS protein expression as determined in hiPSC-CMs treated with vehicle (Control), LAP alone (2.5 μM), DOX alone (1.0 μM), or both LAP-plus-DOX. Cells were labeled with anti-iNOS, or an isotype-matched antibody. Percentage of iNOS-expressing cells are displayed as representative flow cytometry histogram plots (left) and quantitative analysis (right). (B) iNOS activity, as a function of total nitric oxide product formation (nitrate + nitrite), was measured using a colorimetric assay. (C) Proportion of viable, apoptotic and necrotic cells, based on flow cytometry analysis of 7-AAD and Annexin V co-labeling. (D) Quantification of fold changes in apoptosis upon L-NIL co-administration. (E) Quantification of L-NIL-mediated iNOS inhibition based on total nitric oxide product formation. (F) Mitochondrial function of hiPSC-CMs cultured in each experimental group. Oxygen consumption rate (OCR) of hiPSC-CMs demonstrating basal OCR and spare OCR respiratory capacity measured after consecutive addition of oligomycin, FCCP, and antimycin. (G) Flow cytometry analysis of HER2 expression in hiPSC-CMs. (H) Effects of L-NIL on LAP-plus-DOX-induced HER2 activity, PI3K/Akt activation, IκBα phosphorylation, and nuclear translocation of NF-κB p65 subunit. All data are presented as mean ± SD (n≥3). * P

    Article Snippet: Compound exposure and toxicity evaluation hPSC-CMs were treated with either LAP (Selleckchem) or DOX (Selleckchem) alone, or in combination with each other, in the presence or absence of N6 - (1-iminoethyl)-L-lysine (L-NIL; Cayman) for 24, 48 or 72 h as indicated.

    Techniques: Inhibition, Derivative Assay, Expressing, Labeling, Flow Cytometry, Cytometry, Activity Assay, Colorimetric Assay, Cell Culture, Activation Assay, Translocation Assay

    L-NIL-mediated iNOS inhibition alleviates LAP-plus-DOX-induced cardiotoxicity in a murine SK-BR-3 breast cancer xenograft model . (A) Immunoblots indicating iNOS and (B) NO levels in the myocardium. Cardiotoxicity was evaluated by (C) animal survival rate, (D) body weight and heart weight, (E) echocardiography, (F) catheter-derived hemodynamic measurements, (G) TUNEL-expressing cTnI + cells (white arrows), (H) collagen I expression, (I) plasma levels of cTnI, and (J) gene expression of stress markers. Scale bars indicate 10 μm in (G) and 50 μm in (H), respectively. Control = vehicle; Low = low dose of LAP (5 mg/kg) + DOX (5 mg/kg); High = high dose of LAP (10 mg/kg) + DOX (10 mg/kg); L-NIL = high dose of LAP + DOX + L-NIL (10 mg/kg). LVEF: left-ventricular ejection fraction; LVFS: left ventricular fractional shortening; LVEDV: left ventricular end-diastolic volume; LVESV: left ventricular end-systolic volume; LVESP: left ventricular end-systolic pressure; LVEDP: left ventricular end-diastolic pressure; ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide. All data are presented as mean ± SD (n≥4). * P

    Journal: Theranostics

    Article Title: The HER2 inhibitor lapatinib potentiates doxorubicin-induced cardiotoxicity through iNOS signaling

    doi: 10.7150/thno.23207

    Figure Lengend Snippet: L-NIL-mediated iNOS inhibition alleviates LAP-plus-DOX-induced cardiotoxicity in a murine SK-BR-3 breast cancer xenograft model . (A) Immunoblots indicating iNOS and (B) NO levels in the myocardium. Cardiotoxicity was evaluated by (C) animal survival rate, (D) body weight and heart weight, (E) echocardiography, (F) catheter-derived hemodynamic measurements, (G) TUNEL-expressing cTnI + cells (white arrows), (H) collagen I expression, (I) plasma levels of cTnI, and (J) gene expression of stress markers. Scale bars indicate 10 μm in (G) and 50 μm in (H), respectively. Control = vehicle; Low = low dose of LAP (5 mg/kg) + DOX (5 mg/kg); High = high dose of LAP (10 mg/kg) + DOX (10 mg/kg); L-NIL = high dose of LAP + DOX + L-NIL (10 mg/kg). LVEF: left-ventricular ejection fraction; LVFS: left ventricular fractional shortening; LVEDV: left ventricular end-diastolic volume; LVESV: left ventricular end-systolic volume; LVESP: left ventricular end-systolic pressure; LVEDP: left ventricular end-diastolic pressure; ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide. All data are presented as mean ± SD (n≥4). * P

    Article Snippet: Compound exposure and toxicity evaluation hPSC-CMs were treated with either LAP (Selleckchem) or DOX (Selleckchem) alone, or in combination with each other, in the presence or absence of N6 - (1-iminoethyl)-L-lysine (L-NIL; Cayman) for 24, 48 or 72 h as indicated.

    Techniques: Inhibition, Western Blot, Derivative Assay, TUNEL Assay, Expressing, Aqueous Normal-phase Chromatography

    Human iPSC-derived cardiomyocytes are capable of modeling the synergistic cardiotoxicity of lapatinib (LAP)-plus-doxorubicin (DOX) combination treatment. (A) Representative phase contrast (left) and sarcomeric protein immunostaining (right) images of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). ACTN1: actinin alpha 1. Scale bars: 100 μm and 20 μm. (B-C) Flow cytometry analysis of cardiac troponin I (cTnI) and human epidermal growth factor receptor-2 (HER2) expression in hiPSC-CMs. (D) Effect of DOX treatment (after 24 h) on viability of hiPSC-CM and RUES2-CM using a TetraZ-based dye assay. (E) Quantification of cell viability in a representative hiPSC-CM line treated with LAP-plus-DOX, LAP-plus-trastuzumab, or LAP-plus-afatinib at different pharmacological plasma level concentrations. (F) Effects of LAP (2.5 μM) on DOX (1 μM)-induced hiPSC-CM, RUES2-CM, HUVEC, and HEK293 toxicity at different time-points based on quantification of cell viability. (G) Representative images of early (annexin V + ) and late (annexin V + 7-AAD + ) apoptotic hiPSC-CMs 24 h after treatment with LAP-plus-DOX using image-in-flow cytometry. (H) Pro-apoptotic effects of LAP-plus-DOX on hPSC-CMs based on flow cytometry analysis of annexin V + and 7-AAD + cells. All data are presented as mean ± SD (n≥3). * P

    Journal: Theranostics

    Article Title: The HER2 inhibitor lapatinib potentiates doxorubicin-induced cardiotoxicity through iNOS signaling

    doi: 10.7150/thno.23207

    Figure Lengend Snippet: Human iPSC-derived cardiomyocytes are capable of modeling the synergistic cardiotoxicity of lapatinib (LAP)-plus-doxorubicin (DOX) combination treatment. (A) Representative phase contrast (left) and sarcomeric protein immunostaining (right) images of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). ACTN1: actinin alpha 1. Scale bars: 100 μm and 20 μm. (B-C) Flow cytometry analysis of cardiac troponin I (cTnI) and human epidermal growth factor receptor-2 (HER2) expression in hiPSC-CMs. (D) Effect of DOX treatment (after 24 h) on viability of hiPSC-CM and RUES2-CM using a TetraZ-based dye assay. (E) Quantification of cell viability in a representative hiPSC-CM line treated with LAP-plus-DOX, LAP-plus-trastuzumab, or LAP-plus-afatinib at different pharmacological plasma level concentrations. (F) Effects of LAP (2.5 μM) on DOX (1 μM)-induced hiPSC-CM, RUES2-CM, HUVEC, and HEK293 toxicity at different time-points based on quantification of cell viability. (G) Representative images of early (annexin V + ) and late (annexin V + 7-AAD + ) apoptotic hiPSC-CMs 24 h after treatment with LAP-plus-DOX using image-in-flow cytometry. (H) Pro-apoptotic effects of LAP-plus-DOX on hPSC-CMs based on flow cytometry analysis of annexin V + and 7-AAD + cells. All data are presented as mean ± SD (n≥3). * P

    Article Snippet: Compound exposure and toxicity evaluation hPSC-CMs were treated with either LAP (Selleckchem) or DOX (Selleckchem) alone, or in combination with each other, in the presence or absence of N6 - (1-iminoethyl)-L-lysine (L-NIL; Cayman) for 24, 48 or 72 h as indicated.

    Techniques: Derivative Assay, Immunostaining, Flow Cytometry, Cytometry, Expressing

    L-NIL-mediated iNOS inhibition attenuates myocardial apoptosis and systolic dysfunction in mice treated with LAP-plus-DOX . (A) Representative flow cytometry scatter plots and (B) statistical analysis of annexin V + and 7-AAD + cells from digested hearts of mice treated with different dose-regimens of LAP-plus-DOX. (C) Caspase-3 activity assay performed on ventricular extracts from hearts of mice treated with different drug-regimens for 0 to 7 days as indicated. (D) TUNEL immunostaining of left-ventricular sections at 7 days after drug administration. Percentages of TUNEL-expressing cTnI + cells (white arrows) are displayed as representative images and cumulative data. TUNEL, red; cTnI, green; DAPI, blue. Scale bar: 10 μm. (E) Echocardiographic measurements of mice treated with different drug-regimens taken 7 days after commencement of treatment. (F) Hematoxylin and eosin-staining (left) and immunohistochemical localization of iNOS expression (right) in myocardial sections. Scale bar: 50 μm. (G) Western blots showing iNOS expression and (H) NO levels in the myocardium. In Figures (D-H): Control = vehicle; LAP = lapatinib 10 mg/kg; DOX = doxorubicin 5 mg/kg; Combo = LAP 10 mg/kg + DOX 5 mg/kg; L-NIL = vehicle + L-NIL 10 mg/kg; Combo + L-NIL = LAP 10 mg/kg + DOX 5 mg/kg + L-NIL 10 mg/kg. LVEF: left-ventricular ejection fraction; FS: fractional shortening; EDV: end-diastolic volume; ESV: end-systolic volume. All data are presented as mean ± SD (n≥4). * P

    Journal: Theranostics

    Article Title: The HER2 inhibitor lapatinib potentiates doxorubicin-induced cardiotoxicity through iNOS signaling

    doi: 10.7150/thno.23207

    Figure Lengend Snippet: L-NIL-mediated iNOS inhibition attenuates myocardial apoptosis and systolic dysfunction in mice treated with LAP-plus-DOX . (A) Representative flow cytometry scatter plots and (B) statistical analysis of annexin V + and 7-AAD + cells from digested hearts of mice treated with different dose-regimens of LAP-plus-DOX. (C) Caspase-3 activity assay performed on ventricular extracts from hearts of mice treated with different drug-regimens for 0 to 7 days as indicated. (D) TUNEL immunostaining of left-ventricular sections at 7 days after drug administration. Percentages of TUNEL-expressing cTnI + cells (white arrows) are displayed as representative images and cumulative data. TUNEL, red; cTnI, green; DAPI, blue. Scale bar: 10 μm. (E) Echocardiographic measurements of mice treated with different drug-regimens taken 7 days after commencement of treatment. (F) Hematoxylin and eosin-staining (left) and immunohistochemical localization of iNOS expression (right) in myocardial sections. Scale bar: 50 μm. (G) Western blots showing iNOS expression and (H) NO levels in the myocardium. In Figures (D-H): Control = vehicle; LAP = lapatinib 10 mg/kg; DOX = doxorubicin 5 mg/kg; Combo = LAP 10 mg/kg + DOX 5 mg/kg; L-NIL = vehicle + L-NIL 10 mg/kg; Combo + L-NIL = LAP 10 mg/kg + DOX 5 mg/kg + L-NIL 10 mg/kg. LVEF: left-ventricular ejection fraction; FS: fractional shortening; EDV: end-diastolic volume; ESV: end-systolic volume. All data are presented as mean ± SD (n≥4). * P

    Article Snippet: Compound exposure and toxicity evaluation hPSC-CMs were treated with either LAP (Selleckchem) or DOX (Selleckchem) alone, or in combination with each other, in the presence or absence of N6 - (1-iminoethyl)-L-lysine (L-NIL; Cayman) for 24, 48 or 72 h as indicated.

    Techniques: Inhibition, Mouse Assay, Flow Cytometry, Cytometry, Caspase-3 Activity Assay, TUNEL Assay, Immunostaining, Expressing, Staining, Immunohistochemistry, Western Blot

    L-NIL-mediated iNOS inhibition maintains anti-cancer potency of LAP-plus-DOX combined treatment in a murine SK-BR-3 breast cancer xenograft model . (A) Tumor progression displayed as representative serial, non-invasive In Vivo Imaging System images. (B) Time-course chart showing tumor size as determined by bioluminescent signal intensity in the region of interest, expressed in photons per second. Control = vehicle; Low = low dose of LAP (5 mg/kg) + DOX (5 mg/kg); High = high dose of LAP (10 mg/kg) + DOX (10 mg/kg); L-NIL = high dose of LAP + DOX + L-NIL (10 mg/kg). In (A), red arrows indicate the time points of drug administration. All data are presented as mean ± SD (n≥4). * P

    Journal: Theranostics

    Article Title: The HER2 inhibitor lapatinib potentiates doxorubicin-induced cardiotoxicity through iNOS signaling

    doi: 10.7150/thno.23207

    Figure Lengend Snippet: L-NIL-mediated iNOS inhibition maintains anti-cancer potency of LAP-plus-DOX combined treatment in a murine SK-BR-3 breast cancer xenograft model . (A) Tumor progression displayed as representative serial, non-invasive In Vivo Imaging System images. (B) Time-course chart showing tumor size as determined by bioluminescent signal intensity in the region of interest, expressed in photons per second. Control = vehicle; Low = low dose of LAP (5 mg/kg) + DOX (5 mg/kg); High = high dose of LAP (10 mg/kg) + DOX (10 mg/kg); L-NIL = high dose of LAP + DOX + L-NIL (10 mg/kg). In (A), red arrows indicate the time points of drug administration. All data are presented as mean ± SD (n≥4). * P

    Article Snippet: Compound exposure and toxicity evaluation hPSC-CMs were treated with either LAP (Selleckchem) or DOX (Selleckchem) alone, or in combination with each other, in the presence or absence of N6 - (1-iminoethyl)-L-lysine (L-NIL; Cayman) for 24, 48 or 72 h as indicated.

    Techniques: Inhibition, In Vivo Imaging

    ADAP enhances TGF-β1/TβRI signaling via TRAF6-TAK1-SMAD3. (A) Wild type controls, ADAP-overexpressing Jurkat cells or ADAP deficient Jurkat cells (JDAP) were transfected with the (CAGA) 12 -luciferase and (SBE) 4 -luciferase reporters respectively. After exogenous TGF-β1 treatment (5ng/mL), the luciferase activity was assessed. Data are representative of two independent experiments. (B) Wild type or ADAP -/- CD8 + OT1 CTLs were generated with 10nM OVA 257-264 peptide stimulation, then treated with 5ng/mL exogenous TGF-β1 as the indicated time points. The levels of pSMAD3 (ser 423/425), total SMAD3 and β-actin were examined. The relative ratio between p-SMAD3 and β-actin was shown. Data are representative of two independent experiments. (C) WT and ADAP KO CD8 + CTLs were treated with 5ng/mL TGF-β1 for 30min, followed by preparation of cytoplasmic and nuclear extracts. Equal amounts of cytoplasmic and nuclear extracts were loaded for western blotting assay with antibodies against SMAD3. SP1 expression in nuclear extracts and tubulin expression in cytoplasmic extracts were used as controls. Data are representative of two independent experiments. (D) Wild type or ADAP -/- CD8 + OT1 Tg cells were stimulated with 10nM OVA 257-264 peptide and 5ng/mL exogenous TGF-β1 as the indicated time points. The levels of p-p38, p-JNK, p-p85 (Tyr458), p-AKT (Ser473) and β-actin were examined. Data are representative of two independent experiments. (E) Cell lysates from OT-I CD8 + CTLs were immunoprecipitated with anti-TRAF6 antibody, followed by immunoblotting with antibodies against ADAP, TAK1 and TRAF6. Data are representative of two independent experiments. (F)The mRNA levels of TGF-β1 were examined in CD8 + CTLs from wild type or ADAP -/- OT1 Tg mice that were generated with 10nM OVA peptide and 5ng/mL exogenous TGF-β1 in the presence or absence of the 2uM TAK1 inhibitor 5Z-7-oxozeaenol. Data are representative of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: The Immune Adaptor ADAP Regulates Reciprocal TGF-β1-Integrin Crosstalk to Protect from Influenza Virus Infection

    doi: 10.1371/journal.ppat.1004824

    Figure Lengend Snippet: ADAP enhances TGF-β1/TβRI signaling via TRAF6-TAK1-SMAD3. (A) Wild type controls, ADAP-overexpressing Jurkat cells or ADAP deficient Jurkat cells (JDAP) were transfected with the (CAGA) 12 -luciferase and (SBE) 4 -luciferase reporters respectively. After exogenous TGF-β1 treatment (5ng/mL), the luciferase activity was assessed. Data are representative of two independent experiments. (B) Wild type or ADAP -/- CD8 + OT1 CTLs were generated with 10nM OVA 257-264 peptide stimulation, then treated with 5ng/mL exogenous TGF-β1 as the indicated time points. The levels of pSMAD3 (ser 423/425), total SMAD3 and β-actin were examined. The relative ratio between p-SMAD3 and β-actin was shown. Data are representative of two independent experiments. (C) WT and ADAP KO CD8 + CTLs were treated with 5ng/mL TGF-β1 for 30min, followed by preparation of cytoplasmic and nuclear extracts. Equal amounts of cytoplasmic and nuclear extracts were loaded for western blotting assay with antibodies against SMAD3. SP1 expression in nuclear extracts and tubulin expression in cytoplasmic extracts were used as controls. Data are representative of two independent experiments. (D) Wild type or ADAP -/- CD8 + OT1 Tg cells were stimulated with 10nM OVA 257-264 peptide and 5ng/mL exogenous TGF-β1 as the indicated time points. The levels of p-p38, p-JNK, p-p85 (Tyr458), p-AKT (Ser473) and β-actin were examined. Data are representative of two independent experiments. (E) Cell lysates from OT-I CD8 + CTLs were immunoprecipitated with anti-TRAF6 antibody, followed by immunoblotting with antibodies against ADAP, TAK1 and TRAF6. Data are representative of two independent experiments. (F)The mRNA levels of TGF-β1 were examined in CD8 + CTLs from wild type or ADAP -/- OT1 Tg mice that were generated with 10nM OVA peptide and 5ng/mL exogenous TGF-β1 in the presence or absence of the 2uM TAK1 inhibitor 5Z-7-oxozeaenol. Data are representative of three independent experiments.

    Article Snippet: Flow cytometry Lymphocytes from each organ was stained with various combinations of mAbs to CD4-FITC/APC, CD8α-PE/APC, C D3α-PE, and B220-FITC (eBiosciences), CD103-APC, LAP (TGF-β1)-PE (Clone TW7-20B9, Biolegend).

    Techniques: Transfection, Luciferase, Activity Assay, Generated, Western Blot, Expressing, Immunoprecipitation, Mouse Assay

    ADAP deficiency reduces TGF-β1-induced CD103 expression in CD8+ T cells. Wild type and ADAP -/- mice were infected with the H5N1 virus (n = 3). At day 10 post infection, surface CD103 expression levels on CD8 + T cells from BAL or MLNs (A, B) or the mRNA levels of CD103 or E-cadherin of lungs (C) were measured. Data are representative of two independent experiments. (D) The mRNA levels of CD103 were examined in 10nM OVA 257-264 -stimulated CD8 + CTLs from wild type or ADAP -/- OT1 Tg mice. Data are representative of two independent experiments. (E) Surface CD103 expression levels were examined from wild type or ADAP -/- CD8 + T cells after treated with anti-CD3/CD28 (2ug/mL) and exogenous TGF-β1 (5ng/mL). Data are representative of two independent experiments. Data are representative of three independent experiments. (F) Surface levels of CD103 were examined from anti-CD3/CD28 and exogenous TGF-β1-stimulated wild type or ADAP -/- CD8 + T cells, in the absence or presence of the TAK1 inhibitor (5Z-7-oxo; 2uM) or the TβRI inhibitor (SB431542; 10uM) respectively. Data are representative of two independent experiments. (G) Surface levels of CD103 were examined from wild type or TRAF6 +/- CD8 + T cells after stimulated with anti-CD3/28 antibody and exogenous TGF-β1. Data are representative of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: The Immune Adaptor ADAP Regulates Reciprocal TGF-β1-Integrin Crosstalk to Protect from Influenza Virus Infection

    doi: 10.1371/journal.ppat.1004824

    Figure Lengend Snippet: ADAP deficiency reduces TGF-β1-induced CD103 expression in CD8+ T cells. Wild type and ADAP -/- mice were infected with the H5N1 virus (n = 3). At day 10 post infection, surface CD103 expression levels on CD8 + T cells from BAL or MLNs (A, B) or the mRNA levels of CD103 or E-cadherin of lungs (C) were measured. Data are representative of two independent experiments. (D) The mRNA levels of CD103 were examined in 10nM OVA 257-264 -stimulated CD8 + CTLs from wild type or ADAP -/- OT1 Tg mice. Data are representative of two independent experiments. (E) Surface CD103 expression levels were examined from wild type or ADAP -/- CD8 + T cells after treated with anti-CD3/CD28 (2ug/mL) and exogenous TGF-β1 (5ng/mL). Data are representative of two independent experiments. Data are representative of three independent experiments. (F) Surface levels of CD103 were examined from anti-CD3/CD28 and exogenous TGF-β1-stimulated wild type or ADAP -/- CD8 + T cells, in the absence or presence of the TAK1 inhibitor (5Z-7-oxo; 2uM) or the TβRI inhibitor (SB431542; 10uM) respectively. Data are representative of two independent experiments. (G) Surface levels of CD103 were examined from wild type or TRAF6 +/- CD8 + T cells after stimulated with anti-CD3/28 antibody and exogenous TGF-β1. Data are representative of three independent experiments.

    Article Snippet: Flow cytometry Lymphocytes from each organ was stained with various combinations of mAbs to CD4-FITC/APC, CD8α-PE/APC, C D3α-PE, and B220-FITC (eBiosciences), CD103-APC, LAP (TGF-β1)-PE (Clone TW7-20B9, Biolegend).

    Techniques: Expressing, Mouse Assay, Infection

    ADAP -/- mice enhanced mortality with defective expression of TGF-β1 and CD103 in response to H1N1 infection. Wild type and ADAP -/- mice were infected with 10 LD 50 of A/PR8 to examine survival rates (A), HE staining of lungs (B), lung weight (C), the relative PR8 mRNA levels (D), amount of lung infiltrating CD8 + T cells (E), concentrations of total and active TGF-1 in lungs (F), the mRNA levels of the indicated cytokines (G) and chemokines (H), and surface CD103 levels on lung infiltrating CD8 + T cells (I) at day 8 post infection (WT: n = 7; ADAP -/- : n = 4). Data are representative of two independent experiments.

    Journal: PLoS Pathogens

    Article Title: The Immune Adaptor ADAP Regulates Reciprocal TGF-β1-Integrin Crosstalk to Protect from Influenza Virus Infection

    doi: 10.1371/journal.ppat.1004824

    Figure Lengend Snippet: ADAP -/- mice enhanced mortality with defective expression of TGF-β1 and CD103 in response to H1N1 infection. Wild type and ADAP -/- mice were infected with 10 LD 50 of A/PR8 to examine survival rates (A), HE staining of lungs (B), lung weight (C), the relative PR8 mRNA levels (D), amount of lung infiltrating CD8 + T cells (E), concentrations of total and active TGF-1 in lungs (F), the mRNA levels of the indicated cytokines (G) and chemokines (H), and surface CD103 levels on lung infiltrating CD8 + T cells (I) at day 8 post infection (WT: n = 7; ADAP -/- : n = 4). Data are representative of two independent experiments.

    Article Snippet: Flow cytometry Lymphocytes from each organ was stained with various combinations of mAbs to CD4-FITC/APC, CD8α-PE/APC, C D3α-PE, and B220-FITC (eBiosciences), CD103-APC, LAP (TGF-β1)-PE (Clone TW7-20B9, Biolegend).

    Techniques: Mouse Assay, Expressing, Infection, Staining

    ADAP regulates TGF-β1 production in T cells via an autocrine manner. (A and B) Surface staining of LAP (TGF-β1) and the relative mRNA levels of TGF-β1 in CD8 + CTLs from wild type or ADAP -/- OT1 Tg mice, which were generated with 10nM OVA 257-264 -peptide stimulation for 3 days. Data are representative of three independent experiments. (C) Human C8166 T cells overexpressing GFP or ADAP were stimulated with 2ug/mL plate bound anti-CD3/CD28 antibodies, followed by surface staining of LAP (TGF-β1). Data are representative of two independent experiments. (D) Wild type or ADAP -/- splenocytes were stimulated with 2ug/mL plate bound anti-CD3/CD28 antibodies, followed by surface staining of CD8 and LAP (TGF-β1). Data are representative of three independent experiments. (E) After stimulation with 5ng/mL TGF-β1 and 10nM OVA 257-264 -peptide for 6hrs, Wild type or ADAP -/- splenocytes were washed extensively and then incubated for further 18hr. Supernatants were collected to detect total TGF-β with ELISA. (F) In the presence or absence of the inhibitors SD208 and SB431542 (10uM respectively), the mRNA levels of TGF-β1 were examined in wild type or ADAP -/- splenocytes after stimulated with 10nM OVA 257-264 -peptide with or without 5ng/mL exogenous TGF-β1. Data are representative of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: The Immune Adaptor ADAP Regulates Reciprocal TGF-β1-Integrin Crosstalk to Protect from Influenza Virus Infection

    doi: 10.1371/journal.ppat.1004824

    Figure Lengend Snippet: ADAP regulates TGF-β1 production in T cells via an autocrine manner. (A and B) Surface staining of LAP (TGF-β1) and the relative mRNA levels of TGF-β1 in CD8 + CTLs from wild type or ADAP -/- OT1 Tg mice, which were generated with 10nM OVA 257-264 -peptide stimulation for 3 days. Data are representative of three independent experiments. (C) Human C8166 T cells overexpressing GFP or ADAP were stimulated with 2ug/mL plate bound anti-CD3/CD28 antibodies, followed by surface staining of LAP (TGF-β1). Data are representative of two independent experiments. (D) Wild type or ADAP -/- splenocytes were stimulated with 2ug/mL plate bound anti-CD3/CD28 antibodies, followed by surface staining of CD8 and LAP (TGF-β1). Data are representative of three independent experiments. (E) After stimulation with 5ng/mL TGF-β1 and 10nM OVA 257-264 -peptide for 6hrs, Wild type or ADAP -/- splenocytes were washed extensively and then incubated for further 18hr. Supernatants were collected to detect total TGF-β with ELISA. (F) In the presence or absence of the inhibitors SD208 and SB431542 (10uM respectively), the mRNA levels of TGF-β1 were examined in wild type or ADAP -/- splenocytes after stimulated with 10nM OVA 257-264 -peptide with or without 5ng/mL exogenous TGF-β1. Data are representative of three independent experiments.

    Article Snippet: Flow cytometry Lymphocytes from each organ was stained with various combinations of mAbs to CD4-FITC/APC, CD8α-PE/APC, C D3α-PE, and B220-FITC (eBiosciences), CD103-APC, LAP (TGF-β1)-PE (Clone TW7-20B9, Biolegend).

    Techniques: Staining, Mouse Assay, Generated, Incubation, Enzyme-linked Immunosorbent Assay

    ADAP-/- mice enhanced mortality with reduced TGF-β1 production in response to H5N1 virus infection. Wild type and ADAP -/- mice were infected with the H5N1 strain 10 6 EID 50 of GX/12 to examine their bodyweight changes survival rates (A), lung features (B, C), number of lung infiltrating CD8 + T cells (D) and TGF-1 concentrations or mRNA levels in lungs (E) or serum TGF-1 concentrations (F) at day 10 post infection (Mock group: n = 5; GX/12 infected group: n = 10. Data are representative of at least two independent experiments.

    Journal: PLoS Pathogens

    Article Title: The Immune Adaptor ADAP Regulates Reciprocal TGF-β1-Integrin Crosstalk to Protect from Influenza Virus Infection

    doi: 10.1371/journal.ppat.1004824

    Figure Lengend Snippet: ADAP-/- mice enhanced mortality with reduced TGF-β1 production in response to H5N1 virus infection. Wild type and ADAP -/- mice were infected with the H5N1 strain 10 6 EID 50 of GX/12 to examine their bodyweight changes survival rates (A), lung features (B, C), number of lung infiltrating CD8 + T cells (D) and TGF-1 concentrations or mRNA levels in lungs (E) or serum TGF-1 concentrations (F) at day 10 post infection (Mock group: n = 5; GX/12 infected group: n = 10. Data are representative of at least two independent experiments.

    Article Snippet: Flow cytometry Lymphocytes from each organ was stained with various combinations of mAbs to CD4-FITC/APC, CD8α-PE/APC, C D3α-PE, and B220-FITC (eBiosciences), CD103-APC, LAP (TGF-β1)-PE (Clone TW7-20B9, Biolegend).

    Techniques: Mouse Assay, Infection

    β-LAP inhibited the phosphorylation of MAPKs and AKT and DNA binding of NF-κB and AP-1 in LPS-stimulated BV2 cells. a Western blots for MAPKs and AKT activities. Cell extracts were prepared from BV2 cells pretreated with β-LAP (0.5, 1, and 2 μM, for 1 h), followed by LPS (100 ng/ml, for 1 h), and then subjected to immunoblot analysis using antibodies against the phospho- or total forms of JNK, ERK, p38 MAPK, and Akt. The autoradiograms are representative of three independent experiments. b Quantification of Western blot data. Levels of the phosphorylated forms of MAPKs and AKT were normalized with respect to the level of each total form and expressed as relative fold changes vs. the control group. Data are the means ± SEM for three independent experiments. * P

    Journal: Journal of Neuroinflammation

    Article Title: β-Lapachone suppresses neuroinflammation by modulating the expression of cytokines and matrix metalloproteinases in activated microglia

    doi: 10.1186/s12974-015-0355-z

    Figure Lengend Snippet: β-LAP inhibited the phosphorylation of MAPKs and AKT and DNA binding of NF-κB and AP-1 in LPS-stimulated BV2 cells. a Western blots for MAPKs and AKT activities. Cell extracts were prepared from BV2 cells pretreated with β-LAP (0.5, 1, and 2 μM, for 1 h), followed by LPS (100 ng/ml, for 1 h), and then subjected to immunoblot analysis using antibodies against the phospho- or total forms of JNK, ERK, p38 MAPK, and Akt. The autoradiograms are representative of three independent experiments. b Quantification of Western blot data. Levels of the phosphorylated forms of MAPKs and AKT were normalized with respect to the level of each total form and expressed as relative fold changes vs. the control group. Data are the means ± SEM for three independent experiments. * P

    Article Snippet: As to the BBB permeability of β-LAP, Huntingdon Life Sciences (UK) has reported that a minimal concentration of β-LAP penetrates into the rat brain, compared with other organs, under normal conditions (unpublished report).

    Techniques: Binding Assay, Western Blot

    β-LAP inhibited ROS production via suppression of NADPH oxidase subunits. a BV2 cells or b primary microglia were pretreated with β-LAP (0.5, 1, and 2 μM, for 1 h), followed by LPS (100 or 10 ng/ml, for 16 h), and stained with 50 μM H 2 DCF-DA. DCF fluorescence intensities were measured using a microplate fluorometer. The data are expressed as the means ± SEM of three independent experiments. * P

    Journal: Journal of Neuroinflammation

    Article Title: β-Lapachone suppresses neuroinflammation by modulating the expression of cytokines and matrix metalloproteinases in activated microglia

    doi: 10.1186/s12974-015-0355-z

    Figure Lengend Snippet: β-LAP inhibited ROS production via suppression of NADPH oxidase subunits. a BV2 cells or b primary microglia were pretreated with β-LAP (0.5, 1, and 2 μM, for 1 h), followed by LPS (100 or 10 ng/ml, for 16 h), and stained with 50 μM H 2 DCF-DA. DCF fluorescence intensities were measured using a microplate fluorometer. The data are expressed as the means ± SEM of three independent experiments. * P

    Article Snippet: As to the BBB permeability of β-LAP, Huntingdon Life Sciences (UK) has reported that a minimal concentration of β-LAP penetrates into the rat brain, compared with other organs, under normal conditions (unpublished report).

    Techniques: Staining, Fluorescence

    β-LAP increased HO-1 and NQO1 expression via upregulation of Nrf2/ARE and PKA/CREB pathways in LPS-stimulated microglia. a Western blot analysis shows the effects of β-LAP on HO-1 and NQO1 protein expression. Cell lysates were obtained from BV2 cells treated with β-LAP (0.5, 1, and 2 μM) with or without LPS (100 ng/ml) for HO-1 or NQO1 (6–16 h). b RT-PCR was performed to determine the HO-1 and NQO1 mRNA expression. Cells were treated with β-LAP (0.5, 1, and 2 μM) for 1 h prior to treatment with LPS (100 ng/ml, for 6 h) and analyzed. Quantification data are shown in the graph ( n = 3). c EMSA for Nrf2. Nuclear extracts were prepared from BV2 cells treated with LPS (100 ng/ml, for 3 h) or LPS + β-LAP (1 and 2 μM, pretreatment for 1 h) and incubated with the ARE probe. The arrow indicates a DNA–protein complex of Nrf2. d Effect of β-LAP on ARE-luc reporter gene activity. Cells transfected with the reporter plasmid (ARE-luc) were treated with β-LAP (0.5, 1, and 2 μM) with or without LPS (100 ng/ml) for 6 h, and the reporter gene assay was performed. e Effect of β-LAP on the phosphorylation of CREB. Cell lysates were obtained from BV2 cells treated with β-LAP (0.5, 1, and 2 μM) with or without LPS (100 ng/ml) for 1 h. Quantification data are shown in the graph ( n = 3). f EMSA for CREB. Nuclear extracts were prepared from BV2 cells treated with LPS (100 ng/ml, for 3 h) or LPS + β-LAP (1 and 2 μM, pretreatment for 1 h) and incubated with the CRE probe. The bracket indicates a DNA–protein complex of CREB. g Effect of β-LAP on CRE-luc activity. Cells transfected with the reporter plasmid (CRE-luc) were treated with β-LAP (0.5, 1, and 2 μM), with or without LPS (100 ng/ml) for 6 h, and the reporter gene assay was performed. Data are the means ± SEM of three independent experiments. * P

    Journal: Journal of Neuroinflammation

    Article Title: β-Lapachone suppresses neuroinflammation by modulating the expression of cytokines and matrix metalloproteinases in activated microglia

    doi: 10.1186/s12974-015-0355-z

    Figure Lengend Snippet: β-LAP increased HO-1 and NQO1 expression via upregulation of Nrf2/ARE and PKA/CREB pathways in LPS-stimulated microglia. a Western blot analysis shows the effects of β-LAP on HO-1 and NQO1 protein expression. Cell lysates were obtained from BV2 cells treated with β-LAP (0.5, 1, and 2 μM) with or without LPS (100 ng/ml) for HO-1 or NQO1 (6–16 h). b RT-PCR was performed to determine the HO-1 and NQO1 mRNA expression. Cells were treated with β-LAP (0.5, 1, and 2 μM) for 1 h prior to treatment with LPS (100 ng/ml, for 6 h) and analyzed. Quantification data are shown in the graph ( n = 3). c EMSA for Nrf2. Nuclear extracts were prepared from BV2 cells treated with LPS (100 ng/ml, for 3 h) or LPS + β-LAP (1 and 2 μM, pretreatment for 1 h) and incubated with the ARE probe. The arrow indicates a DNA–protein complex of Nrf2. d Effect of β-LAP on ARE-luc reporter gene activity. Cells transfected with the reporter plasmid (ARE-luc) were treated with β-LAP (0.5, 1, and 2 μM) with or without LPS (100 ng/ml) for 6 h, and the reporter gene assay was performed. e Effect of β-LAP on the phosphorylation of CREB. Cell lysates were obtained from BV2 cells treated with β-LAP (0.5, 1, and 2 μM) with or without LPS (100 ng/ml) for 1 h. Quantification data are shown in the graph ( n = 3). f EMSA for CREB. Nuclear extracts were prepared from BV2 cells treated with LPS (100 ng/ml, for 3 h) or LPS + β-LAP (1 and 2 μM, pretreatment for 1 h) and incubated with the CRE probe. The bracket indicates a DNA–protein complex of CREB. g Effect of β-LAP on CRE-luc activity. Cells transfected with the reporter plasmid (CRE-luc) were treated with β-LAP (0.5, 1, and 2 μM), with or without LPS (100 ng/ml) for 6 h, and the reporter gene assay was performed. Data are the means ± SEM of three independent experiments. * P

    Article Snippet: As to the BBB permeability of β-LAP, Huntingdon Life Sciences (UK) has reported that a minimal concentration of β-LAP penetrates into the rat brain, compared with other organs, under normal conditions (unpublished report).

    Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Incubation, Activity Assay, Transfection, Plasmid Preparation, Reporter Gene Assay

    Effects of β-LAP on iNOS and pro-/anti-inflammatory cytokines in LPS-stimulated BV2 cells and primary cultured microglia. BV2 cells ( a ) or primary cultured microglia ( b ) were pretreated with β-LAP (0.5, 1, and 2 μM) for 1 h and incubated with LPS (100 ng/ml for BV2, 10 ng/ml for primary microglia). After incubation for 16 h, the conditioned media were collected, and the amounts of NO, TNF-α, IL-1β, IL-6, and IL-10 were measured using Griess reagent or ELISA. The data are the mean ± SEM of three independent experiments. * P

    Journal: Journal of Neuroinflammation

    Article Title: β-Lapachone suppresses neuroinflammation by modulating the expression of cytokines and matrix metalloproteinases in activated microglia

    doi: 10.1186/s12974-015-0355-z

    Figure Lengend Snippet: Effects of β-LAP on iNOS and pro-/anti-inflammatory cytokines in LPS-stimulated BV2 cells and primary cultured microglia. BV2 cells ( a ) or primary cultured microglia ( b ) were pretreated with β-LAP (0.5, 1, and 2 μM) for 1 h and incubated with LPS (100 ng/ml for BV2, 10 ng/ml for primary microglia). After incubation for 16 h, the conditioned media were collected, and the amounts of NO, TNF-α, IL-1β, IL-6, and IL-10 were measured using Griess reagent or ELISA. The data are the mean ± SEM of three independent experiments. * P

    Article Snippet: As to the BBB permeability of β-LAP, Huntingdon Life Sciences (UK) has reported that a minimal concentration of β-LAP penetrates into the rat brain, compared with other organs, under normal conditions (unpublished report).

    Techniques: Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

    β-LAP suppressed the LPS-induced expression and enzymatic activity of MMP-3, MMP-8, and MMP-9, whereas it enhanced TIMP-2 expression. BV2 cells ( a ) or primary microglia ( b ) were pretreated with β-LAP (0.5, 1, and 2 μM, for 1 h), followed by LPS (100 or 10 ng/ml), and total RNA was isolated at 6 h after LPS treatments. The mRNA expressions of MMPs and TIMP-2 were analyzed by RT-PCR. Representative gels are shown in the left panel , and quantification data are shown in the right panel ( n = 3). c Western blot analysis was performed using conditioned medium (CM) or cell lysates of BV2 cells pretreated with β-LAP (0.5, 1, and 2 μM, for 1 h), followed by LPS (100 ng/ml) for 16 h. Levels of MMP-3, MMP-8, and MMP-9, and TIMP-2 protein expression were normalized using β-actin and were expressed as relative fold changes in comparison with control samples. d The enzymatic activities of MMPs in the CM were detected using MMP activity assay kits. BV2 cells were pretreated with β-LAP (0.5, 1, and 2 μM, for 1 h), followed by LPS (100 ng/ml, for 24 h), and the CM was collected to measure MMP activity. MMP activity units were expressed as a change in fluorescence intensity. Values are expressed as the means ± SEM for three independent experiments. * P

    Journal: Journal of Neuroinflammation

    Article Title: β-Lapachone suppresses neuroinflammation by modulating the expression of cytokines and matrix metalloproteinases in activated microglia

    doi: 10.1186/s12974-015-0355-z

    Figure Lengend Snippet: β-LAP suppressed the LPS-induced expression and enzymatic activity of MMP-3, MMP-8, and MMP-9, whereas it enhanced TIMP-2 expression. BV2 cells ( a ) or primary microglia ( b ) were pretreated with β-LAP (0.5, 1, and 2 μM, for 1 h), followed by LPS (100 or 10 ng/ml), and total RNA was isolated at 6 h after LPS treatments. The mRNA expressions of MMPs and TIMP-2 were analyzed by RT-PCR. Representative gels are shown in the left panel , and quantification data are shown in the right panel ( n = 3). c Western blot analysis was performed using conditioned medium (CM) or cell lysates of BV2 cells pretreated with β-LAP (0.5, 1, and 2 μM, for 1 h), followed by LPS (100 ng/ml) for 16 h. Levels of MMP-3, MMP-8, and MMP-9, and TIMP-2 protein expression were normalized using β-actin and were expressed as relative fold changes in comparison with control samples. d The enzymatic activities of MMPs in the CM were detected using MMP activity assay kits. BV2 cells were pretreated with β-LAP (0.5, 1, and 2 μM, for 1 h), followed by LPS (100 ng/ml, for 24 h), and the CM was collected to measure MMP activity. MMP activity units were expressed as a change in fluorescence intensity. Values are expressed as the means ± SEM for three independent experiments. * P

    Article Snippet: As to the BBB permeability of β-LAP, Huntingdon Life Sciences (UK) has reported that a minimal concentration of β-LAP penetrates into the rat brain, compared with other organs, under normal conditions (unpublished report).

    Techniques: Expressing, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Fluorescence

    β-LAP suppressed MMP-3, MMP-8, and MMP-9 expression in the LPS-induced systemic inflammation mouse brain. Changes in protein expression of MMP-3 ( a ), MMP-8 ( b ), and MMP-9 ( c ) were determined in LPS (24 h)-injected mouse brains. The number of activated Iba1 + cells with thick and densely stained processes was markedly increased in the cortex at 24 h after systemic LPS treatment (5 mg/kg, i.p.), compared to saline groups. MMP-3, MMP-8, and MMP-9 expression in the cortex of LPS-injected mouse was reduced by treatment with β-LAP (10 mg/kg, i.p., daily for 4 days). Representative images of MMP-positive cells and double-positive cells (MMP, green ; Iba-1, red ), as determined by immunohistochemistry. Representative images ( a – c ) and quantification of the data ( d ). Values represent the number of double-immunopositive cells. Scale bar , 100 μm. n = 3 per group. * P

    Journal: Journal of Neuroinflammation

    Article Title: β-Lapachone suppresses neuroinflammation by modulating the expression of cytokines and matrix metalloproteinases in activated microglia

    doi: 10.1186/s12974-015-0355-z

    Figure Lengend Snippet: β-LAP suppressed MMP-3, MMP-8, and MMP-9 expression in the LPS-induced systemic inflammation mouse brain. Changes in protein expression of MMP-3 ( a ), MMP-8 ( b ), and MMP-9 ( c ) were determined in LPS (24 h)-injected mouse brains. The number of activated Iba1 + cells with thick and densely stained processes was markedly increased in the cortex at 24 h after systemic LPS treatment (5 mg/kg, i.p.), compared to saline groups. MMP-3, MMP-8, and MMP-9 expression in the cortex of LPS-injected mouse was reduced by treatment with β-LAP (10 mg/kg, i.p., daily for 4 days). Representative images of MMP-positive cells and double-positive cells (MMP, green ; Iba-1, red ), as determined by immunohistochemistry. Representative images ( a – c ) and quantification of the data ( d ). Values represent the number of double-immunopositive cells. Scale bar , 100 μm. n = 3 per group. * P

    Article Snippet: As to the BBB permeability of β-LAP, Huntingdon Life Sciences (UK) has reported that a minimal concentration of β-LAP penetrates into the rat brain, compared with other organs, under normal conditions (unpublished report).

    Techniques: Expressing, Injection, Staining, Immunohistochemistry

    β-LAP reduced neuroinflammation induced by systemic LPS administration. a Immunofluorescence labeling of Iba1 ( red ) and quantification of the number of activated Iba1-positive cells 3 h after systemic LPS treatment (5 mg/kg, i.p.). Nuclei are counterstained with DAPI ( blue ). Microglial activation in the cortex and dentate gyrus (DG) of LPS-injected mouse was reduced by β-LAP (10 mg/kg, i.p., daily for 4 days) treatment. Representative images were obtained from one set of experiments, and the three experiments were performed independently. Upper images are the results of Iba1 + DAPI staining with an original magnification of ×40. Lower images are the results of Iba1 staining with an original magnification of ×400. b , c β-LAP reduced the mRNA expression of proinflammatory cytokines (IL-6, IL-1β, TNF-α), iNOS, and MMPs (MMP-3, MMP-8, and MMP-9) in the cortex of LPS-injected mice (5 mg/kg, 3 h). Representative gels are shown in the left panel , and quantification data are shown in the right panel ( n = 3 in each group). Results are representative RT-PCR data in the cortex 3 h after LPS treatment. Values are expressed as the means ± SEM for three independent experiments. * P

    Journal: Journal of Neuroinflammation

    Article Title: β-Lapachone suppresses neuroinflammation by modulating the expression of cytokines and matrix metalloproteinases in activated microglia

    doi: 10.1186/s12974-015-0355-z

    Figure Lengend Snippet: β-LAP reduced neuroinflammation induced by systemic LPS administration. a Immunofluorescence labeling of Iba1 ( red ) and quantification of the number of activated Iba1-positive cells 3 h after systemic LPS treatment (5 mg/kg, i.p.). Nuclei are counterstained with DAPI ( blue ). Microglial activation in the cortex and dentate gyrus (DG) of LPS-injected mouse was reduced by β-LAP (10 mg/kg, i.p., daily for 4 days) treatment. Representative images were obtained from one set of experiments, and the three experiments were performed independently. Upper images are the results of Iba1 + DAPI staining with an original magnification of ×40. Lower images are the results of Iba1 staining with an original magnification of ×400. b , c β-LAP reduced the mRNA expression of proinflammatory cytokines (IL-6, IL-1β, TNF-α), iNOS, and MMPs (MMP-3, MMP-8, and MMP-9) in the cortex of LPS-injected mice (5 mg/kg, 3 h). Representative gels are shown in the left panel , and quantification data are shown in the right panel ( n = 3 in each group). Results are representative RT-PCR data in the cortex 3 h after LPS treatment. Values are expressed as the means ± SEM for three independent experiments. * P

    Article Snippet: As to the BBB permeability of β-LAP, Huntingdon Life Sciences (UK) has reported that a minimal concentration of β-LAP penetrates into the rat brain, compared with other organs, under normal conditions (unpublished report).

    Techniques: Immunofluorescence, Labeling, Activation Assay, Injection, Staining, Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    GARP expression on human ASCs. (A): Human ASCs and human platelets were stained for GARP expression and analyzed on a flow cytometer. In order to compare the relative GARP levels on hASCs and platelets we calculated the GARP MFI/Isotype MFI ratio (upper panel). GARP (gray histograms) and isotype control (white histograms) stainings of hASCs and platelets are shown (lower panel). (B–D): Human ASCs were transduced with lentiviral vectors expressing a nonspecific shRNA (LV-CTRL) or human GARP-specific shRNAs (LV#18 and LV#19). The general vector copy number/hASC for the LVs used was one to two copies per cell. (B): Representative dot plots of LAP/TGF-β1/GARP costaining on LV-CTRL (left) and LV#19 (right) hASCs are shown. (C, D): A quantification of GARP (C) and LAP/TGF-β1 (D) expression levels on NT, LV-CTRL, LV#18, and LV#19 hASCs are shown as mean (SEM) of three independent experiments. *, p

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: Mesenchymal Stromal Cells Express GARP/LRRC32 on Their Surface: Effects on Their Biology and Immunomodulatory Capacity

    doi: 10.1002/stem.1821

    Figure Lengend Snippet: GARP expression on human ASCs. (A): Human ASCs and human platelets were stained for GARP expression and analyzed on a flow cytometer. In order to compare the relative GARP levels on hASCs and platelets we calculated the GARP MFI/Isotype MFI ratio (upper panel). GARP (gray histograms) and isotype control (white histograms) stainings of hASCs and platelets are shown (lower panel). (B–D): Human ASCs were transduced with lentiviral vectors expressing a nonspecific shRNA (LV-CTRL) or human GARP-specific shRNAs (LV#18 and LV#19). The general vector copy number/hASC for the LVs used was one to two copies per cell. (B): Representative dot plots of LAP/TGF-β1/GARP costaining on LV-CTRL (left) and LV#19 (right) hASCs are shown. (C, D): A quantification of GARP (C) and LAP/TGF-β1 (D) expression levels on NT, LV-CTRL, LV#18, and LV#19 hASCs are shown as mean (SEM) of three independent experiments. *, p

    Article Snippet: Cells were incubated with 7AAD (Sigma-Aldrich) and 2.4G2 (for mASCs; eBioscience, San Diego, CA, http://www.eBioscience.com ) followed by anti-mouse LAP/TGF-β1 (TW7-16B4) or anti-human LAP/TGF-β1 (TW4-6H10) (Biolegend, San Diego, CA, http://www.biolegend.com ) followed by goat anti-mouse IgG-APC (Jackson Immunoresearch, West Grove, PA, http://www.jacksonimmuno.com ) or a donkey anti-mouse IgG-Alexa488 (Molecular Probes, Carlsbad, CA, http://www.lifetechnologies.com ), respectively.

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry, Transduction, shRNA, Plasmid Preparation

    MSCs express GARP and LAP/TGF-β1 on their surface. (A): RT-qPCR analysis of GARP expression in human (left) and mouse (right) MSCs. hASCs and hBM-MSCs were analyzed together with human negative (293T) and positive (HUVEC) cell lines. Murine MSCs from adipose tissue (mASCs) and the MSC-line OP9 are shown in the right panel. As controls for murine samples we used BM-derived macrophages, mouse liver, and spleen. (B): Total protein from hASCs, mASCs, murine thymocytes, and 293T cells were analyzed by Western blot using an anti-mouse/human GARP antibody. (C): EDTA-harvested human and murine ASCs were stained for GARP (top) and LAP/TGF-β1 (bottom) surface expression and analyzed by flow cytometry. (D): Fat tissue from BALB/c mice was treated with collagenase type I and the resulting cell suspensions were stained for GARP and sca-1 expression before (day 0) and 1, 2, and 6 days after in vitro culturing and analyzed by flow cytometry. (A) and (D) show mean (SEM) of three independent experiments; (B) and (C) show a representative experiments out of > 3. Abbreviations: GARP, glycoprotein A repetitions predominant; hASC, human mesenchymal stromal cells from adipose tissue; hBM, human bone marrow; LAP, latency-associated peptide; MSC, mesenchymal stromal cell; TGF-β1, transforming growth factor-β1.

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: Mesenchymal Stromal Cells Express GARP/LRRC32 on Their Surface: Effects on Their Biology and Immunomodulatory Capacity

    doi: 10.1002/stem.1821

    Figure Lengend Snippet: MSCs express GARP and LAP/TGF-β1 on their surface. (A): RT-qPCR analysis of GARP expression in human (left) and mouse (right) MSCs. hASCs and hBM-MSCs were analyzed together with human negative (293T) and positive (HUVEC) cell lines. Murine MSCs from adipose tissue (mASCs) and the MSC-line OP9 are shown in the right panel. As controls for murine samples we used BM-derived macrophages, mouse liver, and spleen. (B): Total protein from hASCs, mASCs, murine thymocytes, and 293T cells were analyzed by Western blot using an anti-mouse/human GARP antibody. (C): EDTA-harvested human and murine ASCs were stained for GARP (top) and LAP/TGF-β1 (bottom) surface expression and analyzed by flow cytometry. (D): Fat tissue from BALB/c mice was treated with collagenase type I and the resulting cell suspensions were stained for GARP and sca-1 expression before (day 0) and 1, 2, and 6 days after in vitro culturing and analyzed by flow cytometry. (A) and (D) show mean (SEM) of three independent experiments; (B) and (C) show a representative experiments out of > 3. Abbreviations: GARP, glycoprotein A repetitions predominant; hASC, human mesenchymal stromal cells from adipose tissue; hBM, human bone marrow; LAP, latency-associated peptide; MSC, mesenchymal stromal cell; TGF-β1, transforming growth factor-β1.

    Article Snippet: Cells were incubated with 7AAD (Sigma-Aldrich) and 2.4G2 (for mASCs; eBioscience, San Diego, CA, http://www.eBioscience.com ) followed by anti-mouse LAP/TGF-β1 (TW7-16B4) or anti-human LAP/TGF-β1 (TW4-6H10) (Biolegend, San Diego, CA, http://www.biolegend.com ) followed by goat anti-mouse IgG-APC (Jackson Immunoresearch, West Grove, PA, http://www.jacksonimmuno.com ) or a donkey anti-mouse IgG-Alexa488 (Molecular Probes, Carlsbad, CA, http://www.lifetechnologies.com ), respectively.

    Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Western Blot, Staining, Flow Cytometry, Cytometry, Mouse Assay, In Vitro

    Glycoprotein A repetitions predominant modulates the secretion and activation of TGF-β1. (A, B): NT, LV-CTRL, LV#3, and LV#6 mASCs were cultured for 48 hours and the levels of TGF-β1 were measured in acid-activated (A) or non-acid treated (B) supernatants by ELISA. (C, D): NT, LV-CTRL, and LV#6 mASCs were cultured for 2 days in the presence (white bars) or absence (black bars) of SB431542 (10 µM). Total RNA was reverse transcribed and the expression levels of IL-11 (C) and cnn-1 (D) were analyzed by qPCR. Data are shown as mean (SEM) of four independent experiments. *, p

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: Mesenchymal Stromal Cells Express GARP/LRRC32 on Their Surface: Effects on Their Biology and Immunomodulatory Capacity

    doi: 10.1002/stem.1821

    Figure Lengend Snippet: Glycoprotein A repetitions predominant modulates the secretion and activation of TGF-β1. (A, B): NT, LV-CTRL, LV#3, and LV#6 mASCs were cultured for 48 hours and the levels of TGF-β1 were measured in acid-activated (A) or non-acid treated (B) supernatants by ELISA. (C, D): NT, LV-CTRL, and LV#6 mASCs were cultured for 2 days in the presence (white bars) or absence (black bars) of SB431542 (10 µM). Total RNA was reverse transcribed and the expression levels of IL-11 (C) and cnn-1 (D) were analyzed by qPCR. Data are shown as mean (SEM) of four independent experiments. *, p

    Article Snippet: Cells were incubated with 7AAD (Sigma-Aldrich) and 2.4G2 (for mASCs; eBioscience, San Diego, CA, http://www.eBioscience.com ) followed by anti-mouse LAP/TGF-β1 (TW7-16B4) or anti-human LAP/TGF-β1 (TW4-6H10) (Biolegend, San Diego, CA, http://www.biolegend.com ) followed by goat anti-mouse IgG-APC (Jackson Immunoresearch, West Grove, PA, http://www.jacksonimmuno.com ) or a donkey anti-mouse IgG-Alexa488 (Molecular Probes, Carlsbad, CA, http://www.lifetechnologies.com ), respectively.

    Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction

    GARP binds LAP/TGF-β1 to the surface of mASCs. (A): Murine ASCs were surface stained for GARP-PE and LAP-Alexa488 or isotype controls (bottom panels) as described in Materials and Methods. Stainings are shown separately (panels I and II) and overlapping (panel III). A cut mask representing colocalized GARP/LAP/TGF-β1 is shown in panel IV. Cells were imaged using a Zeiss LSM 710 confocal microscope. (B): Murine ASCs were transduced with lentiviral vectors expressing a nonspecific shRNA (LV-CTRL) or GARP-specific shRNAs (LV#3 and LV#6) and GARP silencing in mASCs was assessed by qPCR (left panel) and flow cytometry (right panel). The general vector copy number/mASC for the different LVs used was two to three copies per cell. (C): LV-CTRL and LV#6 mASCs were stained for GARP (left panels) or GARP and LAP/TGF-β1 (right panels). One representative experiment out of three is shown. (D): The LAP/TGF-β1 expression levels (MFI) on NT, LV-CTRL, LV#3, and LV#6 mASCs were measured on a flow cytometer. Results are shown as mean (SEM) of three independent experiments. *, p

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: Mesenchymal Stromal Cells Express GARP/LRRC32 on Their Surface: Effects on Their Biology and Immunomodulatory Capacity

    doi: 10.1002/stem.1821

    Figure Lengend Snippet: GARP binds LAP/TGF-β1 to the surface of mASCs. (A): Murine ASCs were surface stained for GARP-PE and LAP-Alexa488 or isotype controls (bottom panels) as described in Materials and Methods. Stainings are shown separately (panels I and II) and overlapping (panel III). A cut mask representing colocalized GARP/LAP/TGF-β1 is shown in panel IV. Cells were imaged using a Zeiss LSM 710 confocal microscope. (B): Murine ASCs were transduced with lentiviral vectors expressing a nonspecific shRNA (LV-CTRL) or GARP-specific shRNAs (LV#3 and LV#6) and GARP silencing in mASCs was assessed by qPCR (left panel) and flow cytometry (right panel). The general vector copy number/mASC for the different LVs used was two to three copies per cell. (C): LV-CTRL and LV#6 mASCs were stained for GARP (left panels) or GARP and LAP/TGF-β1 (right panels). One representative experiment out of three is shown. (D): The LAP/TGF-β1 expression levels (MFI) on NT, LV-CTRL, LV#3, and LV#6 mASCs were measured on a flow cytometer. Results are shown as mean (SEM) of three independent experiments. *, p

    Article Snippet: Cells were incubated with 7AAD (Sigma-Aldrich) and 2.4G2 (for mASCs; eBioscience, San Diego, CA, http://www.eBioscience.com ) followed by anti-mouse LAP/TGF-β1 (TW7-16B4) or anti-human LAP/TGF-β1 (TW4-6H10) (Biolegend, San Diego, CA, http://www.biolegend.com ) followed by goat anti-mouse IgG-APC (Jackson Immunoresearch, West Grove, PA, http://www.jacksonimmuno.com ) or a donkey anti-mouse IgG-Alexa488 (Molecular Probes, Carlsbad, CA, http://www.lifetechnologies.com ), respectively.

    Techniques: Staining, Microscopy, Transduction, Expressing, shRNA, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Plasmid Preparation

    Effects of acute leptin suppression on activity in Tet-off hyperleptinemic transgenic obob mice. ( A ) Strategy followed to obtain transgenic mice chronically over-expressing hleptin on an obob background. LAP-tTa/TRE-hleptin/ obob mice are skinny since hleptin is over-produced until doxycycline (DOX) is administered. ( B ) In Tg obob mice, plasma hleptin levels were suppressed 1, 3, 6, and 13 days after beginning chronic administration DOX in the food at concentrations of 0.1, 0.5, and 2 g/kg. After DOX suppression, hleptin can be turned on again by switching back to regular chow. The “recovery” time necessary to document detectable hleptin in the plasma (1M = 1 month, 2M = 2 months) is a function of the DOX concentration in the food and the duration of the administration as shown after a 13 days of DOX. ( C ) Administration of DOX to LAP-tTa/Tre-hleptin/ obob at 8 weeks of age was accompanied by an increase in body weight. ( D ) The top panel compares an 8 week old LAP-tTa/Tre-hleptin/ obob mouse ( left ) and a littermate obob control ( center ) before DOX. The bottom panel shows the same mice 5 weeks after beginning DOX. ( E–F ) Effect of acute leptin suppression on activity in Tg obob mice. ( E ) After beginning DOX, a steady decrease in HCA is observed in Tg obob mice (n = 5) as shown by linear regression analysis, becoming significant (p≤0.05) after 7 days of DOX and continuing through the end of the experiment (p

    Journal: PLoS ONE

    Article Title: Contrasting Effects of Leptin on Food Anticipatory and Total Locomotor Activity

    doi: 10.1371/journal.pone.0023364

    Figure Lengend Snippet: Effects of acute leptin suppression on activity in Tet-off hyperleptinemic transgenic obob mice. ( A ) Strategy followed to obtain transgenic mice chronically over-expressing hleptin on an obob background. LAP-tTa/TRE-hleptin/ obob mice are skinny since hleptin is over-produced until doxycycline (DOX) is administered. ( B ) In Tg obob mice, plasma hleptin levels were suppressed 1, 3, 6, and 13 days after beginning chronic administration DOX in the food at concentrations of 0.1, 0.5, and 2 g/kg. After DOX suppression, hleptin can be turned on again by switching back to regular chow. The “recovery” time necessary to document detectable hleptin in the plasma (1M = 1 month, 2M = 2 months) is a function of the DOX concentration in the food and the duration of the administration as shown after a 13 days of DOX. ( C ) Administration of DOX to LAP-tTa/Tre-hleptin/ obob at 8 weeks of age was accompanied by an increase in body weight. ( D ) The top panel compares an 8 week old LAP-tTa/Tre-hleptin/ obob mouse ( left ) and a littermate obob control ( center ) before DOX. The bottom panel shows the same mice 5 weeks after beginning DOX. ( E–F ) Effect of acute leptin suppression on activity in Tg obob mice. ( E ) After beginning DOX, a steady decrease in HCA is observed in Tg obob mice (n = 5) as shown by linear regression analysis, becoming significant (p≤0.05) after 7 days of DOX and continuing through the end of the experiment (p

    Article Snippet: In synthesis, LAP-tTA C57Bl6J mice were purchased from Jackson laboratories (Bar Harbor, ME) and crossed with TRE-hleptin Tg mice generated in C57Bl6J background by pronuclear injection.

    Techniques: Activity Assay, Transgenic Assay, Mouse Assay, Expressing, Produced, Concentration Assay, High Content Screening

    (a,b) Western blot detection of PEDF and thrombospondin‐1 in cell conditioned medium. 30 μg proteome were used with a 12.5% reducing SDS‐PAGE. (c) Western blot detection of LAP/TGFβ‐1 in cell conditioned medium. 8 μg proteome were used with a 12.5% reducing SDS‐PAGE. The primary antibody was directed against the LAP domain (prodomain). In line with previous reports (Dubois et al., 1995), the ∼55 kDa band is thought to represent proTGFβ‐1 whereas the predominant ∼40 kDa band is thought to represent LAP. The elevated size in comparison to the primary sequence is thought to stem from glycosylation (Brunner et al., 1992; Dubois et al., 1995). (d) Western blot detection of mature TGFβ‐1 (disulfide linked dimer) in cell conditioned medium. 30 μg proteome were used with a 12.5% non‐reducing SDS‐PAGE. The primary antibody was directed against the actual TGFβ‐1 domain. In addition, N‐terminal degradomics showed TGFβ‐1 activation (see corresponding section). (e) Western blot detection of L1CAM in cell conditioned medium (CCM) and total cell lysate, including cell surface proteins. 8 μg (CCM) and 12 μg proteome (total cell lysate), respectively, were separated with a 7.5% reducing SDS‐PAGE. The molecular weight of 220 kDa for L1CAM is higher than expected but in line with previous reports (Li and Galileo, 2010).

    Journal: Molecular Oncology

    Article Title: Secretome and degradome profiling shows that Kallikrein‐related peptidases 4, 5, 6, and 7 induce TGFβ‐1 signaling in ovarian cancer cells), Secretome and degradome profiling shows that Kallikrein‐related peptidases 4, 5, 6, and 7 induce TGFβ‐1 signaling in ovarian cancer cells

    doi: 10.1016/j.molonc.2013.09.003

    Figure Lengend Snippet: (a,b) Western blot detection of PEDF and thrombospondin‐1 in cell conditioned medium. 30 μg proteome were used with a 12.5% reducing SDS‐PAGE. (c) Western blot detection of LAP/TGFβ‐1 in cell conditioned medium. 8 μg proteome were used with a 12.5% reducing SDS‐PAGE. The primary antibody was directed against the LAP domain (prodomain). In line with previous reports (Dubois et al., 1995), the ∼55 kDa band is thought to represent proTGFβ‐1 whereas the predominant ∼40 kDa band is thought to represent LAP. The elevated size in comparison to the primary sequence is thought to stem from glycosylation (Brunner et al., 1992; Dubois et al., 1995). (d) Western blot detection of mature TGFβ‐1 (disulfide linked dimer) in cell conditioned medium. 30 μg proteome were used with a 12.5% non‐reducing SDS‐PAGE. The primary antibody was directed against the actual TGFβ‐1 domain. In addition, N‐terminal degradomics showed TGFβ‐1 activation (see corresponding section). (e) Western blot detection of L1CAM in cell conditioned medium (CCM) and total cell lysate, including cell surface proteins. 8 μg (CCM) and 12 μg proteome (total cell lysate), respectively, were separated with a 7.5% reducing SDS‐PAGE. The molecular weight of 220 kDa for L1CAM is higher than expected but in line with previous reports (Li and Galileo, 2010).

    Article Snippet: Primary antibodies [PEDF (1:1000, R & D Systems, AF1177), L1CAM (1:1000, R & D Systems, AF277); LAP (TGFβ‐1 prodomain) (1:1000, R & D Systems, AF246NA); tubulin (1:1000, Sigma, T6199), TGFβ‐1 (1:200, R & D Systems, AF7555)] were incubated for 18 h at 4 °C.

    Techniques: Western Blot, SDS Page, Sequencing, Activation Assay, Molecular Weight

    αvβ8 integrin and Band 4.1B localize to adhesion sites when astrocytes are plated on the αvβ8 ligand, LAP-TGFβ1. ( A-C ) Primary astrocytes express endogenous αvβ8 and Band 4.1B. ( A ) Astrocytes express

    Journal:

    Article Title: An interaction between ?v?8 integrin and Band 4.1B via a highly conserved region of the Band 4.1 C-terminal domain

    doi: 10.1073/pnas.0506068102

    Figure Lengend Snippet: αvβ8 integrin and Band 4.1B localize to adhesion sites when astrocytes are plated on the αvβ8 ligand, LAP-TGFβ1. ( A-C ) Primary astrocytes express endogenous αvβ8 and Band 4.1B. ( A ) Astrocytes express

    Article Snippet: Alternatively, astrocytes cultured from neonatal mice as described ( ) were plated onto coverslips coated with 10 μg/ml LAP-TGFβ1 (Research Diagnostics, Flanders, NJ) or laminin (Sigma) for 90 min.

    Techniques:

    Inhibition of hMADS cells adipogenesis by activin A. A and B : hMADS3 cells were induced to undergo adipocyte differentiation in the absence or presence of the indicated concentrations of activin A. Twelve days later, adipogenesis was assessed by Oil red O for lipid droplets staining ( 34 ) and by GPDH activity ( 35 ). C : hMADS3 cells were induced to undergo adipocyte differentiation and treated with 100 ng/ml activin A for the indicated time intervals. GPDH activity was determined at day 12. Results are means of three culture wells (24-well plates). Values are means ± SEM ( N = 3). *Significant differences in GPDH activities in treated cells vs. controls. Similar results were obtained with hMADS2 cells and with hMADS7–B7 and -B9 clones. D : Effects of activin A on the expression of adipogenic genes. hMADS3 cells were induced to undergo differentiation in the absence or presence of 100 ng/ml activin A. RNAs were prepared 6 days after induction of differentiation and expression was investigated by semiquantitative PCR. E : Effects of activin A on the expression of C/EBPb-LAP and -LIP isoforms. hMADS3 cells were induced to undergo differentiation in the absence or presence of 100 ng/ml activin A. Proteins were prepared 6 days after induction of differentiation. LAP and LIP isoforms were examined by Western blot analysis using 25 μg total proteins per lane and anti-C/EBPβ antibodies. Similar results were obtained when proteins were prepared 3 days after induction of differentiation (supplementary Fig. S6). The approximate molecular weight of C/EBPβ isoforms is indicated. The 14 kDa C/EBPβ proteolytic degradation product was not detected. Tubulin was used as a loading control. Samples were run on the same gel. (A high-quality digital representation of this figure is available in the online issue.)

    Journal: Diabetes

    Article Title: Activin A Plays a Critical Role in Proliferation and Differentiation of Human Adipose Progenitors

    doi: 10.2337/db10-0013

    Figure Lengend Snippet: Inhibition of hMADS cells adipogenesis by activin A. A and B : hMADS3 cells were induced to undergo adipocyte differentiation in the absence or presence of the indicated concentrations of activin A. Twelve days later, adipogenesis was assessed by Oil red O for lipid droplets staining ( 34 ) and by GPDH activity ( 35 ). C : hMADS3 cells were induced to undergo adipocyte differentiation and treated with 100 ng/ml activin A for the indicated time intervals. GPDH activity was determined at day 12. Results are means of three culture wells (24-well plates). Values are means ± SEM ( N = 3). *Significant differences in GPDH activities in treated cells vs. controls. Similar results were obtained with hMADS2 cells and with hMADS7–B7 and -B9 clones. D : Effects of activin A on the expression of adipogenic genes. hMADS3 cells were induced to undergo differentiation in the absence or presence of 100 ng/ml activin A. RNAs were prepared 6 days after induction of differentiation and expression was investigated by semiquantitative PCR. E : Effects of activin A on the expression of C/EBPb-LAP and -LIP isoforms. hMADS3 cells were induced to undergo differentiation in the absence or presence of 100 ng/ml activin A. Proteins were prepared 6 days after induction of differentiation. LAP and LIP isoforms were examined by Western blot analysis using 25 μg total proteins per lane and anti-C/EBPβ antibodies. Similar results were obtained when proteins were prepared 3 days after induction of differentiation (supplementary Fig. S6). The approximate molecular weight of C/EBPβ isoforms is indicated. The 14 kDa C/EBPβ proteolytic degradation product was not detected. Tubulin was used as a loading control. Samples were run on the same gel. (A high-quality digital representation of this figure is available in the online issue.)

    Article Snippet: Ecotropic retrovirus vector expressing C/EBPβ-LIP (Addgene plasmid 15714), C/EBPβ-LAP (Addgene plasmid 15712), Klf4 (Addgene plasmid 17219), or green fluorescent protein (GFP) (kindly provided by Dr. Kitamura, University of Tokyo, Tokyo, Japan) were produced in PLAT-E cells (Cell Biolabs).

    Techniques: Inhibition, Staining, Activity Assay, Clone Assay, Expressing, Polymerase Chain Reaction, Western Blot, Molecular Weight

    Suppression of antiadipogenic effects of activin A by C/EBPβ-LAP forced expression. A : hMADS3-EcoRec cells were infected with retroviral vectors expressing GFP, C/EBPβ-LAP, or C/EBPβ-LIP and induced to differentiate into adipocytes in the absence or presence of 100 ng/ml activin A. Adipogenesis was assessed 6 days later by GPDH activities. GPDH activity obtained in the absence of activin A for each transduced cells was taken as 100%. Results are means of three culture wells (24-well plates). Values are means ± SEM ( n = 3). *Significant differences between treated versus untreated cells. B : Western blot analysis of FABP4 in the absence or presence of activin A. Samples were run on the same gel.

    Journal: Diabetes

    Article Title: Activin A Plays a Critical Role in Proliferation and Differentiation of Human Adipose Progenitors

    doi: 10.2337/db10-0013

    Figure Lengend Snippet: Suppression of antiadipogenic effects of activin A by C/EBPβ-LAP forced expression. A : hMADS3-EcoRec cells were infected with retroviral vectors expressing GFP, C/EBPβ-LAP, or C/EBPβ-LIP and induced to differentiate into adipocytes in the absence or presence of 100 ng/ml activin A. Adipogenesis was assessed 6 days later by GPDH activities. GPDH activity obtained in the absence of activin A for each transduced cells was taken as 100%. Results are means of three culture wells (24-well plates). Values are means ± SEM ( n = 3). *Significant differences between treated versus untreated cells. B : Western blot analysis of FABP4 in the absence or presence of activin A. Samples were run on the same gel.

    Article Snippet: Ecotropic retrovirus vector expressing C/EBPβ-LIP (Addgene plasmid 15714), C/EBPβ-LAP (Addgene plasmid 15712), Klf4 (Addgene plasmid 17219), or green fluorescent protein (GFP) (kindly provided by Dr. Kitamura, University of Tokyo, Tokyo, Japan) were produced in PLAT-E cells (Cell Biolabs).

    Techniques: Expressing, Infection, Activity Assay, Western Blot

    NMCAB protects MDM from HIV-1 infection over time MDM were treated with NMCAB, CAB LAP or NCAB containing 100 µM drug for 8h. At days 0, 2, 5,10, and 15 post drug loading, MDM were challenged with HIV-1 ADA at 0.1 MOI for 4 h. Uninfected cells without treatment served as negative controls (Control); HIV-1 infected cells without any treatment served as positive controls (HIV-1). Samples were collected for antiretroviral activity test seven days after viral challenge. ( A ) HIV RT activities were measured in culture media. Results are shown as percentage of RT activities as compared to HIV-1 infected MDM. Data are expressed as mean ± SD for n = 6 samples per group. ( B ) Cells were fixed in paraformaldehyde and stained for HIV-1p24 antigen (brown). Scale bar: 100 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Journal: Biomaterials

    Article Title: Creation of a nanoformulated cabotegravir prodrug with improved antiretroviral profiles

    doi: 10.1016/j.biomaterials.2017.10.023

    Figure Lengend Snippet: NMCAB protects MDM from HIV-1 infection over time MDM were treated with NMCAB, CAB LAP or NCAB containing 100 µM drug for 8h. At days 0, 2, 5,10, and 15 post drug loading, MDM were challenged with HIV-1 ADA at 0.1 MOI for 4 h. Uninfected cells without treatment served as negative controls (Control); HIV-1 infected cells without any treatment served as positive controls (HIV-1). Samples were collected for antiretroviral activity test seven days after viral challenge. ( A ) HIV RT activities were measured in culture media. Results are shown as percentage of RT activities as compared to HIV-1 infected MDM. Data are expressed as mean ± SD for n = 6 samples per group. ( B ) Cells were fixed in paraformaldehyde and stained for HIV-1p24 antigen (brown). Scale bar: 100 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: An important milestone in recent years to improve ART adherence is long-acting parenteral (LAP) antiretroviral drugs (ARVs) [ ].

    Techniques: Infection, Activity Assay, Staining

    Myc and YAP cooperate in inducing liver growth and tumorigenesis. ( A ) Liver weight assessed at 5 wk of induction. Data are reported as percentage relative to total body weight. ( B ) Kaplan-Meier disease-free survival analysis. ( C ) Western blotting analysis of YAP and Myc levels in LAP-tTA tet-YAP mice at the pretumoral stage (4 wk of YAP activation) and in tumors. Vinculin (vin) was used as aninternal control for equal loading. ( D ) Box plot of the expression level of MDSR genes up-regulated in the liver upon YAP and/or Myc induction. ( Inset at the right ) Ranked heat map. ( E , top panel) Heat map of Myc and YAP/TAZ gene signatures based on the expression data of breast cancers (TCGA_BRCA). The heat map was clustered by breast cancer subtypes (basal-like, normal-like, and Luminal/Her2 + ). ( Bottom panel) The statistical track shows the logarithmic plot of P -values for each gene. (Red bars) Genes up in basal-like; (green bars) genes up in Luminal/Her + .

    Journal: Genes & Development

    Article Title: Transcriptional integration of mitogenic and mechanical signals by Myc and YAP

    doi: 10.1101/gad.301184.117

    Figure Lengend Snippet: Myc and YAP cooperate in inducing liver growth and tumorigenesis. ( A ) Liver weight assessed at 5 wk of induction. Data are reported as percentage relative to total body weight. ( B ) Kaplan-Meier disease-free survival analysis. ( C ) Western blotting analysis of YAP and Myc levels in LAP-tTA tet-YAP mice at the pretumoral stage (4 wk of YAP activation) and in tumors. Vinculin (vin) was used as aninternal control for equal loading. ( D ) Box plot of the expression level of MDSR genes up-regulated in the liver upon YAP and/or Myc induction. ( Inset at the right ) Ranked heat map. ( E , top panel) Heat map of Myc and YAP/TAZ gene signatures based on the expression data of breast cancers (TCGA_BRCA). The heat map was clustered by breast cancer subtypes (basal-like, normal-like, and Luminal/Her2 + ). ( Bottom panel) The statistical track shows the logarithmic plot of P -values for each gene. (Red bars) Genes up in basal-like; (green bars) genes up in Luminal/Her + .

    Article Snippet: For liver-specific transgene expression, mice were crossed with LAP-tTA mice expressing the tTA tetracycline transactivator under the control of the LAP promoter [B6.Cg-Tg( tTALap )5Bjd/J; purchased from Jackson Laboratories].

    Techniques: Western Blot, Mouse Assay, Activation Assay, Expressing