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  • 99
    ATCC thp 1 cells thp 1 cells
    ARE inhibited TNF-α-stimulated adhesion of <t>THP-1</t> cells to SVEC cells. A, B. Quantitation of THP-1 cells adhering to untreated SVEC cells and to SVEC cells treated for 4 h with 10 ng/ml TNF-α in the absence or presence of ARE (30, 60 or 120 μg/ml) for up to 2, 4, 6, or 8 h. For details of the adhesion assay see Materials and methods. The number of adherent THP-1 cells was determined using a counting slide. Data are representative of at least three independent experiments and are shown as mean values ± S.D. *P
    Thp 1 Cells Thp 1 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore thp 1 cells thp 1 cells
    hCD13 expression on epithelial cells is necessary and sufficient for CD13‐mediated phagocytosis. (A) HEK293 cells transduced with lentiviral particles containing plasmids for CD13 expression pLenti‐suCMV(hANPEP)‐Rsv(RFP‐Bsd; clones A4 and A6) and transfection control pLenti‐suCMV(empty)‐Rsv(RFP‐Bsd; clone M6) were incubated with 2 µg Fab fragments of mAb452 (anti‐CD13) and 8 µg Fab fragments of mAb32.2 (anti‐FcγRI) or without antibody for 30 minutes at 4°C. Cells were then washed and incubated with secondary goat anti‐mouse FITC. A representative histogram of the expression of hCD13 on these clones is shown. (B) <t>THP‐1</t> cells or (C) HEK293 clones A6, A4, and M6 were incubated with EFab452, EFab32.2, or Ec for 120 minutes at 37°C. Noningested erythrocytes were lysed, and samples were analyzed by flow cytometry to determine the percentage of CFSE‐positive cells. Average of CFSE‐positive cells of 5 independent experiments is shown. ** P
    Thp 1 Cells Thp 1 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 836 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    InvivoGen thp 1 cells thp 1 cells
    Arginine methylation regulates c-Myc transcriptional activation activity. A , c-Myc down-regulates PRMT1 gene expression. Left , Western blot analysis of PRMT1 protein levels in <t>THP-1</t> cells expressing c-Myc. Right , relative mRNA of PRMT1 in THP-1 cells expressing wild-type c-Myc. Data are presented as mean ± S.D. **, p
    Thp 1 Cells Thp 1 Cells, supplied by InvivoGen, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Keygen Biotech thp 1 cells thp 1 cells
    Arginine methylation regulates c-Myc transcriptional activation activity. A , c-Myc down-regulates PRMT1 gene expression. Left , Western blot analysis of PRMT1 protein levels in <t>THP-1</t> cells expressing c-Myc. Right , relative mRNA of PRMT1 in THP-1 cells expressing wild-type c-Myc. Data are presented as mean ± S.D. **, p
    Thp 1 Cells Thp 1 Cells, supplied by Keygen Biotech, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher thp 1 cells thp 1 cells
    miR-223 target ARNT expression and signalling. ( a ) Ingenuity analysis of putative miR-223 targets as determined in silico . Signalling pathways with the highest scores are indicated. ( b ) Luciferase reporter assays of HEK-293 cells transduced with pre-miR-223 or a control (mock), and transfected with the wild-type ikk α- or the arnt 3′UTR reporter constructs. Luciferase activity was normalised to that of mock-transfected cells. Data shown as mean ± SEM of triplicates in one representative experiment ( n = 3). ( c ) Immunoblot analysis of extracts of HEK-293 cells expressing control or pre-miR-223, probed with anti-ARNT and -hnRNP U (nuclear loading control); the hnRNP U/ARNT ratio in mock-transfected cells is equal to 1. For ( b , c ), data are representative of at least three experiments. ( d ) CYP1A1 mRNA levels in mock and pre-miR-223-expressing <t>THP-1</t> cells treated with B a P or vehicle. Data shown as mean ± SEM of RQ from triplicates in three independent experiments. *p
    Thp 1 Cells Thp 1 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    National Centre for Cell Science thp 1 cells
    ( A ) Rv3131 stimulates the secretion of pro-inflammatory cytokines in <t>THP-1</t> cells. THP-1 cells differentiated by PMA were stimulated with 10–2500 ng/ml of rRv3131 or LPS (100 ng/ml) or proteinase K digested rRv3131 for 24 and 48 h. Supernatants were collected and the levels of secreted cytokines were estimated by ELISA. Data represent the mean ± SEM of three technical replicates. *p
    Thp 1 Cells, supplied by National Centre for Cell Science, used in various techniques. Bioz Stars score: 92/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    DSMZ thp 1 cells
    Conditioned medium from monocytic cells stimulates migration of phOBs. In order to investigate the influence of immune cell-conditioned medium on phOBs migration, a scratch assay and an under agar spot assay were performed. phOBs ( N ≥ 4, n ≥ 4) are stimulated with conditioned medium from <t>THP-1</t> cells (representing monocytes), PMA-stimulated adherent THP-1 cells (representing macrophages), and DMSO-stimulated HL-60 cells (representing granulocytes). ( a ) For the scratch assay, gap closure was determined from microscopic images (100 − gap area 40h /gap area 0h × 100) with the help of the ImageJ software. ( b ) Representative microscopic images for the scratch assay (20× magnification). ( c ) For the under agar spot assay, the number of cells in the spot area was determined from microscopic images with the help of the ImageJ software. ( d ) Representative microscopic images for the under agar spot assay (12.5× magnification). Cells were visualized with Calcein-AM stain for living cells. Data are represented in bar diagrams (mean ± 95% C.I.). * p
    Thp 1 Cells, supplied by DSMZ, used in various techniques. Bioz Stars score: 93/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    InvivoGen thp1 blue cd14 cd14 thp 1 cells
    Effects of DDMG-1 on hTNF-α, hIL-8, hIL-6, and hIL-1β production and cell proliferation induced by LPS-stimulated <t>CD14</t> + <t>-THP-1</t> cells. (a) Effects of DDMG-1 on hTNF-α, hIL-8, hIL-6, and hIL-1β production in LPS-stimulated or non-stimulated CD14 + -THP-1 cells. CD14 + -THP-1 cells (1 × 10 6 cells/mL) were untreated or treated with LPS (1 μg/mL) at the indicated concentrations of compounds for 24 h. hTNF-α, hIL-8, hIL-6, and hIL-1β levels in the culture supernatant were measured by ELISA, as described in Materials and Methods. This experiment was repeated three times on different days. Figures were created based on the results. *p
    Thp1 Blue Cd14 Cd14 Thp 1 Cells, supplied by InvivoGen, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DS Pharma Biomedical thp 1 cells
    CCK-8S suppresses both expression of TNF-α and chemotaxis in <t>THP-1</t> cells. A : THP-1 cells were cultured under different conditions for 72 h. CCK-8S inhibited HG-induced TNF-α expression in THP-1 cells ( n = 5 each). Values are presented as the ratio of HG group. *** P
    Thp 1 Cells, supplied by DS Pharma Biomedical, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare thp 1 cells
    SAB inhibits oxLDL-induced upregulation of CD36 mRNA in RAW 264.7 cells (A), <t>THP-1</t> cells (B) and primary macrophages (C). Cells were exposed to the indicated concentration of SAB in the presence or absence of 25 μg/mL oxLDL, and the CD36 mRNA
    Thp 1 Cells, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 395 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam thp 1 cells
    SAMHD1 knockdown and Vpx enhance the dNTP pool in PMA-treated <t>THP-1</t> cells. (a) THP-1, engineered to stably express shRNA scrambled (control) or shRNA specifically targeting SAMHD1, were differentiated overnight with PMA. Where indicated, the cells were incubated for 2 h with Vpx-containing and control VLP (+/−Vpx). Cell extracts were analyzed by western blot with the indicated antibodies. (b) dNTP concentrations were determined by the single nucleotide incorporation assay described in Diamond et al . The experiment was performed in duplicate and one representative result is shown. (c) dNTP triphosphohydrolase activity of recombinant SAMHD1. dNTPase activity assay was performed as described in the methods section in the presence of the indicated dNTP, 1 μCi of the corresponding γ- 32 P-dNTP and 1 μM of wild type SAMHD1. Where indicated 200 μM of unlabeled dGTP was added. The non-labeled standards were visualized by UV shadowing and γ- 32 P-labeled nucleotides were visualized using a phosphorimager (Fluorescent Image Analyzer FLA3000 (Fuji).
    Thp 1 Cells, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Addgene inc thp 1 cells plenticrisprv2
    SAMHD1 knockdown and Vpx enhance the dNTP pool in PMA-treated <t>THP-1</t> cells. (a) THP-1, engineered to stably express shRNA scrambled (control) or shRNA specifically targeting SAMHD1, were differentiated overnight with PMA. Where indicated, the cells were incubated for 2 h with Vpx-containing and control VLP (+/−Vpx). Cell extracts were analyzed by western blot with the indicated antibodies. (b) dNTP concentrations were determined by the single nucleotide incorporation assay described in Diamond et al . The experiment was performed in duplicate and one representative result is shown. (c) dNTP triphosphohydrolase activity of recombinant SAMHD1. dNTPase activity assay was performed as described in the methods section in the presence of the indicated dNTP, 1 μCi of the corresponding γ- 32 P-dNTP and 1 μM of wild type SAMHD1. Where indicated 200 μM of unlabeled dGTP was added. The non-labeled standards were visualized by UV shadowing and γ- 32 P-labeled nucleotides were visualized using a phosphorimager (Fluorescent Image Analyzer FLA3000 (Fuji).
    Thp 1 Cells Plenticrisprv2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ScienCell human thp 1 cells
    Catalpol increased telomerase activity and inhibited macrophage senescence. 6–8-week-old male LDLr −/− mice were administered with a standard diet with catalpol (0 and 100 mg/kg) or HFD with catalpol (0, 100, and 200 mg/kg). <t>THP-1</t> cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation. Then, macrophages were treated with oxLDL or catalpol (0, 5, 20, and 80 μ M) for 24 h as indicated. Catalpol increased telomerase activity in (a) LDLr −/− mice and (b) oxLDL-treated macrophages, n = 10. ∗∗ p
    Human Thp 1 Cells, supplied by ScienCell, 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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    Image Search Results


    ARE inhibited TNF-α-stimulated adhesion of THP-1 cells to SVEC cells. A, B. Quantitation of THP-1 cells adhering to untreated SVEC cells and to SVEC cells treated for 4 h with 10 ng/ml TNF-α in the absence or presence of ARE (30, 60 or 120 μg/ml) for up to 2, 4, 6, or 8 h. For details of the adhesion assay see Materials and methods. The number of adherent THP-1 cells was determined using a counting slide. Data are representative of at least three independent experiments and are shown as mean values ± S.D. *P

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Anti-atherosclerotic function of Astragali Radix extract: downregulation of adhesion molecules in vitro and in vivo

    doi: 10.1186/1472-6882-12-54

    Figure Lengend Snippet: ARE inhibited TNF-α-stimulated adhesion of THP-1 cells to SVEC cells. A, B. Quantitation of THP-1 cells adhering to untreated SVEC cells and to SVEC cells treated for 4 h with 10 ng/ml TNF-α in the absence or presence of ARE (30, 60 or 120 μg/ml) for up to 2, 4, 6, or 8 h. For details of the adhesion assay see Materials and methods. The number of adherent THP-1 cells was determined using a counting slide. Data are representative of at least three independent experiments and are shown as mean values ± S.D. *P

    Article Snippet: Culture of THP-1 cells THP-1 cells were purchased from the American Type Culture Collection and grown in RPMI-1640 media (Sigma) containing 10 % FCS, 100 μg/ml streptomycin, 100 IU/ml penicillin, 250 ng/ml fungizone, 1 mM glutamine, 5 × 10–5 M 2-mercaptoethanol, and routinely subcultured three times per week at a ratio of 1:5.

    Techniques: Quantitation Assay, Cell Adhesion Assay

    Subcellular location of NLRP3 inflammasome requires F-actin but not active polymerization. ( A,B ) Primed THP-1 cells were activated with ( A ) ATP and ( B ) nigericin after pretreatment with cytochalasin D (CytoD) or latrunculin B (LatB). Cytosolic (CYT) and cytoskeletal (CSK) fractions were analyzed by Western blot. The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of subcellular location of ASC and NLRP3 in primed THP-1 cells treated or not with cytochalasin D (CytoD) and latrunculin B (LatB) prior to activation with ATP or nigericin for 6 h; Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments.

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: Subcellular location of NLRP3 inflammasome requires F-actin but not active polymerization. ( A,B ) Primed THP-1 cells were activated with ( A ) ATP and ( B ) nigericin after pretreatment with cytochalasin D (CytoD) or latrunculin B (LatB). Cytosolic (CYT) and cytoskeletal (CSK) fractions were analyzed by Western blot. The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of subcellular location of ASC and NLRP3 in primed THP-1 cells treated or not with cytochalasin D (CytoD) and latrunculin B (LatB) prior to activation with ATP or nigericin for 6 h; Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments.

    Article Snippet: Cells THP-1 cells (ATCC) were grown in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 50 μg/ml streptomycin, 50 U/ml penicillin, 2 mM glutamine (medium) and 0.05 mM β-mercaptoethanol.

    Techniques: Western Blot, Confocal Microscopy, Activation Assay

    F-actin downregulates NLRP3 inflammasome activity. ( A ) Actin polymerization was performed with 9.6 μM of pyrene-labeled G-actin containing ATP- or nigericin-activated THP-1 cells lysate. The polymerization was initiated by addition of actin polymerization buffer (arrow). Representative experiments out of 3 are presented. ( B ) Area under the curve (AUC) represented in ( A ) was calculated using GraphPad Prism version 6. Data are means ± SEM of at least 3 independent. ( C,D ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and then activated by ATP ( C ) and nigericin ( D ) for 6 h. Data are means ± SEM of at least 3 independent. ( E,F ) IL-1β production in culture supernatants of LPS-primed primary human monocytes pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and stimulated or not with ATP ( E ) or nigericin ( F ) for 15 min. Data are means ± SEM of at least 3 independent. ( G,H ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); total, uncleaved caspase-1 in cell lysate is presented in bottom panels. The cropped blots were run under the same experimental conditions. The asterisk indicates nonspecific crossreactive bands. Data are means ± SEM of at least 4 independent experiments (upper panels) or representative of 3 independent experiments (bottom panels). Statistical significance was determined by Mann-Whitney U analysis. See also Figures S1 and S2 .

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: F-actin downregulates NLRP3 inflammasome activity. ( A ) Actin polymerization was performed with 9.6 μM of pyrene-labeled G-actin containing ATP- or nigericin-activated THP-1 cells lysate. The polymerization was initiated by addition of actin polymerization buffer (arrow). Representative experiments out of 3 are presented. ( B ) Area under the curve (AUC) represented in ( A ) was calculated using GraphPad Prism version 6. Data are means ± SEM of at least 3 independent. ( C,D ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and then activated by ATP ( C ) and nigericin ( D ) for 6 h. Data are means ± SEM of at least 3 independent. ( E,F ) IL-1β production in culture supernatants of LPS-primed primary human monocytes pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and stimulated or not with ATP ( E ) or nigericin ( F ) for 15 min. Data are means ± SEM of at least 3 independent. ( G,H ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); total, uncleaved caspase-1 in cell lysate is presented in bottom panels. The cropped blots were run under the same experimental conditions. The asterisk indicates nonspecific crossreactive bands. Data are means ± SEM of at least 4 independent experiments (upper panels) or representative of 3 independent experiments (bottom panels). Statistical significance was determined by Mann-Whitney U analysis. See also Figures S1 and S2 .

    Article Snippet: Cells THP-1 cells (ATCC) were grown in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 50 μg/ml streptomycin, 50 U/ml penicillin, 2 mM glutamine (medium) and 0.05 mM β-mercaptoethanol.

    Techniques: Activity Assay, Labeling, Enzyme-linked Immunosorbent Assay, Western Blot, MANN-WHITNEY

    Ca 2+ increase the NLRP3 inflammasome activation by enhancing the severing of F-actin by FliI. ( A ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of CaCl 2 (Ca 2+ ) and stimulated with nigericin. Data are means ± SEM of at least 3 independent. ( B ) Caspase-1 in supernatants (SN) of THP-1 cells pretreated with 1.6 mM CaCl 2 and activated by nigericin as indicated and assessed by ELISA. Data are means ± SEM of at least 3 independent. ( C ) Actin depolymerization was performed with 0.2 μg/ml of pyrene-labeled F-actin containing nigericin-activated THP-1 cells lysate. Representative pictures of 3 independent experiments. ( D ) Area under the curve (AUC) represented in ( C ) was calculated using GraphPad Prism version 6. Data are represented as mean ± SEM of at least 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. See Figure S4 .

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: Ca 2+ increase the NLRP3 inflammasome activation by enhancing the severing of F-actin by FliI. ( A ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of CaCl 2 (Ca 2+ ) and stimulated with nigericin. Data are means ± SEM of at least 3 independent. ( B ) Caspase-1 in supernatants (SN) of THP-1 cells pretreated with 1.6 mM CaCl 2 and activated by nigericin as indicated and assessed by ELISA. Data are means ± SEM of at least 3 independent. ( C ) Actin depolymerization was performed with 0.2 μg/ml of pyrene-labeled F-actin containing nigericin-activated THP-1 cells lysate. Representative pictures of 3 independent experiments. ( D ) Area under the curve (AUC) represented in ( C ) was calculated using GraphPad Prism version 6. Data are represented as mean ± SEM of at least 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. See Figure S4 .

    Article Snippet: Cells THP-1 cells (ATCC) were grown in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 50 μg/ml streptomycin, 50 U/ml penicillin, 2 mM glutamine (medium) and 0.05 mM β-mercaptoethanol.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Labeling, MANN-WHITNEY

    FliI and LRRFIP2 enable inhibition of NLRP3 inflammasome activity via co-localization with F-actin. ( A ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( B ) Western blot analysis of FliI and LRRFIP2 expression in THP-1 cells stably transduced with lentivirus carrying FliI and LRRFIP2 shRNA. Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells transduced with FliI and LRRFIP2 shRNA. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( D ) IL-1β production in culture supernatants of THP-1 cells transduced with FliI and LRRFIP2 shRNA and unstimulated or stimulated with ATP and nigericin. Data are means ± SEM of at least 3 independent. ( E ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); the presence of caspase-1 was assessed by Western blot into cells lysate. Data are represented as mean ± SEM of at least 3 independent experiments or representative of 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. The cropped blots were run under the same experimental conditions. See Figure S3 .

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: FliI and LRRFIP2 enable inhibition of NLRP3 inflammasome activity via co-localization with F-actin. ( A ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( B ) Western blot analysis of FliI and LRRFIP2 expression in THP-1 cells stably transduced with lentivirus carrying FliI and LRRFIP2 shRNA. Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells transduced with FliI and LRRFIP2 shRNA. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( D ) IL-1β production in culture supernatants of THP-1 cells transduced with FliI and LRRFIP2 shRNA and unstimulated or stimulated with ATP and nigericin. Data are means ± SEM of at least 3 independent. ( E ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); the presence of caspase-1 was assessed by Western blot into cells lysate. Data are represented as mean ± SEM of at least 3 independent experiments or representative of 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. The cropped blots were run under the same experimental conditions. See Figure S3 .

    Article Snippet: Cells THP-1 cells (ATCC) were grown in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 50 μg/ml streptomycin, 50 U/ml penicillin, 2 mM glutamine (medium) and 0.05 mM β-mercaptoethanol.

    Techniques: Inhibition, Activity Assay, Confocal Microscopy, Western Blot, Expressing, Stable Transfection, Transduction, shRNA, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    NLRP3 inflammasome interacts with F-actin in ATP- and nigericin-treated THP-1 cells. ( A,B ) Primed THP-1 cells were activated with 5 mM ATP or 1 μM nigericin (NIG) for 6 h. Cytosolic (CYT) and cytoskeletal (CSK) fractions of ( A ) ATP- and ( B ) nigericin-activated cells were subjected to Western blot and analyzed for the presence of ASC, NLRP3, pro-IL-1β and vimentin (control for cytoskeletal fraction). The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells; nuclei are stained in blue. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Data are representative of 3 independent experiments. ( D ) Primed THP-1 cells were activated with 5 mM ATP or 1 μM nigericin (NIG) for 6 h, immunoprecipitated with anti-actin and subjected to Western blot and analyzed for the presence of ASC, NLRP3 and actin. The cropped blots were run under the same experimental conditions; Data are representative of 2 independent experiments.

    Journal: Scientific Reports

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    doi: 10.1038/srep29834

    Figure Lengend Snippet: NLRP3 inflammasome interacts with F-actin in ATP- and nigericin-treated THP-1 cells. ( A,B ) Primed THP-1 cells were activated with 5 mM ATP or 1 μM nigericin (NIG) for 6 h. Cytosolic (CYT) and cytoskeletal (CSK) fractions of ( A ) ATP- and ( B ) nigericin-activated cells were subjected to Western blot and analyzed for the presence of ASC, NLRP3, pro-IL-1β and vimentin (control for cytoskeletal fraction). The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells; nuclei are stained in blue. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Data are representative of 3 independent experiments. ( D ) Primed THP-1 cells were activated with 5 mM ATP or 1 μM nigericin (NIG) for 6 h, immunoprecipitated with anti-actin and subjected to Western blot and analyzed for the presence of ASC, NLRP3 and actin. The cropped blots were run under the same experimental conditions; Data are representative of 2 independent experiments.

    Article Snippet: Cells THP-1 cells (ATCC) were grown in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 50 μg/ml streptomycin, 50 U/ml penicillin, 2 mM glutamine (medium) and 0.05 mM β-mercaptoethanol.

    Techniques: Western Blot, Confocal Microscopy, Staining, Immunoprecipitation

    EL ISA results for relative response of TNF- α and MCP-1 in LPS-treated THP-1 cells. Each extract was tested at three different concentrations (50 μ g/ml, 10 μ g/ml, and 1 μ g/ml), and the results are presented as mean with SD displayed as positive bars ( n = 3). (a) TNF- α secretion relative to control. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Antioxidant and Anti-Inflammatory Activities in Extracts from Minke Whale (Balaenoptera acutorostrata) Blubber

    doi: 10.1155/2017/3835851

    Figure Lengend Snippet: EL ISA results for relative response of TNF- α and MCP-1 in LPS-treated THP-1 cells. Each extract was tested at three different concentrations (50 μ g/ml, 10 μ g/ml, and 1 μ g/ml), and the results are presented as mean with SD displayed as positive bars ( n = 3). (a) TNF- α secretion relative to control. ∗ p

    Article Snippet: Differentiated THP-1 Cells THP-1 (number TIB-202, ATCC) is a monocyte cell line derived from a patient with acute monocytic leukemia.

    Techniques:

    Illustration showing Al 2 O 3 thin film coating of MWCNT by ALD and the effect on mononuclear cell cytokine production in vitro and pro-fibrogenic responses in the lungs of mice in vivo . Al 2 O 3 -coated MWCNTs (A-MWCNT) increased IL-1β secreted by THP-1 cells and PBMCs in vitro , but decreased IL-6, OPN, and TNF-α protein levels compared to uncoated (U)-MWCNTs. In vivo , ALD coating reduced lung mRNA levels of OPN and IL-6 but had no effect (NE) on lung mRNA levels of IL-1β or TNF-α at either 1 or 28 days post-exposure and reduced protein levels of IL-1β, OPN, and TNF-α measured in BALF at 1 or 28 days. ALD coating significantly reduced MWCNT-induced lung fibrosis in mice. Also, A-MWCNTs undergo breakage along the radial axis, which could potentially increase lung clearance of MWCNTs and reduce the potential for fibrosis.

    Journal: PLoS ONE

    Article Title: Atomic Layer Deposition Coating of Carbon Nanotubes with Aluminum Oxide Alters Pro-Fibrogenic Cytokine Expression by Human Mononuclear Phagocytes In Vitro and Reduces Lung Fibrosis in Mice In Vivo

    doi: 10.1371/journal.pone.0106870

    Figure Lengend Snippet: Illustration showing Al 2 O 3 thin film coating of MWCNT by ALD and the effect on mononuclear cell cytokine production in vitro and pro-fibrogenic responses in the lungs of mice in vivo . Al 2 O 3 -coated MWCNTs (A-MWCNT) increased IL-1β secreted by THP-1 cells and PBMCs in vitro , but decreased IL-6, OPN, and TNF-α protein levels compared to uncoated (U)-MWCNTs. In vivo , ALD coating reduced lung mRNA levels of OPN and IL-6 but had no effect (NE) on lung mRNA levels of IL-1β or TNF-α at either 1 or 28 days post-exposure and reduced protein levels of IL-1β, OPN, and TNF-α measured in BALF at 1 or 28 days. ALD coating significantly reduced MWCNT-induced lung fibrosis in mice. Also, A-MWCNTs undergo breakage along the radial axis, which could potentially increase lung clearance of MWCNTs and reduce the potential for fibrosis.

    Article Snippet: Human THP-1 Cells THP-1 cells were purchased from ATCC (Manassas, VA) and cultured according to a standard protocol used by the NIEHS Nano GO Consortium .

    Techniques: In Vitro, Mouse Assay, In Vivo

    Cytokine protein levels secreted by THP-1 cells 24 hr after exposure to MWCNTs coated with Al 2 O 3 by ALD. THP-1 cells were exposed to low (10 µg/ml) and high (100 µg/ml) doses of uncoated MWCNTs (U-MWCNT) or atomic layer deposition (ALD)-coated MWCNTs (A-MWCNT) functionalized with increasing layers of Al 2 O 3 achieved by 10, 50, or 100 ALD cycles (A-MWCNT). ELISAs were performed on cell supernatants for A ) IL-1β, B ) OPN, C ) IL-6 and D ) TNF-α. Data are representative graphs of three separate experiments and are expressed as means ± SEM. P values are indicated for each graph. *Significant compared to control. ∧Significant effect between A-MWCNT and U-MWCNT at the same dose.

    Journal: PLoS ONE

    Article Title: Atomic Layer Deposition Coating of Carbon Nanotubes with Aluminum Oxide Alters Pro-Fibrogenic Cytokine Expression by Human Mononuclear Phagocytes In Vitro and Reduces Lung Fibrosis in Mice In Vivo

    doi: 10.1371/journal.pone.0106870

    Figure Lengend Snippet: Cytokine protein levels secreted by THP-1 cells 24 hr after exposure to MWCNTs coated with Al 2 O 3 by ALD. THP-1 cells were exposed to low (10 µg/ml) and high (100 µg/ml) doses of uncoated MWCNTs (U-MWCNT) or atomic layer deposition (ALD)-coated MWCNTs (A-MWCNT) functionalized with increasing layers of Al 2 O 3 achieved by 10, 50, or 100 ALD cycles (A-MWCNT). ELISAs were performed on cell supernatants for A ) IL-1β, B ) OPN, C ) IL-6 and D ) TNF-α. Data are representative graphs of three separate experiments and are expressed as means ± SEM. P values are indicated for each graph. *Significant compared to control. ∧Significant effect between A-MWCNT and U-MWCNT at the same dose.

    Article Snippet: Human THP-1 Cells THP-1 cells were purchased from ATCC (Manassas, VA) and cultured according to a standard protocol used by the NIEHS Nano GO Consortium .

    Techniques:

    Transmission electron microscopy of human macrophages engulfing MWCNTs. A ) Lower magnification (x18000) of uncoated, sonicated MWCNTs contained in vesicles within a THP-1 macrophage. B ) Higher magnification (x56000) of uncoated, sonicated MWCNTs within a vesicle of a THP-1 macrophage. C ) High magnification (x56000) of a single (unsonicated) MWCNT within the cytoplasm of a THP-1 macrophage. D ) Lower magnification (x7100) of a THP-1 macrophage with sonicated Al 2 O 3 coated MWCNTs (50 ALD cycles) in the cytoplasm. E ) Higher magnification (x56000) of a cluster of sonicated Al 2 O 3 -coated MWCNT fragments (50 ALD cycles within the cytoplasm of a THP-1 macrophage. F ) High magnification (x36000) of a THP-1 macrophage with Al 2 O 3 coated MWCNTs (50 ALD cycles –unsonicated) in the cytoplasm in close proximity to the nucleus. G ) Low magnification (x7100) of a macrophage with Al 2 O 3 -coated (100ALD cycles) sonicated MWCNTs in the cytoplasm. H ) Inset showing a higher magnification (x56000) of a cluster of sonicated Al 2 O 3 -coated MWCNTs (100 ALD cycles) within the cytoplasm of a macrophage. I ) High magnification (x56000) showing unsonicated Al 2 O 3 -coated (100 ALD cycles) MWCNTs in the cytoplasm of a THP-1 macrophage. J ) High magnification inverted image of ALD-coated MWCNTs from panel H showing fracture points of breakage (arrows). K ) Al 2 O 3 coated MWCNT length (nm) as a function of ALD cycle post sonication. Data represent mean values (±SEM) of 20 measurements of nanotubes thickness and length.

    Journal: PLoS ONE

    Article Title: Atomic Layer Deposition Coating of Carbon Nanotubes with Aluminum Oxide Alters Pro-Fibrogenic Cytokine Expression by Human Mononuclear Phagocytes In Vitro and Reduces Lung Fibrosis in Mice In Vivo

    doi: 10.1371/journal.pone.0106870

    Figure Lengend Snippet: Transmission electron microscopy of human macrophages engulfing MWCNTs. A ) Lower magnification (x18000) of uncoated, sonicated MWCNTs contained in vesicles within a THP-1 macrophage. B ) Higher magnification (x56000) of uncoated, sonicated MWCNTs within a vesicle of a THP-1 macrophage. C ) High magnification (x56000) of a single (unsonicated) MWCNT within the cytoplasm of a THP-1 macrophage. D ) Lower magnification (x7100) of a THP-1 macrophage with sonicated Al 2 O 3 coated MWCNTs (50 ALD cycles) in the cytoplasm. E ) Higher magnification (x56000) of a cluster of sonicated Al 2 O 3 -coated MWCNT fragments (50 ALD cycles within the cytoplasm of a THP-1 macrophage. F ) High magnification (x36000) of a THP-1 macrophage with Al 2 O 3 coated MWCNTs (50 ALD cycles –unsonicated) in the cytoplasm in close proximity to the nucleus. G ) Low magnification (x7100) of a macrophage with Al 2 O 3 -coated (100ALD cycles) sonicated MWCNTs in the cytoplasm. H ) Inset showing a higher magnification (x56000) of a cluster of sonicated Al 2 O 3 -coated MWCNTs (100 ALD cycles) within the cytoplasm of a macrophage. I ) High magnification (x56000) showing unsonicated Al 2 O 3 -coated (100 ALD cycles) MWCNTs in the cytoplasm of a THP-1 macrophage. J ) High magnification inverted image of ALD-coated MWCNTs from panel H showing fracture points of breakage (arrows). K ) Al 2 O 3 coated MWCNT length (nm) as a function of ALD cycle post sonication. Data represent mean values (±SEM) of 20 measurements of nanotubes thickness and length.

    Article Snippet: Human THP-1 Cells THP-1 cells were purchased from ATCC (Manassas, VA) and cultured according to a standard protocol used by the NIEHS Nano GO Consortium .

    Techniques: Transmission Assay, Electron Microscopy, Sonication

    Cytokine protein levels secreted by THP-1 cells 24 hr after exposure to Al 2 O 3 or carbon black nanoparticles (CB NP). THP-1 cells were exposed to low (10 µg/ml) and high (100 µg/ml) doses of Al 2 O 3 or CB NP. ELISAs were performed on cell supernatants for ELISAs were performed on cell supernatants for A) IL-1β, B) OPN, C) IL-6 and D) TNF-α. Data are representative graphs of three separate experiments and are expressed as means ± SEM. Asterisk directly above bar indicate comparison to control, whereas asterisk over connecting bar indicate comparison between CB NP and Al 2 O 3 NP. * P

    Journal: PLoS ONE

    Article Title: Atomic Layer Deposition Coating of Carbon Nanotubes with Aluminum Oxide Alters Pro-Fibrogenic Cytokine Expression by Human Mononuclear Phagocytes In Vitro and Reduces Lung Fibrosis in Mice In Vivo

    doi: 10.1371/journal.pone.0106870

    Figure Lengend Snippet: Cytokine protein levels secreted by THP-1 cells 24 hr after exposure to Al 2 O 3 or carbon black nanoparticles (CB NP). THP-1 cells were exposed to low (10 µg/ml) and high (100 µg/ml) doses of Al 2 O 3 or CB NP. ELISAs were performed on cell supernatants for ELISAs were performed on cell supernatants for A) IL-1β, B) OPN, C) IL-6 and D) TNF-α. Data are representative graphs of three separate experiments and are expressed as means ± SEM. Asterisk directly above bar indicate comparison to control, whereas asterisk over connecting bar indicate comparison between CB NP and Al 2 O 3 NP. * P

    Article Snippet: Human THP-1 Cells THP-1 cells were purchased from ATCC (Manassas, VA) and cultured according to a standard protocol used by the NIEHS Nano GO Consortium .

    Techniques:

    Pretreatment with 5,6-EET increased the adhesiveness of HCAECs to THP-1 cells. HCAECs were untreated ( A ) or treated with vehicle ( B ), 0.25 nM ( C ), 2.5 nM (D ), or 25 nM ( E ) 5,6-EET for 24 h. The amount of THP-1 cell adherence is represented as relative fluorescence units. Pretreatment with 0.25 nM, 2.5 nM, or 25 nM 5,6-EET significantly and dose-dependently increased the adhesiveness of HCAECs to THP-1 cells ( P

    Journal: Scientific Reports

    Article Title: Association of Arachidonic Acid-derived Lipid Mediators with Subsequent Onset of Acute Myocardial Infarction in Patients with Coronary Artery Disease

    doi: 10.1038/s41598-020-65014-z

    Figure Lengend Snippet: Pretreatment with 5,6-EET increased the adhesiveness of HCAECs to THP-1 cells. HCAECs were untreated ( A ) or treated with vehicle ( B ), 0.25 nM ( C ), 2.5 nM (D ), or 25 nM ( E ) 5,6-EET for 24 h. The amount of THP-1 cell adherence is represented as relative fluorescence units. Pretreatment with 0.25 nM, 2.5 nM, or 25 nM 5,6-EET significantly and dose-dependently increased the adhesiveness of HCAECs to THP-1 cells ( P

    Article Snippet: THP-1 cell culture THP-1 cells were purchased from ATCC (Manassas, VA, USA), and incubated at 37 °C in 5% CO2 .

    Techniques: Fluorescence

    Pretreatment with 14,15-EET increased the adhesiveness of HCAECs to THP-1 cells. HCAECs were untreated ( A ) or treated with vehicle ( B ), 0.25 nM ( C ), 2.5 nM ( D ), or 25 nM ( E ) 14,15-EET for 24 h. The amount of THP-1 cell adherence is represented as relative fluorescence units. Pretreatment with 0.25 nM, 2.5 nM, or 25 nM 5,6-EET significantly and dose-dependently increased the adhesiveness of HCAECs to THP-1 cells ( P

    Journal: Scientific Reports

    Article Title: Association of Arachidonic Acid-derived Lipid Mediators with Subsequent Onset of Acute Myocardial Infarction in Patients with Coronary Artery Disease

    doi: 10.1038/s41598-020-65014-z

    Figure Lengend Snippet: Pretreatment with 14,15-EET increased the adhesiveness of HCAECs to THP-1 cells. HCAECs were untreated ( A ) or treated with vehicle ( B ), 0.25 nM ( C ), 2.5 nM ( D ), or 25 nM ( E ) 14,15-EET for 24 h. The amount of THP-1 cell adherence is represented as relative fluorescence units. Pretreatment with 0.25 nM, 2.5 nM, or 25 nM 5,6-EET significantly and dose-dependently increased the adhesiveness of HCAECs to THP-1 cells ( P

    Article Snippet: THP-1 cell culture THP-1 cells were purchased from ATCC (Manassas, VA, USA), and incubated at 37 °C in 5% CO2 .

    Techniques: Fluorescence

    Mechanism of Action of a JQ1-VHL PROTAC (A) Schematic representation of the mechanism of action of a PROTAC. (B) Scatterplots of protein fold changes (FC) observed in mature (upper panel) and nascent (lower panel) forms of proteins in THP-1 cells treated for 6 hr with the BET inhibitor JQ1-Az (10 μM), the JQ1-VHL PROTAC (1 and 10 μM), or VHL alkyne (10 μM) relative to vehicle control in two biological replicate experiments. Red closed circles indicate statistically significant regulation (p

    Journal: Cell

    Article Title: Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis

    doi: 10.1016/j.cell.2018.02.030

    Figure Lengend Snippet: Mechanism of Action of a JQ1-VHL PROTAC (A) Schematic representation of the mechanism of action of a PROTAC. (B) Scatterplots of protein fold changes (FC) observed in mature (upper panel) and nascent (lower panel) forms of proteins in THP-1 cells treated for 6 hr with the BET inhibitor JQ1-Az (10 μM), the JQ1-VHL PROTAC (1 and 10 μM), or VHL alkyne (10 μM) relative to vehicle control in two biological replicate experiments. Red closed circles indicate statistically significant regulation (p

    Article Snippet: THP-1 cells THP-1 cell cultures (ATCC TIB-202, male) were established in normal growth medium (RPMI1640 + 10% FBS) or RPMI-based SILAC-L and SILAC-H medium.

    Techniques:

    Off-Target Effects of JQ1 and the JQ1-VHL-PROTAC (A) Imaging of nuclear RNA content by fluorescence in situ hybridization (FISH) and confocal microscopy. THP-1 cells were treated with vehicle, JQ1-Az (10 μM), JQ1-VHL-PROTAC (at 1 and 10 μM), or I-BET-151-VHL-PROTAC (10 μM) for 6 hr, fixed, and processed for FISH using Cy3-labeled oligo-dT50. Nuclei were stained by Hoechst. Representative fluorescent images recorded after excitation at 514 nm (Cy3, gray, upper panel) are shown. The lower panel displays an overlay of Cy3 staining (gray) and Hoechst staining (cyan). Scale bar, 20 μm. (B) Bar chart displaying the ratio of mean fluorescence intensity of the FISH probe (Cy3 channel) between nucleus (defined by Hoechst staining) and cytosol (cell borders as defined by WGA staining) was calculated for single cells (706–1,215 cells per condition). Mean fluorescence of treated samples is normalized to control vehicle. SEM is shown. The experiment was repeated three times ( Figures S3 A and S3B). (C) Scheme of 2D thermal proteome profiling (2D-TPP) experiments. (D) 2D-TPP results for JQ1 and I-BET151. Sigmoidal curves show dose-dependent changes in thermal stability for selected proteins. pEC 50 is defined as – log 10 (EC 50 ). (E) Dose-dependent effects of cellular JQ1 treatment on the thermal stability of five proteins involved in cholesterol biosynthesis revealed by 2D-TPP. The table shows pEC 50 s for dose-dependent stabilization; the pathway is displayed in the center, and enzymes are marked in blue. Curves depict dose-dependent stabilization in JQ1-treated cells for indicated enzymes.

    Journal: Cell

    Article Title: Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis

    doi: 10.1016/j.cell.2018.02.030

    Figure Lengend Snippet: Off-Target Effects of JQ1 and the JQ1-VHL-PROTAC (A) Imaging of nuclear RNA content by fluorescence in situ hybridization (FISH) and confocal microscopy. THP-1 cells were treated with vehicle, JQ1-Az (10 μM), JQ1-VHL-PROTAC (at 1 and 10 μM), or I-BET-151-VHL-PROTAC (10 μM) for 6 hr, fixed, and processed for FISH using Cy3-labeled oligo-dT50. Nuclei were stained by Hoechst. Representative fluorescent images recorded after excitation at 514 nm (Cy3, gray, upper panel) are shown. The lower panel displays an overlay of Cy3 staining (gray) and Hoechst staining (cyan). Scale bar, 20 μm. (B) Bar chart displaying the ratio of mean fluorescence intensity of the FISH probe (Cy3 channel) between nucleus (defined by Hoechst staining) and cytosol (cell borders as defined by WGA staining) was calculated for single cells (706–1,215 cells per condition). Mean fluorescence of treated samples is normalized to control vehicle. SEM is shown. The experiment was repeated three times ( Figures S3 A and S3B). (C) Scheme of 2D thermal proteome profiling (2D-TPP) experiments. (D) 2D-TPP results for JQ1 and I-BET151. Sigmoidal curves show dose-dependent changes in thermal stability for selected proteins. pEC 50 is defined as – log 10 (EC 50 ). (E) Dose-dependent effects of cellular JQ1 treatment on the thermal stability of five proteins involved in cholesterol biosynthesis revealed by 2D-TPP. The table shows pEC 50 s for dose-dependent stabilization; the pathway is displayed in the center, and enzymes are marked in blue. Curves depict dose-dependent stabilization in JQ1-treated cells for indicated enzymes.

    Article Snippet: THP-1 cells THP-1 cell cultures (ATCC TIB-202, male) were established in normal growth medium (RPMI1640 + 10% FBS) or RPMI-based SILAC-L and SILAC-H medium.

    Techniques: Imaging, Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Confocal Microscopy, Labeling, Staining, Whole Genome Amplification

    Assessment of Reproducibility of Biological Replicates and Technical Validation of the Quantification, Related to Figure 2 (A–E) Samples were analyzed on a Q-executive (using MS 2 for reporter ion quantification) as well as on a Fusion Lumos (using SPS MS 3 for reporter ion quantification) mass spectrometer to assess the reproducibility of biological replicates and as a technical validation of the quantification. (A) Annotated MS 2 and MS 3 spectra for the BRD2 peptide VVHIIQAR. (B) The first two scatterplots compare protein fold changes (FC) between two biological replicates using MS 2 and MS 3 based quantification respectively, the third scatterplot compares fold changes calculated using either MS 2 or MS 3 based quantification performed on aliquots from the same samples. The density plots show the difference of protein fold changes between the biological replicates in the first and second plot and the difference between protein fold changes from MS 2 and MS 3 based quantification in the third plot. (C) Protein fold changes (FC) observed in mature (upper panel) and nascent (lower panel) forms of proteins in THP-1 cells treated for 24 h with 10 μM JQ1-VHL PROTAC relative to vehicle, for BRD2, BRD3 and BRD4 using MS 2 and MS 3 quantification in two biological replicates. (D) Protein fold changes (FC) observed in mature (upper panel) and nascent (lower panel) forms of proteins in THP-1 cells treated for 24 h with 10 μM JQ1-Az relative to vehicle, for BRD2, BRD3 and BRD4 using MS 2 and MS 3 quantification in two biological replicates. (E) Scatterplots showing protein fold changes (FC) observed in mature forms (upper panel), nascent forms (middle panel) and the total proteome level estimated from the summed-up reporter ion abundances over mature and nascent proteins (lower panel) of proteins in THP-1 cells treated for 24 h with JQ1-Az (10 μM), JQ1-VHL PROTAC (1 and 10 μM) or VHL alkyne (10 μM) relative to vehicle treated cells. Red closed circles indicate statistically significant regulation (p

    Journal: Cell

    Article Title: Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis

    doi: 10.1016/j.cell.2018.02.030

    Figure Lengend Snippet: Assessment of Reproducibility of Biological Replicates and Technical Validation of the Quantification, Related to Figure 2 (A–E) Samples were analyzed on a Q-executive (using MS 2 for reporter ion quantification) as well as on a Fusion Lumos (using SPS MS 3 for reporter ion quantification) mass spectrometer to assess the reproducibility of biological replicates and as a technical validation of the quantification. (A) Annotated MS 2 and MS 3 spectra for the BRD2 peptide VVHIIQAR. (B) The first two scatterplots compare protein fold changes (FC) between two biological replicates using MS 2 and MS 3 based quantification respectively, the third scatterplot compares fold changes calculated using either MS 2 or MS 3 based quantification performed on aliquots from the same samples. The density plots show the difference of protein fold changes between the biological replicates in the first and second plot and the difference between protein fold changes from MS 2 and MS 3 based quantification in the third plot. (C) Protein fold changes (FC) observed in mature (upper panel) and nascent (lower panel) forms of proteins in THP-1 cells treated for 24 h with 10 μM JQ1-VHL PROTAC relative to vehicle, for BRD2, BRD3 and BRD4 using MS 2 and MS 3 quantification in two biological replicates. (D) Protein fold changes (FC) observed in mature (upper panel) and nascent (lower panel) forms of proteins in THP-1 cells treated for 24 h with 10 μM JQ1-Az relative to vehicle, for BRD2, BRD3 and BRD4 using MS 2 and MS 3 quantification in two biological replicates. (E) Scatterplots showing protein fold changes (FC) observed in mature forms (upper panel), nascent forms (middle panel) and the total proteome level estimated from the summed-up reporter ion abundances over mature and nascent proteins (lower panel) of proteins in THP-1 cells treated for 24 h with JQ1-Az (10 μM), JQ1-VHL PROTAC (1 and 10 μM) or VHL alkyne (10 μM) relative to vehicle treated cells. Red closed circles indicate statistically significant regulation (p

    Article Snippet: THP-1 cells THP-1 cell cultures (ATCC TIB-202, male) were established in normal growth medium (RPMI1640 + 10% FBS) or RPMI-based SILAC-L and SILAC-H medium.

    Techniques: Mass Spectrometry

    Off-Target Effects of JQ1 and the JQ1-VHL-PROTAC, Related to Figure 3 (A) Fluorescence in situ hybridization (FISH) of polyA+RNA detected by a Cy3-labeled oligo-dT50 probe. THP-1 cells were treated with either vehicle, JQ1-Az (10μM), JQ1-VHL-PROTAC (at 1 or 10 μM) or I-BET-151-VHL-PROTAC (10 μM) for 6 hr (3 independent experiments are displayed). Cellular localization of polyA+RNA was visualized with confocal microscopy. Representative fluorescent images recorded after excitation at 514 nm (Cy3, gray) are shown. Lower panel displays an overlay of Cy3 staining (gray) and Hoechst nuclear staining (cyan) Scale bar, 20 μm. (B) Ratio of mean fluorescence intensity of FISH probe (Cy3 channel) between nucleus (defined by Hoechst staining) and cytosol (cell borders were defined by WGA staining, not shown) was calculated for single cells using the CellProfiler software (520-790 cells for experiment 1, 546-791 cells for experiment 2 and 278-578 cells for experiment 3 were quantified per condition) from experiment displayed in (A). SEM is shown. (C) Schematic representation of the TREX complex components. Protein names in red text are found significantly regulated compared to vehicle control upon treatment with JQ1-VHL PROTAC. (D) Thermal proteome profiling (TPP) experiment in HepG2 cells after incubation with 15 μM JQ1 (Tm difference in degree Celsius of JQ1 versus vehicle). Proteins with strongest stabilization in two independent replicates are highlighted. Red color indicates significant changes (E) Thermal stabilization of BRD4, SOAT1 and CYP4F12 induced by 15 μM JQ1 in HepG2 cells displayed as normalized non-denatured protein fraction. (F) 2D-thermal proteome profiling of JQ1 in THP1 cell lysate. Dose-dependent stabilization of BRD2, 3, 4 and SOAT1 are displayed (normalized apparent stability and pEC 50 are reported) (G) Thermal shift assay of recombinant FYTTD1 with JQ1.

    Journal: Cell

    Article Title: Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis

    doi: 10.1016/j.cell.2018.02.030

    Figure Lengend Snippet: Off-Target Effects of JQ1 and the JQ1-VHL-PROTAC, Related to Figure 3 (A) Fluorescence in situ hybridization (FISH) of polyA+RNA detected by a Cy3-labeled oligo-dT50 probe. THP-1 cells were treated with either vehicle, JQ1-Az (10μM), JQ1-VHL-PROTAC (at 1 or 10 μM) or I-BET-151-VHL-PROTAC (10 μM) for 6 hr (3 independent experiments are displayed). Cellular localization of polyA+RNA was visualized with confocal microscopy. Representative fluorescent images recorded after excitation at 514 nm (Cy3, gray) are shown. Lower panel displays an overlay of Cy3 staining (gray) and Hoechst nuclear staining (cyan) Scale bar, 20 μm. (B) Ratio of mean fluorescence intensity of FISH probe (Cy3 channel) between nucleus (defined by Hoechst staining) and cytosol (cell borders were defined by WGA staining, not shown) was calculated for single cells using the CellProfiler software (520-790 cells for experiment 1, 546-791 cells for experiment 2 and 278-578 cells for experiment 3 were quantified per condition) from experiment displayed in (A). SEM is shown. (C) Schematic representation of the TREX complex components. Protein names in red text are found significantly regulated compared to vehicle control upon treatment with JQ1-VHL PROTAC. (D) Thermal proteome profiling (TPP) experiment in HepG2 cells after incubation with 15 μM JQ1 (Tm difference in degree Celsius of JQ1 versus vehicle). Proteins with strongest stabilization in two independent replicates are highlighted. Red color indicates significant changes (E) Thermal stabilization of BRD4, SOAT1 and CYP4F12 induced by 15 μM JQ1 in HepG2 cells displayed as normalized non-denatured protein fraction. (F) 2D-thermal proteome profiling of JQ1 in THP1 cell lysate. Dose-dependent stabilization of BRD2, 3, 4 and SOAT1 are displayed (normalized apparent stability and pEC 50 are reported) (G) Thermal shift assay of recombinant FYTTD1 with JQ1.

    Article Snippet: THP-1 cells THP-1 cell cultures (ATCC TIB-202, male) were established in normal growth medium (RPMI1640 + 10% FBS) or RPMI-based SILAC-L and SILAC-H medium.

    Techniques: Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Labeling, Confocal Microscopy, Staining, Whole Genome Amplification, Software, Incubation, Thermal Shift Assay, Recombinant

    Toxoplasma gondii infection induces IL-23 and IL-12 expression in human monocytic THP-1 cells. (A) Kinetics of T . gondii induction of p19, p40, and p35 mRNA expression. Human monocytic THP-1 cells were treated with T . gondii at a multiplicity of infection 1 (MOI 1) for the times indicated. RNA was extracted, and semi-quantitative RT-PCR for p19, p40, and p35 was performed. HPRT was used as a loading control. A representative gel of three independent replicates with similar results was shown. (B) MOI-dependent increases in the mRNA expression of p19, p40, and p35. RT-PCR analysis for p19, p40, and p35 mRNA expression at 6 h was assessed in THP-1 cells after treatment with T . gondii at a MOI of 0, 0.1, 1, or 10. (C) MOI-dependent increases in IL-23 and IL-12 production. ELISA for IL-23 and IL-12 was performed in THP-1 cells after treatment with T . gondii at a MOI of 0, 0.1, 1, or 10 for 18 h. The results, expressed as means ± SDs of values for triplicate wells, are representative of three separate experiments.* P

    Journal: PLoS ONE

    Article Title: Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

    doi: 10.1371/journal.pone.0141550

    Figure Lengend Snippet: Toxoplasma gondii infection induces IL-23 and IL-12 expression in human monocytic THP-1 cells. (A) Kinetics of T . gondii induction of p19, p40, and p35 mRNA expression. Human monocytic THP-1 cells were treated with T . gondii at a multiplicity of infection 1 (MOI 1) for the times indicated. RNA was extracted, and semi-quantitative RT-PCR for p19, p40, and p35 was performed. HPRT was used as a loading control. A representative gel of three independent replicates with similar results was shown. (B) MOI-dependent increases in the mRNA expression of p19, p40, and p35. RT-PCR analysis for p19, p40, and p35 mRNA expression at 6 h was assessed in THP-1 cells after treatment with T . gondii at a MOI of 0, 0.1, 1, or 10. (C) MOI-dependent increases in IL-23 and IL-12 production. ELISA for IL-23 and IL-12 was performed in THP-1 cells after treatment with T . gondii at a MOI of 0, 0.1, 1, or 10 for 18 h. The results, expressed as means ± SDs of values for triplicate wells, are representative of three separate experiments.* P

    Article Snippet: Human monocytic THP-1 cells THP-1 cells are a promonocytic cell line derived from a patient with acute lymphocytic leukemia (ATCC TIB-202) that produce sufficient IL-12 in a T . gondii model [ ].

    Techniques: Infection, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Differential regulation of IL-23 and IL-12 productions in T . gondii -infected THP-1 cells upon treatment with small interfering RNA (siRNA) of TLR2 or TLR4. (A) THP-1 cells were transfected with 25 pmol of siRNA against TLR2 or TLR4 (or control siRNA) for 48 h, the TLR2 and TLR4 protein expression were determined by western blot analysis. (B) THP-1 cells were treated as Fig 3A, the supernatants were harvested after treatment with T . gondii at a MOI 1 for 18 h. The secretion levels of IL-23 and IL-12 were determined using ELISA. The results, expressed as means ± SDs of values for triplicate wells, were representative of three separate experiments.* P

    Journal: PLoS ONE

    Article Title: Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

    doi: 10.1371/journal.pone.0141550

    Figure Lengend Snippet: Differential regulation of IL-23 and IL-12 productions in T . gondii -infected THP-1 cells upon treatment with small interfering RNA (siRNA) of TLR2 or TLR4. (A) THP-1 cells were transfected with 25 pmol of siRNA against TLR2 or TLR4 (or control siRNA) for 48 h, the TLR2 and TLR4 protein expression were determined by western blot analysis. (B) THP-1 cells were treated as Fig 3A, the supernatants were harvested after treatment with T . gondii at a MOI 1 for 18 h. The secretion levels of IL-23 and IL-12 were determined using ELISA. The results, expressed as means ± SDs of values for triplicate wells, were representative of three separate experiments.* P

    Article Snippet: Human monocytic THP-1 cells THP-1 cells are a promonocytic cell line derived from a patient with acute lymphocytic leukemia (ATCC TIB-202) that produce sufficient IL-12 in a T . gondii model [ ].

    Techniques: Infection, Small Interfering RNA, Transfection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

    Differential regulation of IL-23 and IL-12 productions in T . gondii -infected THP-1 cells upon pretreatment with PI3K inhibitors. PI3K inhibitor LY294002 (LY) or wortmannin (WM) was added to THP-1 cells at the concentrations indicated at 1 h before infection with T . gondii at a MOI of 1. The supernatants were harvested after 18 h for cytokine assessment using ELISA. One representative experiment performed in triplicate is shown. The IL-23 (A) or IL-12 (B) levels (mean ± SD) following stimulation with T . gondii , as well as IL-23 and IL-12 production in the presence of the inhibitor, are shown. The solvent control was 0.1% DMSO. * P

    Journal: PLoS ONE

    Article Title: Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

    doi: 10.1371/journal.pone.0141550

    Figure Lengend Snippet: Differential regulation of IL-23 and IL-12 productions in T . gondii -infected THP-1 cells upon pretreatment with PI3K inhibitors. PI3K inhibitor LY294002 (LY) or wortmannin (WM) was added to THP-1 cells at the concentrations indicated at 1 h before infection with T . gondii at a MOI of 1. The supernatants were harvested after 18 h for cytokine assessment using ELISA. One representative experiment performed in triplicate is shown. The IL-23 (A) or IL-12 (B) levels (mean ± SD) following stimulation with T . gondii , as well as IL-23 and IL-12 production in the presence of the inhibitor, are shown. The solvent control was 0.1% DMSO. * P

    Article Snippet: Human monocytic THP-1 cells THP-1 cells are a promonocytic cell line derived from a patient with acute lymphocytic leukemia (ATCC TIB-202) that produce sufficient IL-12 in a T . gondii model [ ].

    Techniques: Infection, Enzyme-linked Immunosorbent Assay

    Differential regulation of IL-23 and IL-12 productions in T . gondii -infected THP-1 cells upon pretreatment with specific inhibitor of p38 MAPK, ERK1/2 or JNK. The p38 inhibitor (SB203580), ERK inhibitor (PD98059), or JNK inhibitor (SP600125) was added to THP-1 cells at the concentrations indicated at 1 h before infection with T . gondii . The culture supernatants were harvested after 18 h for cytokine assessment using ELISA. The IL-23 and IL-12 levels following stimulation with T . gondii with or without the presence of p38 MAPK inhibitor (A), ERK1/2 inhibitor (B), or JNK inhibitor (C) are shown. The solvent control was 0.1% DMSO. One representative experiment performed in triplicate is shown.* P

    Journal: PLoS ONE

    Article Title: Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

    doi: 10.1371/journal.pone.0141550

    Figure Lengend Snippet: Differential regulation of IL-23 and IL-12 productions in T . gondii -infected THP-1 cells upon pretreatment with specific inhibitor of p38 MAPK, ERK1/2 or JNK. The p38 inhibitor (SB203580), ERK inhibitor (PD98059), or JNK inhibitor (SP600125) was added to THP-1 cells at the concentrations indicated at 1 h before infection with T . gondii . The culture supernatants were harvested after 18 h for cytokine assessment using ELISA. The IL-23 and IL-12 levels following stimulation with T . gondii with or without the presence of p38 MAPK inhibitor (A), ERK1/2 inhibitor (B), or JNK inhibitor (C) are shown. The solvent control was 0.1% DMSO. One representative experiment performed in triplicate is shown.* P

    Article Snippet: Human monocytic THP-1 cells THP-1 cells are a promonocytic cell line derived from a patient with acute lymphocytic leukemia (ATCC TIB-202) that produce sufficient IL-12 in a T . gondii model [ ].

    Techniques: Infection, Enzyme-linked Immunosorbent Assay

    Hypothetical networks of PI3K/AKT and MAPK signaling pathwaysinvolved in the differential regulation of T . gondii -induced IL-23 and IL-12 production in THP-1 cells. The PI3K/AKT and MAPK pathways are activated by T . gondii -infection via TLR2 and TLR4. AKT activation differentially modulates ERK1/2, p38 MAPK, and JNK activation; PI3K/AKT inhibition suppresses ERK activation, whereas inhibition of PI3K/AKT results in the induction of p38 MAPK and JNK. Bold lines indicate a positive regulation pathway, and dotted lines indicate the negative regulation pathway of IL-23 and IL-12 production.

    Journal: PLoS ONE

    Article Title: Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

    doi: 10.1371/journal.pone.0141550

    Figure Lengend Snippet: Hypothetical networks of PI3K/AKT and MAPK signaling pathwaysinvolved in the differential regulation of T . gondii -induced IL-23 and IL-12 production in THP-1 cells. The PI3K/AKT and MAPK pathways are activated by T . gondii -infection via TLR2 and TLR4. AKT activation differentially modulates ERK1/2, p38 MAPK, and JNK activation; PI3K/AKT inhibition suppresses ERK activation, whereas inhibition of PI3K/AKT results in the induction of p38 MAPK and JNK. Bold lines indicate a positive regulation pathway, and dotted lines indicate the negative regulation pathway of IL-23 and IL-12 production.

    Article Snippet: Human monocytic THP-1 cells THP-1 cells are a promonocytic cell line derived from a patient with acute lymphocytic leukemia (ATCC TIB-202) that produce sufficient IL-12 in a T . gondii model [ ].

    Techniques: Infection, Activation Assay, Inhibition

    Differential regulation of IL-23 and IL-12 productions in T . gondii -infected THP-1 cells by pretreatments of anti-TLR2 and anti-TLR4 monoclonal antibodies. THP-1 cells were preincubated with anti-TLR2, anti-TLR4 or isotype-matched control (IC) (10 μg/mL) and infected with T . gondii sat a MOI 1 subsequently for 18 h. The culture supernatants were harvested and IL-23 (A) and IL-12 (B) productions were measured by ELISA. UI, uninfected control; D, DMSO (0.1%) control; IC, infected control. One representative experiment performed in triplicate is shown.* P

    Journal: PLoS ONE

    Article Title: Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

    doi: 10.1371/journal.pone.0141550

    Figure Lengend Snippet: Differential regulation of IL-23 and IL-12 productions in T . gondii -infected THP-1 cells by pretreatments of anti-TLR2 and anti-TLR4 monoclonal antibodies. THP-1 cells were preincubated with anti-TLR2, anti-TLR4 or isotype-matched control (IC) (10 μg/mL) and infected with T . gondii sat a MOI 1 subsequently for 18 h. The culture supernatants were harvested and IL-23 (A) and IL-12 (B) productions were measured by ELISA. UI, uninfected control; D, DMSO (0.1%) control; IC, infected control. One representative experiment performed in triplicate is shown.* P

    Article Snippet: Human monocytic THP-1 cells THP-1 cells are a promonocytic cell line derived from a patient with acute lymphocytic leukemia (ATCC TIB-202) that produce sufficient IL-12 in a T . gondii model [ ].

    Techniques: Infection, Enzyme-linked Immunosorbent Assay

    Regulation of AKT activation in T . gondii -infected THP-1 cells upon treatment with TLR2 or TLR4-specific antibodies or siRNA. (A) THP-1 cells were infected with T . gondii at MOI 10 for the indicated time points, cells were collected, and the levels of phospho-AKT (Ser473, Thr308) and total AKT were measured by western blot. (B) THP-1 cells were preincubated with the indicated amounts of anti-TLR2, anti-TLR4, or isotype-matched control (IC) 10 (μg/mL) and infected with T . gondii at MOI 10 for 30 min. Cells were collected, and the levels of phospho-AKT (Ser473) and total AKT were measured. (C) THP-1 cells were transfected with siRNA against TLR2 or TLR4 (or control siRNA) for 48 h and infected with T . gondii at MOI 10 for 30 min. Cells were collected, and the levels of phospho-AKT (Ser473) and total AKT were measured. A representative result of three independent replicates is shown.

    Journal: PLoS ONE

    Article Title: Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

    doi: 10.1371/journal.pone.0141550

    Figure Lengend Snippet: Regulation of AKT activation in T . gondii -infected THP-1 cells upon treatment with TLR2 or TLR4-specific antibodies or siRNA. (A) THP-1 cells were infected with T . gondii at MOI 10 for the indicated time points, cells were collected, and the levels of phospho-AKT (Ser473, Thr308) and total AKT were measured by western blot. (B) THP-1 cells were preincubated with the indicated amounts of anti-TLR2, anti-TLR4, or isotype-matched control (IC) 10 (μg/mL) and infected with T . gondii at MOI 10 for 30 min. Cells were collected, and the levels of phospho-AKT (Ser473) and total AKT were measured. (C) THP-1 cells were transfected with siRNA against TLR2 or TLR4 (or control siRNA) for 48 h and infected with T . gondii at MOI 10 for 30 min. Cells were collected, and the levels of phospho-AKT (Ser473) and total AKT were measured. A representative result of three independent replicates is shown.

    Article Snippet: Human monocytic THP-1 cells THP-1 cells are a promonocytic cell line derived from a patient with acute lymphocytic leukemia (ATCC TIB-202) that produce sufficient IL-12 in a T . gondii model [ ].

    Techniques: Activation Assay, Infection, Western Blot, Transfection

    Regulation of p38 MAPK, ERK1/2 or JNK activation in T . gondii -infected THP-1 cells by specific inhibitors. (A)THP-1 cells were infected with T . gondii at MOI 10 for the indicated time and the phosphorylation levels of p38 MAPK, ERK1/2 and JNK was checked by western blot. (B) THP-1 cells were pretreated with the p38 inhibitor (SB203580), ERK inhibitor (PD98059), or JNK inhibitor (SP600125) for the indicated concentration for 1 h and infected with T . gondii at MOI 10 for 30 min. The phosphorylation levels of p38 MAPK, ERK1/2 and JNK was checked. A representative experiment of three independent replicates with similar results is shown.

    Journal: PLoS ONE

    Article Title: Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

    doi: 10.1371/journal.pone.0141550

    Figure Lengend Snippet: Regulation of p38 MAPK, ERK1/2 or JNK activation in T . gondii -infected THP-1 cells by specific inhibitors. (A)THP-1 cells were infected with T . gondii at MOI 10 for the indicated time and the phosphorylation levels of p38 MAPK, ERK1/2 and JNK was checked by western blot. (B) THP-1 cells were pretreated with the p38 inhibitor (SB203580), ERK inhibitor (PD98059), or JNK inhibitor (SP600125) for the indicated concentration for 1 h and infected with T . gondii at MOI 10 for 30 min. The phosphorylation levels of p38 MAPK, ERK1/2 and JNK was checked. A representative experiment of three independent replicates with similar results is shown.

    Article Snippet: Human monocytic THP-1 cells THP-1 cells are a promonocytic cell line derived from a patient with acute lymphocytic leukemia (ATCC TIB-202) that produce sufficient IL-12 in a T . gondii model [ ].

    Techniques: Activation Assay, Infection, Western Blot, Concentration Assay

    Regulation of AKT and MAPKs activations in T . gondii -infected THP-1 cells upon pretreatment with PI3K inhibitors. THP-1 monocytes were preincubated with the PI3K inhibitor LY294002 (LY) or wortmannin (WM) for 1 h, and then were infected with T . gondii at MOI 10 for 30 min. The cellular lysates were separated by SDS-PAGE and analyzed by immunoblotting using phospho-specific primary antibodies against AKT (Ser473), p38 MAPK, ERK1/2, and JNK. To ensure equal protein loading, the blots were stripped and reprobed with antibodies against AKT, p38 MAPK, ERK1/2, and JNK. A representative experiment of three independent replicates with similar results is shown.

    Journal: PLoS ONE

    Article Title: Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

    doi: 10.1371/journal.pone.0141550

    Figure Lengend Snippet: Regulation of AKT and MAPKs activations in T . gondii -infected THP-1 cells upon pretreatment with PI3K inhibitors. THP-1 monocytes were preincubated with the PI3K inhibitor LY294002 (LY) or wortmannin (WM) for 1 h, and then were infected with T . gondii at MOI 10 for 30 min. The cellular lysates were separated by SDS-PAGE and analyzed by immunoblotting using phospho-specific primary antibodies against AKT (Ser473), p38 MAPK, ERK1/2, and JNK. To ensure equal protein loading, the blots were stripped and reprobed with antibodies against AKT, p38 MAPK, ERK1/2, and JNK. A representative experiment of three independent replicates with similar results is shown.

    Article Snippet: Human monocytic THP-1 cells THP-1 cells are a promonocytic cell line derived from a patient with acute lymphocytic leukemia (ATCC TIB-202) that produce sufficient IL-12 in a T . gondii model [ ].

    Techniques: Infection, SDS Page

    hCD13 expression on epithelial cells is necessary and sufficient for CD13‐mediated phagocytosis. (A) HEK293 cells transduced with lentiviral particles containing plasmids for CD13 expression pLenti‐suCMV(hANPEP)‐Rsv(RFP‐Bsd; clones A4 and A6) and transfection control pLenti‐suCMV(empty)‐Rsv(RFP‐Bsd; clone M6) were incubated with 2 µg Fab fragments of mAb452 (anti‐CD13) and 8 µg Fab fragments of mAb32.2 (anti‐FcγRI) or without antibody for 30 minutes at 4°C. Cells were then washed and incubated with secondary goat anti‐mouse FITC. A representative histogram of the expression of hCD13 on these clones is shown. (B) THP‐1 cells or (C) HEK293 clones A6, A4, and M6 were incubated with EFab452, EFab32.2, or Ec for 120 minutes at 37°C. Noningested erythrocytes were lysed, and samples were analyzed by flow cytometry to determine the percentage of CFSE‐positive cells. Average of CFSE‐positive cells of 5 independent experiments is shown. ** P

    Journal: Journal of Leukocyte Biology

    Article Title: CD13 mediates phagocytosis in human monocytic cells

    doi: 10.1189/jlb.2A0914-458R

    Figure Lengend Snippet: hCD13 expression on epithelial cells is necessary and sufficient for CD13‐mediated phagocytosis. (A) HEK293 cells transduced with lentiviral particles containing plasmids for CD13 expression pLenti‐suCMV(hANPEP)‐Rsv(RFP‐Bsd; clones A4 and A6) and transfection control pLenti‐suCMV(empty)‐Rsv(RFP‐Bsd; clone M6) were incubated with 2 µg Fab fragments of mAb452 (anti‐CD13) and 8 µg Fab fragments of mAb32.2 (anti‐FcγRI) or without antibody for 30 minutes at 4°C. Cells were then washed and incubated with secondary goat anti‐mouse FITC. A representative histogram of the expression of hCD13 on these clones is shown. (B) THP‐1 cells or (C) HEK293 clones A6, A4, and M6 were incubated with EFab452, EFab32.2, or Ec for 120 minutes at 37°C. Noningested erythrocytes were lysed, and samples were analyzed by flow cytometry to determine the percentage of CFSE‐positive cells. Average of CFSE‐positive cells of 5 independent experiments is shown. ** P

    Article Snippet: Lentiviral shRNA‐mediated silencing of CD13 in THP‐1 cells THP‐1 cells at 70% confluency in a 96‐well plate were transduced by MISSION Lentiviral Transduction Particles against hCD13 (SHVRS‐NM_001150 Virus titer: > 1 × 106 transducing units/ml; Sigma‐Aldrich).

    Techniques: Expressing, Transduction, Clone Assay, Transfection, Incubation, Flow Cytometry

    Phagocytosis in CD13‐deficient cells. THP‐1 (parental cell), L2 (THP‐1 CD13‐deficient cells), or C+ (THP‐1 transduction control) were incubated with 2 µg Fab fragments of mAb452 (anti‐CD13) and 8 µg Fab fragments of mAb32.2 (anti‐FcγRI) or without antibody for 30 minutes at 4°C. After washing, cells were incubated for 105 minutes at 37°C with CFSE‐labeled F(ab)′ 2 goat anti‐mouse‐opsonized erythrocytes (EBS‐Fab). Noningested erythrocytes were lysed, and samples were analyzed by flow cytometry to determine the percentage of CFSE‐positive cells. (A) Expression of CD13 or FcγRI was determined by flow cytometry by use of a secondary, FITC‐coupled goat‐anti mouse. (B) Representative dot plot of a single experiment. (C) Average of CFSE‐positive cells of 7 independent experiments. *** P

    Journal: Journal of Leukocyte Biology

    Article Title: CD13 mediates phagocytosis in human monocytic cells

    doi: 10.1189/jlb.2A0914-458R

    Figure Lengend Snippet: Phagocytosis in CD13‐deficient cells. THP‐1 (parental cell), L2 (THP‐1 CD13‐deficient cells), or C+ (THP‐1 transduction control) were incubated with 2 µg Fab fragments of mAb452 (anti‐CD13) and 8 µg Fab fragments of mAb32.2 (anti‐FcγRI) or without antibody for 30 minutes at 4°C. After washing, cells were incubated for 105 minutes at 37°C with CFSE‐labeled F(ab)′ 2 goat anti‐mouse‐opsonized erythrocytes (EBS‐Fab). Noningested erythrocytes were lysed, and samples were analyzed by flow cytometry to determine the percentage of CFSE‐positive cells. (A) Expression of CD13 or FcγRI was determined by flow cytometry by use of a secondary, FITC‐coupled goat‐anti mouse. (B) Representative dot plot of a single experiment. (C) Average of CFSE‐positive cells of 7 independent experiments. *** P

    Article Snippet: Lentiviral shRNA‐mediated silencing of CD13 in THP‐1 cells THP‐1 cells at 70% confluency in a 96‐well plate were transduced by MISSION Lentiviral Transduction Particles against hCD13 (SHVRS‐NM_001150 Virus titer: > 1 × 106 transducing units/ml; Sigma‐Aldrich).

    Techniques: Transduction, Incubation, Labeling, Flow Cytometry, Expressing

    CD13‐mediated‐phagocytosis is dependent on actin cytoskeleton rearrangements. THP‐1 cells were preincubated with 200 nM cytochalasin D for 2 hours at 37°C. Then cells were incubated with 2 µg Fab fragments of mAb452 (anti‐CD13) and 8 µg Fab fragments of mAb32.2 (anti‐FcγRI) or without antibody for 30 minutes at 4°C. After washing, cells were incubated for 105 minutes at 37°C with CFSE‐labeled F(ab)′ 2 goat anti‐mouse‐opsonized erythrocytes (EBS‐Fab), always in the presence of cytochalasin D. Noningested erythrocytes were lysed, and samples were analyzed by flow cytometry to determine the percentage of CFSE‐positive cells. (A) Representative dot plot of a single experiment. (B) Average of CFSE‐positive cells of 9 independent experiments. *** P

    Journal: Journal of Leukocyte Biology

    Article Title: CD13 mediates phagocytosis in human monocytic cells

    doi: 10.1189/jlb.2A0914-458R

    Figure Lengend Snippet: CD13‐mediated‐phagocytosis is dependent on actin cytoskeleton rearrangements. THP‐1 cells were preincubated with 200 nM cytochalasin D for 2 hours at 37°C. Then cells were incubated with 2 µg Fab fragments of mAb452 (anti‐CD13) and 8 µg Fab fragments of mAb32.2 (anti‐FcγRI) or without antibody for 30 minutes at 4°C. After washing, cells were incubated for 105 minutes at 37°C with CFSE‐labeled F(ab)′ 2 goat anti‐mouse‐opsonized erythrocytes (EBS‐Fab), always in the presence of cytochalasin D. Noningested erythrocytes were lysed, and samples were analyzed by flow cytometry to determine the percentage of CFSE‐positive cells. (A) Representative dot plot of a single experiment. (B) Average of CFSE‐positive cells of 9 independent experiments. *** P

    Article Snippet: Lentiviral shRNA‐mediated silencing of CD13 in THP‐1 cells THP‐1 cells at 70% confluency in a 96‐well plate were transduced by MISSION Lentiviral Transduction Particles against hCD13 (SHVRS‐NM_001150 Virus titer: > 1 × 106 transducing units/ml; Sigma‐Aldrich).

    Techniques: Incubation, Labeling, Flow Cytometry

    CD13 mediates internalization of erythrocytes in THP‐1 cells. (A) Schematic representation of EBS‐Fabs construction and treatment on cells for phagocytosis assays. THP‐1 cells were incubated with 2 µg Fab fragments of mAb452 (Fab452; anti‐CD13), 8 µg Fab fragments of mAb32.2 (Fab32.2; anti‐FcγRI), and 8 µg IgG1 or without antibody (No Fab) for 30 minutes at 4°C. After washing, cells were incubated for 105 minutes at 37°C or 4°C with CFSE‐labeled F(ab)′ 2 goat anti‐mouse‐opsonized erythrocytes (EBS‐Fab). Noningested erythrocytes were lysed, and samples were analyzed by flow cytometry to determine the percentage of CFSE‐positive cells. (B) Representative dot plot of a single experiment. FSC‐A, Forward‐scatter‐area. (C) Average of CFSE‐positive cells of 25 independent experiments. *** P

    Journal: Journal of Leukocyte Biology

    Article Title: CD13 mediates phagocytosis in human monocytic cells

    doi: 10.1189/jlb.2A0914-458R

    Figure Lengend Snippet: CD13 mediates internalization of erythrocytes in THP‐1 cells. (A) Schematic representation of EBS‐Fabs construction and treatment on cells for phagocytosis assays. THP‐1 cells were incubated with 2 µg Fab fragments of mAb452 (Fab452; anti‐CD13), 8 µg Fab fragments of mAb32.2 (Fab32.2; anti‐FcγRI), and 8 µg IgG1 or without antibody (No Fab) for 30 minutes at 4°C. After washing, cells were incubated for 105 minutes at 37°C or 4°C with CFSE‐labeled F(ab)′ 2 goat anti‐mouse‐opsonized erythrocytes (EBS‐Fab). Noningested erythrocytes were lysed, and samples were analyzed by flow cytometry to determine the percentage of CFSE‐positive cells. (B) Representative dot plot of a single experiment. FSC‐A, Forward‐scatter‐area. (C) Average of CFSE‐positive cells of 25 independent experiments. *** P

    Article Snippet: Lentiviral shRNA‐mediated silencing of CD13 in THP‐1 cells THP‐1 cells at 70% confluency in a 96‐well plate were transduced by MISSION Lentiviral Transduction Particles against hCD13 (SHVRS‐NM_001150 Virus titer: > 1 × 106 transducing units/ml; Sigma‐Aldrich).

    Techniques: Incubation, Labeling, Flow Cytometry

    Infection of human macrophages THP-1 cell line with the different E. coli pathotypes induce a differential cytokine secretion of IL-8, IL-1β, TNF-α, IL-6, and IL-10 . THP-1 cells were differentiated into macrophages-like cells using PMA at a concentration of 10 ng/mL during 48 h. After differentiation, adherent cells were infected with the different E. coli pathotypes (MOI 10) during 2 or 4 h. After infection, cell supernatants were removed and analyzed using Human inflammatory cytokines CBA kit. Data were acquired on a BD™ FACS Fortessa flow cytometer. Graphs represent cytokine secretion of IL-8 (A) and TNF-α (B) , as IL-1β (C) , IL-6 (D) , and IL-10 (E) . All data are means ± SEM of the three independent experiments. For statistical comparison, the pathotypes were grouped according to their way of interacting with the host cell as in Figure 6 : * p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Pathogenic Lifestyles of E. coli Pathotypes in a Standardized Epithelial Cell Model Influence Inflammatory Signaling Pathways and Cytokines Secretion

    doi: 10.3389/fcimb.2016.00120

    Figure Lengend Snippet: Infection of human macrophages THP-1 cell line with the different E. coli pathotypes induce a differential cytokine secretion of IL-8, IL-1β, TNF-α, IL-6, and IL-10 . THP-1 cells were differentiated into macrophages-like cells using PMA at a concentration of 10 ng/mL during 48 h. After differentiation, adherent cells were infected with the different E. coli pathotypes (MOI 10) during 2 or 4 h. After infection, cell supernatants were removed and analyzed using Human inflammatory cytokines CBA kit. Data were acquired on a BD™ FACS Fortessa flow cytometer. Graphs represent cytokine secretion of IL-8 (A) and TNF-α (B) , as IL-1β (C) , IL-6 (D) , and IL-10 (E) . All data are means ± SEM of the three independent experiments. For statistical comparison, the pathotypes were grouped according to their way of interacting with the host cell as in Figure 6 : * p

    Article Snippet: THP-1 cells were differentiated into macrophage-like cells with 10 ng/ml of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) during 48 h.

    Techniques: Infection, Concentration Assay, Crocin Bleaching Assay, FACS, Flow Cytometry, Cytometry

    GRIP1 phosphorylation affects its localization at a subset of genomic sites. THP1 cells ( a ) and BMMΦ ( b ) were treated ±dexamentasone (Dex) ±50 nM flavopiridol (FVP) for 1 h, and the occupancy of GR, pS469-GRIP1 and GRIP1 was analyzed by ChIP-qPCR as in Fig. 1f . c THP1-mutant-MΦ expressing WT or S469A/S487A/S493A/S499A (4A) mGRIP1 were treated ±Dex for 1 h and the expression of indicated genes was analyzed by RT-qPCR as in Fig. 1a and expressed relative to untreated cells for each genotype (=1). Means + SEM are shown ( n ≥ 3, * P

    Journal: Nature Communications

    Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

    doi: 10.1038/s41467-017-01569-2

    Figure Lengend Snippet: GRIP1 phosphorylation affects its localization at a subset of genomic sites. THP1 cells ( a ) and BMMΦ ( b ) were treated ±dexamentasone (Dex) ±50 nM flavopiridol (FVP) for 1 h, and the occupancy of GR, pS469-GRIP1 and GRIP1 was analyzed by ChIP-qPCR as in Fig. 1f . c THP1-mutant-MΦ expressing WT or S469A/S487A/S493A/S499A (4A) mGRIP1 were treated ±Dex for 1 h and the expression of indicated genes was analyzed by RT-qPCR as in Fig. 1a and expressed relative to untreated cells for each genotype (=1). Means + SEM are shown ( n ≥ 3, * P

    Article Snippet: THP1 cells were differentiated by 25 ng ml−1 phorbol myristate acetate (Sigma) for 18–24 h followed by a 48 h to obtain THP1 MΦ.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Mutagenesis, Expressing, Quantitative RT-PCR

    GRIP1 phosphorylation does not potentiate its GR corepressor function. a THP1 cells (small hairpin scrambled (shSCR) or shGRIP1) or BMMΦ (WT or GRIP1 KO) were treated ± 10 ng ml −1 LPS and ± Dex for 1 h and the expression of indicated genes was analyzed by RT-qPCR as described in Fig. 1a . “Fold repression” was defined as the quotient of the transcript level at lipopolysaccharide (LPS) over LPS+dexamethasone (Dex) condition and is shown as Tukey box-and-whisker plots ( n = 4). b THP1 cells expressing WT or S469A/S487A/S493A/S499A (4A) mGRIP1 were treated as in (a) and fold repression of indicated genes is shown. ns, non-significant. c THP1 MΦ or BMMΦ were treated ± Dex ± LPS for 2 h, as indicated, and the levels of GRIP1, GRIP1 phospho-isoforms, GR, CDK9 and HSP90 were evaluated by immunoblotting (quantified in Supplementary Fig. 9d ; full-size blots are shown in Supplementary Fig. 10h ). THP1 MΦ ( d ) or BMMΦ ( e ) treated with ±Dex ±LPS for 1 h were analyzed for GR, GRIP1, CDK9 and phospho-GRIP1 occupancy by ChIP-qPCR as in Fig. 1f (see Supplementary Fig. 9e for more genes). Shown are mean + SD, n ≥ 3, * P

    Journal: Nature Communications

    Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

    doi: 10.1038/s41467-017-01569-2

    Figure Lengend Snippet: GRIP1 phosphorylation does not potentiate its GR corepressor function. a THP1 cells (small hairpin scrambled (shSCR) or shGRIP1) or BMMΦ (WT or GRIP1 KO) were treated ± 10 ng ml −1 LPS and ± Dex for 1 h and the expression of indicated genes was analyzed by RT-qPCR as described in Fig. 1a . “Fold repression” was defined as the quotient of the transcript level at lipopolysaccharide (LPS) over LPS+dexamethasone (Dex) condition and is shown as Tukey box-and-whisker plots ( n = 4). b THP1 cells expressing WT or S469A/S487A/S493A/S499A (4A) mGRIP1 were treated as in (a) and fold repression of indicated genes is shown. ns, non-significant. c THP1 MΦ or BMMΦ were treated ± Dex ± LPS for 2 h, as indicated, and the levels of GRIP1, GRIP1 phospho-isoforms, GR, CDK9 and HSP90 were evaluated by immunoblotting (quantified in Supplementary Fig. 9d ; full-size blots are shown in Supplementary Fig. 10h ). THP1 MΦ ( d ) or BMMΦ ( e ) treated with ±Dex ±LPS for 1 h were analyzed for GR, GRIP1, CDK9 and phospho-GRIP1 occupancy by ChIP-qPCR as in Fig. 1f (see Supplementary Fig. 9e for more genes). Shown are mean + SD, n ≥ 3, * P

    Article Snippet: THP1 cells were differentiated by 25 ng ml−1 phorbol myristate acetate (Sigma) for 18–24 h followed by a 48 h to obtain THP1 MΦ.

    Techniques: Expressing, Quantitative RT-PCR, Whisker Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Macrophage GRIP1 is a GR coactivator of anti-inflammatory genes. GRIP1 was depleted in a THP1 cells (using a human-specific small-hairpin (sh)GRIP1 vs. scrambled (shSCR) control) or b mouse BMMΦ (from GRIP1 KO vs. WT control mice), and the induction of indicated genes by dexamethasone (Dex; 100 nM, 2 h) was analyzed by RT-qPCR, normalized to the expression of β-actin and expressed relative to untreated (=1). GRIP1 depletion efficiency is shown by immunoblotting with HSP90 as a loading control on the left (quantified in Supplementary Fig. 1b ; see Supplementary Fig. 10a for full-size bots). ChIP-seq for GR and GRIP1 was performed in THP1 cells ( c ) or mBMMΦ ( d ) treated ±Dex for 1 h (or 45 min for GR in BMMΦ), as indicated, and peaks were called as described in Methods. (Top) The overlap between peaks for GRIP1±Dex or GRIP1 and GR (Venn diagrams) was determined using subsetByOverlap function from GenomicRanges package (Bioconductor) with the minimum overlap of 1 bp (see “Methods”). (Middle) Ab initio sequence motif overrepresentation in GR–GRIP1 overlapping peaks was determined using MEME-ChIP. Genomic location of GRIP1 binding sites relative to known genomic features was determined by ChIPpeakAnno (Bioconductor). The union of peaks for two GRIP1 replicas in each cell type, the consensus for two THP1 GR replicas and a single mBMMΦ GR experiment are shown (Supplementary Figs. 2 – 4 ). (Bottom) Read density profiles of genes analyzed in a , b show peaks for GR and GRIP1. e CD14 + hMΦ were untreated (U) or treated with Dex for 2 h, and the expression of representative genes was measured as in a . f GR and GRIP1 occupancy at relevant sites in hMΦ was assessed by ChIP-qPCR (normalized to the 28s rDNA housekeeping gene, untreated=1). Samples in a , b and e , f were compared by unpaired two-tailed Student’s t -test. Shown are means+standard deviation (SD) error bars ( n ≥ 3, * P

    Journal: Nature Communications

    Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

    doi: 10.1038/s41467-017-01569-2

    Figure Lengend Snippet: Macrophage GRIP1 is a GR coactivator of anti-inflammatory genes. GRIP1 was depleted in a THP1 cells (using a human-specific small-hairpin (sh)GRIP1 vs. scrambled (shSCR) control) or b mouse BMMΦ (from GRIP1 KO vs. WT control mice), and the induction of indicated genes by dexamethasone (Dex; 100 nM, 2 h) was analyzed by RT-qPCR, normalized to the expression of β-actin and expressed relative to untreated (=1). GRIP1 depletion efficiency is shown by immunoblotting with HSP90 as a loading control on the left (quantified in Supplementary Fig. 1b ; see Supplementary Fig. 10a for full-size bots). ChIP-seq for GR and GRIP1 was performed in THP1 cells ( c ) or mBMMΦ ( d ) treated ±Dex for 1 h (or 45 min for GR in BMMΦ), as indicated, and peaks were called as described in Methods. (Top) The overlap between peaks for GRIP1±Dex or GRIP1 and GR (Venn diagrams) was determined using subsetByOverlap function from GenomicRanges package (Bioconductor) with the minimum overlap of 1 bp (see “Methods”). (Middle) Ab initio sequence motif overrepresentation in GR–GRIP1 overlapping peaks was determined using MEME-ChIP. Genomic location of GRIP1 binding sites relative to known genomic features was determined by ChIPpeakAnno (Bioconductor). The union of peaks for two GRIP1 replicas in each cell type, the consensus for two THP1 GR replicas and a single mBMMΦ GR experiment are shown (Supplementary Figs. 2 – 4 ). (Bottom) Read density profiles of genes analyzed in a , b show peaks for GR and GRIP1. e CD14 + hMΦ were untreated (U) or treated with Dex for 2 h, and the expression of representative genes was measured as in a . f GR and GRIP1 occupancy at relevant sites in hMΦ was assessed by ChIP-qPCR (normalized to the 28s rDNA housekeeping gene, untreated=1). Samples in a , b and e , f were compared by unpaired two-tailed Student’s t -test. Shown are means+standard deviation (SD) error bars ( n ≥ 3, * P

    Article Snippet: THP1 cells were differentiated by 25 ng ml−1 phorbol myristate acetate (Sigma) for 18–24 h followed by a 48 h to obtain THP1 MΦ.

    Techniques: Mouse Assay, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation, Sequencing, Binding Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, Standard Deviation

    GRIP1 is a substrate for dexamethasone (Dex)-induced phosphorylation by CDK9 in macrophages. a hMΦ, THP1 cells or BMMΦ were treated with Dex for the times indicated and the levels of GRIP1 (total or phosphorylated (p) at S469, S487, S493, and S499), GR, CDK9 and HSP90 as a loading control were assessed by immunoblotting (quantified in Supplementary Fig. 5a ). b THP1 cells or mBMMΦ were treated with Dex±flavopiridol (FVP) at indicated concentrations for 2 h and GRIP1 phosphorylation was visualized by immunoblotting (quantified in Supplementary Fig. 5b ). c Immortalized BMMΦ were depleted of CDK9 (single guide (sg)CDK9 vs. scrambled sgSCR control) as described in Methods and treated ±Dex for 1 h. The levels of GRIP1, phospho-(p)GRIP1, GR, CDK9 and HSP90 were assayed by western blot and quantified by densitometry; CDK9 levels or ratios of pGRIP1/GRIP1 in sgCDK9 cells are expressed relative to those in sgSCR cells (=1). Samples were compared by unpaired, two-tailed Student’s t -test; means + standard error of the mean (SEM) are shown ( n ≥ 3, * P

    Journal: Nature Communications

    Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

    doi: 10.1038/s41467-017-01569-2

    Figure Lengend Snippet: GRIP1 is a substrate for dexamethasone (Dex)-induced phosphorylation by CDK9 in macrophages. a hMΦ, THP1 cells or BMMΦ were treated with Dex for the times indicated and the levels of GRIP1 (total or phosphorylated (p) at S469, S487, S493, and S499), GR, CDK9 and HSP90 as a loading control were assessed by immunoblotting (quantified in Supplementary Fig. 5a ). b THP1 cells or mBMMΦ were treated with Dex±flavopiridol (FVP) at indicated concentrations for 2 h and GRIP1 phosphorylation was visualized by immunoblotting (quantified in Supplementary Fig. 5b ). c Immortalized BMMΦ were depleted of CDK9 (single guide (sg)CDK9 vs. scrambled sgSCR control) as described in Methods and treated ±Dex for 1 h. The levels of GRIP1, phospho-(p)GRIP1, GR, CDK9 and HSP90 were assayed by western blot and quantified by densitometry; CDK9 levels or ratios of pGRIP1/GRIP1 in sgCDK9 cells are expressed relative to those in sgSCR cells (=1). Samples were compared by unpaired, two-tailed Student’s t -test; means + standard error of the mean (SEM) are shown ( n ≥ 3, * P

    Article Snippet: THP1 cells were differentiated by 25 ng ml−1 phorbol myristate acetate (Sigma) for 18–24 h followed by a 48 h to obtain THP1 MΦ.

    Techniques: Western Blot, Two Tailed Test

    GRIP1 phospho-isoforms occupy GR target genes in a site-specific manner. THP1 cells ( a ) or BMMΦ ( b ) were incubated with dexamentasone (Dex) for 1 h, and the occupancy of phospho- (p)S469-, pS487-, pS493-, and pS499-GRIP1 as well as CDK9 was assessed at GRIP1-occupied sites from Fig. 1 by ChIP-qPCR as in Fig. 1f . Mean + SEM are shown ( n ≥ 3, * P

    Journal: Nature Communications

    Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

    doi: 10.1038/s41467-017-01569-2

    Figure Lengend Snippet: GRIP1 phospho-isoforms occupy GR target genes in a site-specific manner. THP1 cells ( a ) or BMMΦ ( b ) were incubated with dexamentasone (Dex) for 1 h, and the occupancy of phospho- (p)S469-, pS487-, pS493-, and pS499-GRIP1 as well as CDK9 was assessed at GRIP1-occupied sites from Fig. 1 by ChIP-qPCR as in Fig. 1f . Mean + SEM are shown ( n ≥ 3, * P

    Article Snippet: THP1 cells were differentiated by 25 ng ml−1 phorbol myristate acetate (Sigma) for 18–24 h followed by a 48 h to obtain THP1 MΦ.

    Techniques: Incubation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    GRIP1 phosphorylation potentiates GR-mediated transcriptional activation. a THP1 cells or BMMΦ were treated ±dexamentasone (Dex)±50 nM flavopiridol (FVP) for 2 h and “fold induction” of indicated genes analyzed by RT-qPCR, normalized to β-actin and expressed relative to untreated or FVP alone (=1); mean+SEM are shown; n ≥ 3. b The induction by Dex (30 min) of GR target genes in single guide (sg)CDK9 and scrambled sgSCR control IBMMΦ was analyzed by RT-qPCR as in a ; mean + SD is shown, n = 4. c THP1 cells transduced with SCR shRNA (-) or those depleted of endogenous hGRIP1 (+) from Fig. 1a , were stably transduced with WT or 4A mGRIP1 (see “Methods”), as indicated, and GRIP1 expression tested by immunoblotting with HSP90 is a loading control (left). WT- and S469A/S487A/S493A/S499A (4A)-expressing THP1 cells were treated ±Dex for 2 h and GRIP1 (total and phospho-isoforms), GR, CDK9 and HSP90 were visualized by immunoblotting (right). Quantification was performed as in Fig. 2c and expressed relative to WT (=1); mean + SEM are shown; n ≥ 3. Full-size western blots are shown in Supplementary Fig. 10 g. d WT and 4A THP1 cells were treated ±Dex for 2 h and the expression of GR target genes was analyzed by RT-qPCR as in a ; mean + SEM are shown, n ≥ 3. In each panel * P

    Journal: Nature Communications

    Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

    doi: 10.1038/s41467-017-01569-2

    Figure Lengend Snippet: GRIP1 phosphorylation potentiates GR-mediated transcriptional activation. a THP1 cells or BMMΦ were treated ±dexamentasone (Dex)±50 nM flavopiridol (FVP) for 2 h and “fold induction” of indicated genes analyzed by RT-qPCR, normalized to β-actin and expressed relative to untreated or FVP alone (=1); mean+SEM are shown; n ≥ 3. b The induction by Dex (30 min) of GR target genes in single guide (sg)CDK9 and scrambled sgSCR control IBMMΦ was analyzed by RT-qPCR as in a ; mean + SD is shown, n = 4. c THP1 cells transduced with SCR shRNA (-) or those depleted of endogenous hGRIP1 (+) from Fig. 1a , were stably transduced with WT or 4A mGRIP1 (see “Methods”), as indicated, and GRIP1 expression tested by immunoblotting with HSP90 is a loading control (left). WT- and S469A/S487A/S493A/S499A (4A)-expressing THP1 cells were treated ±Dex for 2 h and GRIP1 (total and phospho-isoforms), GR, CDK9 and HSP90 were visualized by immunoblotting (right). Quantification was performed as in Fig. 2c and expressed relative to WT (=1); mean + SEM are shown; n ≥ 3. Full-size western blots are shown in Supplementary Fig. 10 g. d WT and 4A THP1 cells were treated ±Dex for 2 h and the expression of GR target genes was analyzed by RT-qPCR as in a ; mean + SEM are shown, n ≥ 3. In each panel * P

    Article Snippet: THP1 cells were differentiated by 25 ng ml−1 phorbol myristate acetate (Sigma) for 18–24 h followed by a 48 h to obtain THP1 MΦ.

    Techniques: Activation Assay, Quantitative RT-PCR, Transduction, shRNA, Stable Transfection, Expressing, Western Blot

    Arginine methylation regulates c-Myc transcriptional activation activity. A , c-Myc down-regulates PRMT1 gene expression. Left , Western blot analysis of PRMT1 protein levels in THP-1 cells expressing c-Myc. Right , relative mRNA of PRMT1 in THP-1 cells expressing wild-type c-Myc. Data are presented as mean ± S.D. **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Arginine methylation regulates c-Myc–dependent transcription by altering promoter recruitment of the acetyltransferase p300

    doi: 10.1074/jbc.M117.797928

    Figure Lengend Snippet: Arginine methylation regulates c-Myc transcriptional activation activity. A , c-Myc down-regulates PRMT1 gene expression. Left , Western blot analysis of PRMT1 protein levels in THP-1 cells expressing c-Myc. Right , relative mRNA of PRMT1 in THP-1 cells expressing wild-type c-Myc. Data are presented as mean ± S.D. **, p

    Article Snippet: THP-1 cells (1.5 × 107 ) were cross-linked by the addition of 1% formaldehyde for 10 min.

    Techniques: Methylation, Activation Assay, Activity Assay, Expressing, Western Blot

    Arginine methylation effect on c-Myc promoter binding and co-factor recruitment. A and C , ChIP using anti-c-Myc, anti-p300, anti-HDAC1, anti-H3K27ac antibody, or IgG as a negative control in untreated or AMI-1–treated THP-1 cells. Data are presented as mean percent of input ± S.D.; n = 3–5. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Arginine methylation regulates c-Myc–dependent transcription by altering promoter recruitment of the acetyltransferase p300

    doi: 10.1074/jbc.M117.797928

    Figure Lengend Snippet: Arginine methylation effect on c-Myc promoter binding and co-factor recruitment. A and C , ChIP using anti-c-Myc, anti-p300, anti-HDAC1, anti-H3K27ac antibody, or IgG as a negative control in untreated or AMI-1–treated THP-1 cells. Data are presented as mean percent of input ± S.D.; n = 3–5. *, p

    Article Snippet: THP-1 cells (1.5 × 107 ) were cross-linked by the addition of 1% formaldehyde for 10 min.

    Techniques: Methylation, Binding Assay, Chromatin Immunoprecipitation, Negative Control

    c-Myc is arginine-methylated. A , sites of c-Myc arginine methylation. Mass spectrometry analysis of c-Myc purified from THP-1 cells. MS/MS spectra of methylated and corresponding unmethylated peptides are shown for each site with highest A-scores. B , schematic image of c-Myc domain structure. MYC N , N-terminal transactivation domain; HLH , helix-loop-helix DNA-binding domain; LZ , leucine zipper Max dimerization domain. Image shows the position of methylated arginines relative to known co-factor binding overlapping with methylated arginine sites. C , relative p300–c-Myc binding was measured by ELISA as described under “Materials and methods.” Relative p300–c-Myc binding is plotted against PRMT1 expression in individual patient-derived peripheral blood monocyte samples.

    Journal: The Journal of Biological Chemistry

    Article Title: Arginine methylation regulates c-Myc–dependent transcription by altering promoter recruitment of the acetyltransferase p300

    doi: 10.1074/jbc.M117.797928

    Figure Lengend Snippet: c-Myc is arginine-methylated. A , sites of c-Myc arginine methylation. Mass spectrometry analysis of c-Myc purified from THP-1 cells. MS/MS spectra of methylated and corresponding unmethylated peptides are shown for each site with highest A-scores. B , schematic image of c-Myc domain structure. MYC N , N-terminal transactivation domain; HLH , helix-loop-helix DNA-binding domain; LZ , leucine zipper Max dimerization domain. Image shows the position of methylated arginines relative to known co-factor binding overlapping with methylated arginine sites. C , relative p300–c-Myc binding was measured by ELISA as described under “Materials and methods.” Relative p300–c-Myc binding is plotted against PRMT1 expression in individual patient-derived peripheral blood monocyte samples.

    Article Snippet: THP-1 cells (1.5 × 107 ) were cross-linked by the addition of 1% formaldehyde for 10 min.

    Techniques: Methylation, Mass Spectrometry, Purification, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Derivative Assay

    Extracellular calcium activates the inflammasome via CaSR and GPRC6A. ( a ) Immunoblot (IB) analysis demonstrating the expression of GPRC6A (left panel) and CaSR (right) in human monocytes. In both blots, lanes 1, 2 and 3 represent three different healthy donors. ( b ) Influence of Calhex231 and NPS2143 on the IL-1β secretion of monocytes stimulated with increased ex[Ca 2+ ]+LPS ( n =3). ( c ) IB analysis showing the active form of caspase-1 (p20) in supernatants collected from monocytes stimulated with increased ex[Ca 2+ ]+LPS in the absence or presence of Calhex231 after 16 h of culture. One representative blot out of three experiments performed is shown. ( d ) Ex[Ca 2+ ]-induced IL-1β secretion of CD11b+ mononuclear cells isolated from bone marrow and of peritoneal macrophages from GPRC6A +/+ (wt) and GPRC6A −/− (ko) mice ( n =5). ( e ) Influence of CaSR and GPRC6A inactivation by transfection with siRNA on IL-1β secretion of THP-1 cells in response to ex[Ca 2+ ]+LPS ( n =3). IBs show inhibition of protein expression of GPRC6A and CaSR in cells transfected with the respective siRNA compared with control siRNA. ( f ) IL-1β secretion of monocytes in response to increasing concentration of Al 3+ with or without LPS ( n =3). ( g ) IL-1β secretion of peritoneal macrophages from GPRC6A +/+ (wt) and GPRC6A −/− (ko) mice in response to 0.9mM Al 3+ plus LPS ( n =3). ( h ) IL-1β secretion of THP-1 cells (control), NLRP3-deficient THP-1 cells (THP1defNLRP3) and ASC-deficient THP-1 cells (THP1defASC) stimulated with 0.9 mM Al 3+ plus LPS, for 16 h ( n =3). ( i ) IL-1β release of monocytes stimulated with different GPCR ligands at the indicated concentration alone (white bars) or with GPCR ligands +LPS (black bars) for 16 h. ( j ) IL-1β release from monocytes stimulated with ex[Ca 2+ ]+LPS in the presence of dimethyl sulfoxide (control) or with the phosphoinositide-specific PLC inhibitor U-73122 (5 μM) in dimethyl sulfoxide ( n =3). ( k ) PLC activity in monocytes stimulated for 16 h with LPS alone or with ex[Ca 2+ ]+LPS ( n =5). ( l ) For [Ca 2+ ] i imaging, monocytes were loaded with the Ca 2+ -sensitive indicator dye Fura-2-AM and subsequently stimulated with ex[Ca 2+ ] (1.7 mM). The increase in [Ca 2+ ] i was inhibited by NPS2143. One representative experiment out of three is shown. ( m ) IL-1β release from monocytes stimulated with ex[Ca 2+ ]+LPS in the presence of increasing concentrations of BAPTA-AM ( n =3). * P

    Journal: Nature Communications

    Article Title: Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

    doi: 10.1038/ncomms2339

    Figure Lengend Snippet: Extracellular calcium activates the inflammasome via CaSR and GPRC6A. ( a ) Immunoblot (IB) analysis demonstrating the expression of GPRC6A (left panel) and CaSR (right) in human monocytes. In both blots, lanes 1, 2 and 3 represent three different healthy donors. ( b ) Influence of Calhex231 and NPS2143 on the IL-1β secretion of monocytes stimulated with increased ex[Ca 2+ ]+LPS ( n =3). ( c ) IB analysis showing the active form of caspase-1 (p20) in supernatants collected from monocytes stimulated with increased ex[Ca 2+ ]+LPS in the absence or presence of Calhex231 after 16 h of culture. One representative blot out of three experiments performed is shown. ( d ) Ex[Ca 2+ ]-induced IL-1β secretion of CD11b+ mononuclear cells isolated from bone marrow and of peritoneal macrophages from GPRC6A +/+ (wt) and GPRC6A −/− (ko) mice ( n =5). ( e ) Influence of CaSR and GPRC6A inactivation by transfection with siRNA on IL-1β secretion of THP-1 cells in response to ex[Ca 2+ ]+LPS ( n =3). IBs show inhibition of protein expression of GPRC6A and CaSR in cells transfected with the respective siRNA compared with control siRNA. ( f ) IL-1β secretion of monocytes in response to increasing concentration of Al 3+ with or without LPS ( n =3). ( g ) IL-1β secretion of peritoneal macrophages from GPRC6A +/+ (wt) and GPRC6A −/− (ko) mice in response to 0.9mM Al 3+ plus LPS ( n =3). ( h ) IL-1β secretion of THP-1 cells (control), NLRP3-deficient THP-1 cells (THP1defNLRP3) and ASC-deficient THP-1 cells (THP1defASC) stimulated with 0.9 mM Al 3+ plus LPS, for 16 h ( n =3). ( i ) IL-1β release of monocytes stimulated with different GPCR ligands at the indicated concentration alone (white bars) or with GPCR ligands +LPS (black bars) for 16 h. ( j ) IL-1β release from monocytes stimulated with ex[Ca 2+ ]+LPS in the presence of dimethyl sulfoxide (control) or with the phosphoinositide-specific PLC inhibitor U-73122 (5 μM) in dimethyl sulfoxide ( n =3). ( k ) PLC activity in monocytes stimulated for 16 h with LPS alone or with ex[Ca 2+ ]+LPS ( n =5). ( l ) For [Ca 2+ ] i imaging, monocytes were loaded with the Ca 2+ -sensitive indicator dye Fura-2-AM and subsequently stimulated with ex[Ca 2+ ] (1.7 mM). The increase in [Ca 2+ ] i was inhibited by NPS2143. One representative experiment out of three is shown. ( m ) IL-1β release from monocytes stimulated with ex[Ca 2+ ]+LPS in the presence of increasing concentrations of BAPTA-AM ( n =3). * P

    Article Snippet: NLRP3-deficient, ASC-deficient and control THP-1 cells were purchased from InvivoGen.

    Techniques: Expressing, Isolation, Mouse Assay, Transfection, Inhibition, Concentration Assay, Planar Chromatography, Activity Assay, Imaging

    Extracellular calcium induces monocyte IL-1β release by activating the inflammasome. ( a , b ) IL-1β release of CD14+ monocytes in response to increasing ex[Ca 2+ ] alone ( a ) or to increasing ex[Ca 2+ ] plus LPS ( b ) after 16 h of stimulation ( n =3). ( c ) time course of IL-1β release of CD14+ monocytes after stimulation with LPS alone or with increased ex[Ca 2+ ] (1.7 mM) plus LPS ( n =3). ( d ) PCR (upper panel) and immunoblot (IB, lower panel) for IL-1β in unstimulated monocytes (control) and in monocytes stimulated with LPS, with increased ex[Ca 2+ ] concentration (1.7 mM), or both, after 16 h of culture. Specific bands for IL-1β mRNA, pro-IL-1β p35 and IL-1β p17 as indicated. One representative experiment out of three is shown. ( e ) IB analysis of Caspase-1, which shows the activation of Caspase-1 in supernatants harvested from unstimulated monocytes (control) and in monocytes stimulated with LPS, with increased ex[Ca 2+ ] concentration (1.7 mM), or with both, after 16 h of culture. Specific bands for pro-Caspase-1 (pro-Casp1) and Caspase-1 (Casp1 p20) are indicated, shown is one representative blot out of three experiments performed. ( f ) IL-1β secretion of monocytes stimulated with ex[Ca 2+ ] plus LPS in the absence and presence of the caspase-1 inhibitor Z-WEHD-FMK (5 μM, n =3). ( g ) mIL-1β secretion of monocytes from Caspase-1 +/+ and Caspase-1 −/− NOD mice stimulated with LPS or with ex[Ca 2+ ] plus LPS ( n =3). ( h ) IL-1β secretion of THP-1 cells (control), NLRP3-deficient THP-1 cells (THP1defNLRP3) and ASC-deficient THP-1 cells (THP1defASC) stimulated with increased ex[Ca 2+ ] concentration (1.7 mM) and LPS, after 16 h of culture ( n =3). ( i ) Time course of IL-18 release of CD14+ monocytes after stimulation with LPS alone or with increased ex[Ca 2+ ] (1.7 mM) plus LPS ( n =3). In all experiments, cytokine concentrations were determined in the supernatant by enzyme-linked immunosorbent assay. All bars show mean±s.e.m. Statistical analysis was performed using the two-tailed Student’s t -test; ** P

    Journal: Nature Communications

    Article Title: Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

    doi: 10.1038/ncomms2339

    Figure Lengend Snippet: Extracellular calcium induces monocyte IL-1β release by activating the inflammasome. ( a , b ) IL-1β release of CD14+ monocytes in response to increasing ex[Ca 2+ ] alone ( a ) or to increasing ex[Ca 2+ ] plus LPS ( b ) after 16 h of stimulation ( n =3). ( c ) time course of IL-1β release of CD14+ monocytes after stimulation with LPS alone or with increased ex[Ca 2+ ] (1.7 mM) plus LPS ( n =3). ( d ) PCR (upper panel) and immunoblot (IB, lower panel) for IL-1β in unstimulated monocytes (control) and in monocytes stimulated with LPS, with increased ex[Ca 2+ ] concentration (1.7 mM), or both, after 16 h of culture. Specific bands for IL-1β mRNA, pro-IL-1β p35 and IL-1β p17 as indicated. One representative experiment out of three is shown. ( e ) IB analysis of Caspase-1, which shows the activation of Caspase-1 in supernatants harvested from unstimulated monocytes (control) and in monocytes stimulated with LPS, with increased ex[Ca 2+ ] concentration (1.7 mM), or with both, after 16 h of culture. Specific bands for pro-Caspase-1 (pro-Casp1) and Caspase-1 (Casp1 p20) are indicated, shown is one representative blot out of three experiments performed. ( f ) IL-1β secretion of monocytes stimulated with ex[Ca 2+ ] plus LPS in the absence and presence of the caspase-1 inhibitor Z-WEHD-FMK (5 μM, n =3). ( g ) mIL-1β secretion of monocytes from Caspase-1 +/+ and Caspase-1 −/− NOD mice stimulated with LPS or with ex[Ca 2+ ] plus LPS ( n =3). ( h ) IL-1β secretion of THP-1 cells (control), NLRP3-deficient THP-1 cells (THP1defNLRP3) and ASC-deficient THP-1 cells (THP1defASC) stimulated with increased ex[Ca 2+ ] concentration (1.7 mM) and LPS, after 16 h of culture ( n =3). ( i ) Time course of IL-18 release of CD14+ monocytes after stimulation with LPS alone or with increased ex[Ca 2+ ] (1.7 mM) plus LPS ( n =3). In all experiments, cytokine concentrations were determined in the supernatant by enzyme-linked immunosorbent assay. All bars show mean±s.e.m. Statistical analysis was performed using the two-tailed Student’s t -test; ** P

    Article Snippet: NLRP3-deficient, ASC-deficient and control THP-1 cells were purchased from InvivoGen.

    Techniques: Polymerase Chain Reaction, Concentration Assay, Activation Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Extracellular calcium induces several proinflammatory cytokines and is released from necrotic cells. ( a ) IL-1α release from unprimed (white bars) and LPS-primed (black bars) monocytes in response to stimulation with ex[Ca 2+ ] ( n =3). ( b ) Influence of the specific inhibitors Calhex231 and NPS2143 on IL-1α secretion of monocytes stimulated with increased ex[Ca 2+ ]+LPS ( n =3). ( c ) ex[Ca 2+ ]-induced IL-1α secretion of CD11b+ mononuclear cells isolated from peripheral blood and of peritoneal macrophages from GPRC6A +/+ (wt) and GPRC6A −/− (ko) mice. ( d ) IL-1α secretion of THP-1 cells (control), NLRP3-deficient THP-1 cells (THP1defNLRP3) and ASC-deficient THP-1 cells (THP1defASC) stimulated with increased ex[Ca 2+ ] concentration and LPS, after 16 h of culture ( n =3). ( e ) Secretion of TNF from LPS-stimulated peritoneal macrophages or from macrophages stimulated with ex[Ca 2+ ]+LPS from GPRC6A +/+ (wt) or GPRC6A −/− (ko) mice ( n =3). ( f ) Influence of the specific inhibitor NPS2143 on IL-6 secretion of monocytes stimulated with increased ex[Ca 2+ ]+LPS ( n =3). ( g ) Prostaglandin E2 (PGE 2 ) release from unprimed (white bars) and LPS-primed (black bars) monocytes in response to stimulation with ex[Ca 2+ ] ( n =3). ( h ) Influence of 7-BIO-induced monocyte necrosis on the Ca 2+ concentration in the supernatant ( n =3). ( i ) IL-1β release from LPS-primed CD14+ monocytes cultured alone (white bars) or co-cultured with necrotic autologous CD4+T cells (black bars) in the absence or presence of the specific inhibitor Calhex231 ( n =3). ( j ) mIL-1β secretion of LPS-primed monocytes from GPRC6A +/+ (wt) and GPRC6A −/− (ko) mice cultured alone (LPS) or co-cultured with necrotic autologous CD4+ T cells (Nc+LPS, n =9). All bars show mean±s.e.m. Statistical analysis was performed using the two-tailed Student’s t -test. * P

    Journal: Nature Communications

    Article Title: Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

    doi: 10.1038/ncomms2339

    Figure Lengend Snippet: Extracellular calcium induces several proinflammatory cytokines and is released from necrotic cells. ( a ) IL-1α release from unprimed (white bars) and LPS-primed (black bars) monocytes in response to stimulation with ex[Ca 2+ ] ( n =3). ( b ) Influence of the specific inhibitors Calhex231 and NPS2143 on IL-1α secretion of monocytes stimulated with increased ex[Ca 2+ ]+LPS ( n =3). ( c ) ex[Ca 2+ ]-induced IL-1α secretion of CD11b+ mononuclear cells isolated from peripheral blood and of peritoneal macrophages from GPRC6A +/+ (wt) and GPRC6A −/− (ko) mice. ( d ) IL-1α secretion of THP-1 cells (control), NLRP3-deficient THP-1 cells (THP1defNLRP3) and ASC-deficient THP-1 cells (THP1defASC) stimulated with increased ex[Ca 2+ ] concentration and LPS, after 16 h of culture ( n =3). ( e ) Secretion of TNF from LPS-stimulated peritoneal macrophages or from macrophages stimulated with ex[Ca 2+ ]+LPS from GPRC6A +/+ (wt) or GPRC6A −/− (ko) mice ( n =3). ( f ) Influence of the specific inhibitor NPS2143 on IL-6 secretion of monocytes stimulated with increased ex[Ca 2+ ]+LPS ( n =3). ( g ) Prostaglandin E2 (PGE 2 ) release from unprimed (white bars) and LPS-primed (black bars) monocytes in response to stimulation with ex[Ca 2+ ] ( n =3). ( h ) Influence of 7-BIO-induced monocyte necrosis on the Ca 2+ concentration in the supernatant ( n =3). ( i ) IL-1β release from LPS-primed CD14+ monocytes cultured alone (white bars) or co-cultured with necrotic autologous CD4+T cells (black bars) in the absence or presence of the specific inhibitor Calhex231 ( n =3). ( j ) mIL-1β secretion of LPS-primed monocytes from GPRC6A +/+ (wt) and GPRC6A −/− (ko) mice cultured alone (LPS) or co-cultured with necrotic autologous CD4+ T cells (Nc+LPS, n =9). All bars show mean±s.e.m. Statistical analysis was performed using the two-tailed Student’s t -test. * P

    Article Snippet: NLRP3-deficient, ASC-deficient and control THP-1 cells were purchased from InvivoGen.

    Techniques: Isolation, Mouse Assay, Concentration Assay, Cell Culture, Two Tailed Test

    miR-223 target ARNT expression and signalling. ( a ) Ingenuity analysis of putative miR-223 targets as determined in silico . Signalling pathways with the highest scores are indicated. ( b ) Luciferase reporter assays of HEK-293 cells transduced with pre-miR-223 or a control (mock), and transfected with the wild-type ikk α- or the arnt 3′UTR reporter constructs. Luciferase activity was normalised to that of mock-transfected cells. Data shown as mean ± SEM of triplicates in one representative experiment ( n = 3). ( c ) Immunoblot analysis of extracts of HEK-293 cells expressing control or pre-miR-223, probed with anti-ARNT and -hnRNP U (nuclear loading control); the hnRNP U/ARNT ratio in mock-transfected cells is equal to 1. For ( b , c ), data are representative of at least three experiments. ( d ) CYP1A1 mRNA levels in mock and pre-miR-223-expressing THP-1 cells treated with B a P or vehicle. Data shown as mean ± SEM of RQ from triplicates in three independent experiments. *p

    Journal: Scientific Reports

    Article Title: Notch-regulated miR-223 targets the aryl hydrocarbon receptor pathway and increases cytokine production in macrophages from rheumatoid arthritis patients

    doi: 10.1038/srep20223

    Figure Lengend Snippet: miR-223 target ARNT expression and signalling. ( a ) Ingenuity analysis of putative miR-223 targets as determined in silico . Signalling pathways with the highest scores are indicated. ( b ) Luciferase reporter assays of HEK-293 cells transduced with pre-miR-223 or a control (mock), and transfected with the wild-type ikk α- or the arnt 3′UTR reporter constructs. Luciferase activity was normalised to that of mock-transfected cells. Data shown as mean ± SEM of triplicates in one representative experiment ( n = 3). ( c ) Immunoblot analysis of extracts of HEK-293 cells expressing control or pre-miR-223, probed with anti-ARNT and -hnRNP U (nuclear loading control); the hnRNP U/ARNT ratio in mock-transfected cells is equal to 1. For ( b , c ), data are representative of at least three experiments. ( d ) CYP1A1 mRNA levels in mock and pre-miR-223-expressing THP-1 cells treated with B a P or vehicle. Data shown as mean ± SEM of RQ from triplicates in three independent experiments. *p

    Article Snippet: Transfection and stimulation of THP-1 cells THP-1 cells were transfected with the pre-miR-223 (10 μM; Life Technologies), or with a fluorescently-labeled negative control (5 μM) using Amaxa I and the Monocyte Nucleofector Kit (Lonza).

    Techniques: Expressing, In Silico, Luciferase, Transduction, Transfection, Construct, Activity Assay

    miR-223 reverses AHR/ARNT-induced inhibition of pro-inflammatory cytokines. ELISA analyses for IL-1β, TNF-α and IL-6 levels in the supernatant of mock- and pre-miR-223-transfected THP-1 cells stimulated with vehicle, LPS or LPS + B a P. Values are expressed as the concentration of cytokine (pg/ml) per mg cell extract. Data shown as mean ± SEM of values in three independent experiments. *p

    Journal: Scientific Reports

    Article Title: Notch-regulated miR-223 targets the aryl hydrocarbon receptor pathway and increases cytokine production in macrophages from rheumatoid arthritis patients

    doi: 10.1038/srep20223

    Figure Lengend Snippet: miR-223 reverses AHR/ARNT-induced inhibition of pro-inflammatory cytokines. ELISA analyses for IL-1β, TNF-α and IL-6 levels in the supernatant of mock- and pre-miR-223-transfected THP-1 cells stimulated with vehicle, LPS or LPS + B a P. Values are expressed as the concentration of cytokine (pg/ml) per mg cell extract. Data shown as mean ± SEM of values in three independent experiments. *p

    Article Snippet: Transfection and stimulation of THP-1 cells THP-1 cells were transfected with the pre-miR-223 (10 μM; Life Technologies), or with a fluorescently-labeled negative control (5 μM) using Amaxa I and the Monocyte Nucleofector Kit (Lonza).

    Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Transfection, Concentration Assay

    miR-223 prevents AHR/ARNT-induced Notch3 upregulation. ( a) Notch3 mRNA levels in THP-1 cells expressing control (mock) or pre-miR-223 stimulated with B a P or vehicle. Data shown as mean ± SEM of RQ values (triplicates) from three independent experiments. ( b ) Linear regression analysis of CYP1B1 and Notch3 RQ values in RA and OA samples ( n = 10). ( c ) Proposed model of miR-223 regulation in macrophages; miR-223 activity in RA macrophages indicated in red. *p

    Journal: Scientific Reports

    Article Title: Notch-regulated miR-223 targets the aryl hydrocarbon receptor pathway and increases cytokine production in macrophages from rheumatoid arthritis patients

    doi: 10.1038/srep20223

    Figure Lengend Snippet: miR-223 prevents AHR/ARNT-induced Notch3 upregulation. ( a) Notch3 mRNA levels in THP-1 cells expressing control (mock) or pre-miR-223 stimulated with B a P or vehicle. Data shown as mean ± SEM of RQ values (triplicates) from three independent experiments. ( b ) Linear regression analysis of CYP1B1 and Notch3 RQ values in RA and OA samples ( n = 10). ( c ) Proposed model of miR-223 regulation in macrophages; miR-223 activity in RA macrophages indicated in red. *p

    Article Snippet: Transfection and stimulation of THP-1 cells THP-1 cells were transfected with the pre-miR-223 (10 μM; Life Technologies), or with a fluorescently-labeled negative control (5 μM) using Amaxa I and the Monocyte Nucleofector Kit (Lonza).

    Techniques: Expressing, Activity Assay

    ( A ) Rv3131 stimulates the secretion of pro-inflammatory cytokines in THP-1 cells. THP-1 cells differentiated by PMA were stimulated with 10–2500 ng/ml of rRv3131 or LPS (100 ng/ml) or proteinase K digested rRv3131 for 24 and 48 h. Supernatants were collected and the levels of secreted cytokines were estimated by ELISA. Data represent the mean ± SEM of three technical replicates. *p

    Journal: Scientific Reports

    Article Title: A putative nitroreductase from the DosR regulon of Mycobacterium tuberculosis induces pro-inflammatory cytokine expression via TLR2 signaling pathway

    doi: 10.1038/srep24535

    Figure Lengend Snippet: ( A ) Rv3131 stimulates the secretion of pro-inflammatory cytokines in THP-1 cells. THP-1 cells differentiated by PMA were stimulated with 10–2500 ng/ml of rRv3131 or LPS (100 ng/ml) or proteinase K digested rRv3131 for 24 and 48 h. Supernatants were collected and the levels of secreted cytokines were estimated by ELISA. Data represent the mean ± SEM of three technical replicates. *p

    Article Snippet: A significant increase in the expression of TLR2 ( ) but not TLR4 (data not shown) was observed in THP-1 cells when treated with rRv3131.

    Techniques: Enzyme-linked Immunosorbent Assay

    Rv3131 induces TLR2 expression and NF-κB activation. ( A ) Real time expression of TLR2: RNA isolated from differentiated THP-1 cells treated with rRv3131 (1000 ng) or TSA (250 ng) was reverse transcribed and real time PCR was carried out using gene specific primers. The expression of glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) was used as internal control. Data are presented as mean ± SEM of three independent experiments. ( B ) Surface expression of TLR2 by flow cytometry: PMA-differentiated THP-1 cells were treated with 100 ng of rRv3131 for 24 h followed by incubation with primary and secondary FITC labelled antibodies and analysed by flow cytometry. Data are expressed as mean ± SEM of percentage of cell population/MFI values from three independent experiments. ( C ) TLR2 protein expression: total protein isolated from differentiated THP-1 cells treated with rRv3131 (1000 ng) or LPS (100 ng) was electrophoresed on polyacrylamide gel, transferred onto PVDF membrane and probed with antibodies against TLR2 and β-actin (internal control). Signal corresponding to the intensity of the band was measured using chemiluminiscence. The graph represents relative TLR2 expression levels quantified densitometrically ( D ) NF-κB phosphorylation: the total protein isolated was analysed using western blot with antibodies specific to phosphorylated p65.

    Journal: Scientific Reports

    Article Title: A putative nitroreductase from the DosR regulon of Mycobacterium tuberculosis induces pro-inflammatory cytokine expression via TLR2 signaling pathway

    doi: 10.1038/srep24535

    Figure Lengend Snippet: Rv3131 induces TLR2 expression and NF-κB activation. ( A ) Real time expression of TLR2: RNA isolated from differentiated THP-1 cells treated with rRv3131 (1000 ng) or TSA (250 ng) was reverse transcribed and real time PCR was carried out using gene specific primers. The expression of glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) was used as internal control. Data are presented as mean ± SEM of three independent experiments. ( B ) Surface expression of TLR2 by flow cytometry: PMA-differentiated THP-1 cells were treated with 100 ng of rRv3131 for 24 h followed by incubation with primary and secondary FITC labelled antibodies and analysed by flow cytometry. Data are expressed as mean ± SEM of percentage of cell population/MFI values from three independent experiments. ( C ) TLR2 protein expression: total protein isolated from differentiated THP-1 cells treated with rRv3131 (1000 ng) or LPS (100 ng) was electrophoresed on polyacrylamide gel, transferred onto PVDF membrane and probed with antibodies against TLR2 and β-actin (internal control). Signal corresponding to the intensity of the band was measured using chemiluminiscence. The graph represents relative TLR2 expression levels quantified densitometrically ( D ) NF-κB phosphorylation: the total protein isolated was analysed using western blot with antibodies specific to phosphorylated p65.

    Article Snippet: A significant increase in the expression of TLR2 ( ) but not TLR4 (data not shown) was observed in THP-1 cells when treated with rRv3131.

    Techniques: Expressing, Activation Assay, Isolation, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Incubation, Western Blot

    Conditioned medium from monocytic cells stimulates migration of phOBs. In order to investigate the influence of immune cell-conditioned medium on phOBs migration, a scratch assay and an under agar spot assay were performed. phOBs ( N ≥ 4, n ≥ 4) are stimulated with conditioned medium from THP-1 cells (representing monocytes), PMA-stimulated adherent THP-1 cells (representing macrophages), and DMSO-stimulated HL-60 cells (representing granulocytes). ( a ) For the scratch assay, gap closure was determined from microscopic images (100 − gap area 40h /gap area 0h × 100) with the help of the ImageJ software. ( b ) Representative microscopic images for the scratch assay (20× magnification). ( c ) For the under agar spot assay, the number of cells in the spot area was determined from microscopic images with the help of the ImageJ software. ( d ) Representative microscopic images for the under agar spot assay (12.5× magnification). Cells were visualized with Calcein-AM stain for living cells. Data are represented in bar diagrams (mean ± 95% C.I.). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Immune Cell Induced Migration of Osteoprogenitor Cells Is Mediated by TGF-β Dependent Upregulation of NOX4 and Activation of Focal Adhesion Kinase

    doi: 10.3390/ijms19082239

    Figure Lengend Snippet: Conditioned medium from monocytic cells stimulates migration of phOBs. In order to investigate the influence of immune cell-conditioned medium on phOBs migration, a scratch assay and an under agar spot assay were performed. phOBs ( N ≥ 4, n ≥ 4) are stimulated with conditioned medium from THP-1 cells (representing monocytes), PMA-stimulated adherent THP-1 cells (representing macrophages), and DMSO-stimulated HL-60 cells (representing granulocytes). ( a ) For the scratch assay, gap closure was determined from microscopic images (100 − gap area 40h /gap area 0h × 100) with the help of the ImageJ software. ( b ) Representative microscopic images for the scratch assay (20× magnification). ( c ) For the under agar spot assay, the number of cells in the spot area was determined from microscopic images with the help of the ImageJ software. ( d ) Representative microscopic images for the under agar spot assay (12.5× magnification). Cells were visualized with Calcein-AM stain for living cells. Data are represented in bar diagrams (mean ± 95% C.I.). * p

    Article Snippet: THP-1 cells (ACC-16) and HL-60 cells (ACC-3) were obtained from DSMZ (Leibniz-Institut-Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH).

    Techniques: Migration, Wound Healing Assay, Spot Test, Software, Staining

    Conditioned medium from monocytic cells stimulates TGF-β (Smad2/3) signaling in phOBs. ( a ) phOBs ( N = 4, n = 3), infected with the Smad3/4 reporter adenovirus (Ad5-CAGA9-MLP-Luc), were stimulated with conditioned medium from THP-1 cells (representing monocytes), phorbol 12-myristate 13-acetate (PMA)-stimulated adherent THP-1 cells (representing macrophages), and dimethyl sulfoxide (DMSO)-stimulated HL-60 cells (representing granulocytes). In the presence of active TGF-β luciferase is expressed in the cytoplasm of the cells. After 48 h, the luciferase activity was measured with a microplate reader. ( b – e ) In order to investigate the influence of rhTGF-β 1 (5 ng/mL) on phOBs migration, a scratch assay and an under agar spot assay were performed. ( b ) Representative microscopic images for the scratch assay (20× magnification). ( c ) Gap closure was determined from microscopic images (100 − gap area 40h /gap area 0h × 100) with the help of the ImageJ software. ( d ) For the under agar spot assay, the number of cells in the spot area was determined from microscopic images with the help of the ImageJ software. ( e ) Representative microscopic images for the under agar spot assay (12.5× magnification). Cells were visualized with Calcein-AM stain for living cells. Data are represented in bar diagrams (mean ± 95% C.I.). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Immune Cell Induced Migration of Osteoprogenitor Cells Is Mediated by TGF-β Dependent Upregulation of NOX4 and Activation of Focal Adhesion Kinase

    doi: 10.3390/ijms19082239

    Figure Lengend Snippet: Conditioned medium from monocytic cells stimulates TGF-β (Smad2/3) signaling in phOBs. ( a ) phOBs ( N = 4, n = 3), infected with the Smad3/4 reporter adenovirus (Ad5-CAGA9-MLP-Luc), were stimulated with conditioned medium from THP-1 cells (representing monocytes), phorbol 12-myristate 13-acetate (PMA)-stimulated adherent THP-1 cells (representing macrophages), and dimethyl sulfoxide (DMSO)-stimulated HL-60 cells (representing granulocytes). In the presence of active TGF-β luciferase is expressed in the cytoplasm of the cells. After 48 h, the luciferase activity was measured with a microplate reader. ( b – e ) In order to investigate the influence of rhTGF-β 1 (5 ng/mL) on phOBs migration, a scratch assay and an under agar spot assay were performed. ( b ) Representative microscopic images for the scratch assay (20× magnification). ( c ) Gap closure was determined from microscopic images (100 − gap area 40h /gap area 0h × 100) with the help of the ImageJ software. ( d ) For the under agar spot assay, the number of cells in the spot area was determined from microscopic images with the help of the ImageJ software. ( e ) Representative microscopic images for the under agar spot assay (12.5× magnification). Cells were visualized with Calcein-AM stain for living cells. Data are represented in bar diagrams (mean ± 95% C.I.). * p

    Article Snippet: THP-1 cells (ACC-16) and HL-60 cells (ACC-3) were obtained from DSMZ (Leibniz-Institut-Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH).

    Techniques: Infection, Luciferase, Activity Assay, Migration, Wound Healing Assay, Spot Test, Software, Staining

    Effects of DDMG-1 on hTNF-α, hIL-8, hIL-6, and hIL-1β production and cell proliferation induced by LPS-stimulated CD14 + -THP-1 cells. (a) Effects of DDMG-1 on hTNF-α, hIL-8, hIL-6, and hIL-1β production in LPS-stimulated or non-stimulated CD14 + -THP-1 cells. CD14 + -THP-1 cells (1 × 10 6 cells/mL) were untreated or treated with LPS (1 μg/mL) at the indicated concentrations of compounds for 24 h. hTNF-α, hIL-8, hIL-6, and hIL-1β levels in the culture supernatant were measured by ELISA, as described in Materials and Methods. This experiment was repeated three times on different days. Figures were created based on the results. *p

    Journal: Marine Drugs

    Article Title: Inhibitory Effect of N,N-Didesmethylgrossularine-1 on Inflammatory Cytokine Production in Lipopolysaccharide-Stimulated RAW 264.7 Cells

    doi: 10.3390/md7040589

    Figure Lengend Snippet: Effects of DDMG-1 on hTNF-α, hIL-8, hIL-6, and hIL-1β production and cell proliferation induced by LPS-stimulated CD14 + -THP-1 cells. (a) Effects of DDMG-1 on hTNF-α, hIL-8, hIL-6, and hIL-1β production in LPS-stimulated or non-stimulated CD14 + -THP-1 cells. CD14 + -THP-1 cells (1 × 10 6 cells/mL) were untreated or treated with LPS (1 μg/mL) at the indicated concentrations of compounds for 24 h. hTNF-α, hIL-8, hIL-6, and hIL-1β levels in the culture supernatant were measured by ELISA, as described in Materials and Methods. This experiment was repeated three times on different days. Figures were created based on the results. *p

    Article Snippet: THP1-Blue™-CD14 (CD14+ -THP-1) cells were purchased from InvivoGen.

    Techniques: Enzyme-linked Immunosorbent Assay

    CCK-8S suppresses both expression of TNF-α and chemotaxis in THP-1 cells. A : THP-1 cells were cultured under different conditions for 72 h. CCK-8S inhibited HG-induced TNF-α expression in THP-1 cells ( n = 5 each). Values are presented as the ratio of HG group. *** P

    Journal: Diabetes

    Article Title: Cholecystokinin Plays a Novel Protective Role in Diabetic Kidney Through Anti-inflammatory Actions on Macrophage

    doi: 10.2337/db11-0402

    Figure Lengend Snippet: CCK-8S suppresses both expression of TNF-α and chemotaxis in THP-1 cells. A : THP-1 cells were cultured under different conditions for 72 h. CCK-8S inhibited HG-induced TNF-α expression in THP-1 cells ( n = 5 each). Values are presented as the ratio of HG group. *** P

    Article Snippet: THP-1 cells were obtained from DS Pharma Biomedical (Osaka, Japan) and cultured according to the manufacturer’s instructions.

    Techniques: Expressing, Chemotaxis Assay, Cell Culture

    SAB inhibits oxLDL-induced upregulation of CD36 mRNA in RAW 264.7 cells (A), THP-1 cells (B) and primary macrophages (C). Cells were exposed to the indicated concentration of SAB in the presence or absence of 25 μg/mL oxLDL, and the CD36 mRNA

    Journal: Atherosclerosis

    Article Title: Salvianolic acid B inhibits macrophage uptake of modified low density lipoprotein (mLDL) in a scavenger receptor CD36-dependent manner

    doi: 10.1016/j.atherosclerosis.2012.05.006

    Figure Lengend Snippet: SAB inhibits oxLDL-induced upregulation of CD36 mRNA in RAW 264.7 cells (A), THP-1 cells (B) and primary macrophages (C). Cells were exposed to the indicated concentration of SAB in the presence or absence of 25 μg/mL oxLDL, and the CD36 mRNA

    Article Snippet: Cell culture media and fetal bovine serum (FBS) for RAW 264.7 and THP-1 cells were purchased from Hyclone (Logan, UT).

    Techniques: Concentration Assay

    SAB-induced inhibition of DiI-acLDL uptake was CD36-dependent. (A) The effect of SAB on DiI-acLDL uptake by PMA-induced THP-1 cells with CD36 siRNA using flow cytometry. The experiment was repeated for 4 times. *, p

    Journal: Atherosclerosis

    Article Title: Salvianolic acid B inhibits macrophage uptake of modified low density lipoprotein (mLDL) in a scavenger receptor CD36-dependent manner

    doi: 10.1016/j.atherosclerosis.2012.05.006

    Figure Lengend Snippet: SAB-induced inhibition of DiI-acLDL uptake was CD36-dependent. (A) The effect of SAB on DiI-acLDL uptake by PMA-induced THP-1 cells with CD36 siRNA using flow cytometry. The experiment was repeated for 4 times. *, p

    Article Snippet: Cell culture media and fetal bovine serum (FBS) for RAW 264.7 and THP-1 cells were purchased from Hyclone (Logan, UT).

    Techniques: Inhibition, Flow Cytometry, Cytometry

    The effect of SAB on DiI-acLDL uptake by PMA-induced THP-1 cells and RAW 264.7 cells. (A) RAW 264.7 cells incubated with DiI-acLDL, in the presence or absence of SAB, were visualized and photographed using IN Cell Analyzer 1000 with a 20×objective.

    Journal: Atherosclerosis

    Article Title: Salvianolic acid B inhibits macrophage uptake of modified low density lipoprotein (mLDL) in a scavenger receptor CD36-dependent manner

    doi: 10.1016/j.atherosclerosis.2012.05.006

    Figure Lengend Snippet: The effect of SAB on DiI-acLDL uptake by PMA-induced THP-1 cells and RAW 264.7 cells. (A) RAW 264.7 cells incubated with DiI-acLDL, in the presence or absence of SAB, were visualized and photographed using IN Cell Analyzer 1000 with a 20×objective.

    Article Snippet: Cell culture media and fetal bovine serum (FBS) for RAW 264.7 and THP-1 cells were purchased from Hyclone (Logan, UT).

    Techniques: Incubation

    SAMHD1 knockdown and Vpx enhance the dNTP pool in PMA-treated THP-1 cells. (a) THP-1, engineered to stably express shRNA scrambled (control) or shRNA specifically targeting SAMHD1, were differentiated overnight with PMA. Where indicated, the cells were incubated for 2 h with Vpx-containing and control VLP (+/−Vpx). Cell extracts were analyzed by western blot with the indicated antibodies. (b) dNTP concentrations were determined by the single nucleotide incorporation assay described in Diamond et al . The experiment was performed in duplicate and one representative result is shown. (c) dNTP triphosphohydrolase activity of recombinant SAMHD1. dNTPase activity assay was performed as described in the methods section in the presence of the indicated dNTP, 1 μCi of the corresponding γ- 32 P-dNTP and 1 μM of wild type SAMHD1. Where indicated 200 μM of unlabeled dGTP was added. The non-labeled standards were visualized by UV shadowing and γ- 32 P-labeled nucleotides were visualized using a phosphorimager (Fluorescent Image Analyzer FLA3000 (Fuji).

    Journal: Nature immunology

    Article Title: SAMHD1 restricts HIV-1 by reducing the intracellular pool of deoxynucleotide triphosphates

    doi: 10.1038/ni.2236

    Figure Lengend Snippet: SAMHD1 knockdown and Vpx enhance the dNTP pool in PMA-treated THP-1 cells. (a) THP-1, engineered to stably express shRNA scrambled (control) or shRNA specifically targeting SAMHD1, were differentiated overnight with PMA. Where indicated, the cells were incubated for 2 h with Vpx-containing and control VLP (+/−Vpx). Cell extracts were analyzed by western blot with the indicated antibodies. (b) dNTP concentrations were determined by the single nucleotide incorporation assay described in Diamond et al . The experiment was performed in duplicate and one representative result is shown. (c) dNTP triphosphohydrolase activity of recombinant SAMHD1. dNTPase activity assay was performed as described in the methods section in the presence of the indicated dNTP, 1 μCi of the corresponding γ- 32 P-dNTP and 1 μM of wild type SAMHD1. Where indicated 200 μM of unlabeled dGTP was added. The non-labeled standards were visualized by UV shadowing and γ- 32 P-labeled nucleotides were visualized using a phosphorimager (Fluorescent Image Analyzer FLA3000 (Fuji).

    Article Snippet: SAMHD1 and alpha-tubulin expression in THP-1 cells was controlled using antibodies purchased respectively from Abcam (ab67820) and Sigma (T9026).

    Techniques: Stable Transfection, shRNA, Incubation, Western Blot, Activity Assay, Recombinant, Labeling

    Catalpol increased telomerase activity and inhibited macrophage senescence. 6–8-week-old male LDLr −/− mice were administered with a standard diet with catalpol (0 and 100 mg/kg) or HFD with catalpol (0, 100, and 200 mg/kg). THP-1 cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation. Then, macrophages were treated with oxLDL or catalpol (0, 5, 20, and 80 μ M) for 24 h as indicated. Catalpol increased telomerase activity in (a) LDLr −/− mice and (b) oxLDL-treated macrophages, n = 10. ∗∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Activating the PGC-1α/TERT Pathway by Catalpol Ameliorates Atherosclerosis via Modulating ROS Production, DNA Damage, and Telomere Function: Implications on Mitochondria and Telomere Link

    doi: 10.1155/2018/2876350

    Figure Lengend Snippet: Catalpol increased telomerase activity and inhibited macrophage senescence. 6–8-week-old male LDLr −/− mice were administered with a standard diet with catalpol (0 and 100 mg/kg) or HFD with catalpol (0, 100, and 200 mg/kg). THP-1 cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation. Then, macrophages were treated with oxLDL or catalpol (0, 5, 20, and 80 μ M) for 24 h as indicated. Catalpol increased telomerase activity in (a) LDLr −/− mice and (b) oxLDL-treated macrophages, n = 10. ∗∗ p

    Article Snippet: Human THP-1 cells were purchased from ScienCell company (CA, USA) and cultured in RPMI 1640 medium (Gibco, CA, USA) with 10% ( v / v ) fetal bovine serum (Gibco), 20 mg/mL penicillin, and 20 mg/mL streptomycin and maintained at 37°C in a humidified atmosphere of 5% CO2 and then treated with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, Missouri, USA) for 48 h to induce differentiation into macrophages, and then the in vitro model was established by replacing the mediums with a serum-free medium containing oxLDL (100 μ g/mL) for 24 h as previously described.

    Techniques: Activity Assay, Mouse Assay

    Activation of the PGC-1 α /TERT pathway was involved in the protective effect of catalpol against AS. 6–8-week-old male LDLr −/− mice were administered with a standard diet with catalpol (0 and 100 mg/kg) or HFD with catalpol (0, 100, and 200 mg/kg). (a) Catalpol increased PGC-1 α and TERT expression and decreased p53 expression in HFD-treated LDLr −/− mice, n = 10. (b) THP-1 cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation. Then, macrophages were treated with catalpol (80 μ M) for 0, 3, 6, 12, and 24 h. Catalpol increased PGC-1 α and TERT expression and decreased p53 expression in a time-dependent manner, n = 10. (c) THP-1 cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation. Then, macrophages were treated with catalpol (0, 5, 10, 20, 40, and 80 μ M) for 24 h. Catalpol increased PGC-1 α and TERT expression and decreased p53 expression in a concentration-dependent manner, n = 10. (d) THP-1 cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation. Then, macrophages were treated with oxLDL (0 and 100 μ g/mL) or catalpol (0, 5, 20, and 80 μ M) for 24 h. Catalpol increased PGC-1 α and TERT expression and decreased p53 expression in oxLDL-treated macrophages, n = 10. ∗∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Activating the PGC-1α/TERT Pathway by Catalpol Ameliorates Atherosclerosis via Modulating ROS Production, DNA Damage, and Telomere Function: Implications on Mitochondria and Telomere Link

    doi: 10.1155/2018/2876350

    Figure Lengend Snippet: Activation of the PGC-1 α /TERT pathway was involved in the protective effect of catalpol against AS. 6–8-week-old male LDLr −/− mice were administered with a standard diet with catalpol (0 and 100 mg/kg) or HFD with catalpol (0, 100, and 200 mg/kg). (a) Catalpol increased PGC-1 α and TERT expression and decreased p53 expression in HFD-treated LDLr −/− mice, n = 10. (b) THP-1 cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation. Then, macrophages were treated with catalpol (80 μ M) for 0, 3, 6, 12, and 24 h. Catalpol increased PGC-1 α and TERT expression and decreased p53 expression in a time-dependent manner, n = 10. (c) THP-1 cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation. Then, macrophages were treated with catalpol (0, 5, 10, 20, 40, and 80 μ M) for 24 h. Catalpol increased PGC-1 α and TERT expression and decreased p53 expression in a concentration-dependent manner, n = 10. (d) THP-1 cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation. Then, macrophages were treated with oxLDL (0 and 100 μ g/mL) or catalpol (0, 5, 20, and 80 μ M) for 24 h. Catalpol increased PGC-1 α and TERT expression and decreased p53 expression in oxLDL-treated macrophages, n = 10. ∗∗ p

    Article Snippet: Human THP-1 cells were purchased from ScienCell company (CA, USA) and cultured in RPMI 1640 medium (Gibco, CA, USA) with 10% ( v / v ) fetal bovine serum (Gibco), 20 mg/mL penicillin, and 20 mg/mL streptomycin and maintained at 37°C in a humidified atmosphere of 5% CO2 and then treated with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, Missouri, USA) for 48 h to induce differentiation into macrophages, and then the in vitro model was established by replacing the mediums with a serum-free medium containing oxLDL (100 μ g/mL) for 24 h as previously described.

    Techniques: Activation Assay, Pyrolysis Gas Chromatography, Mouse Assay, Expressing, Concentration Assay

    Catalpol decreases DNA damage and ROS accumulation and ameliorates telomere function in THP-1 macrophages through the PGC-1 α /TERT pathway. THP-1 cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation and then transfected with small nontargeting RNA for si-control or si-TERT and si-PGC-1 α for 12 h, then treated with ox-LDL or catalpol for an additional 24 h and harvested and analyzed by Western blot analysis. (a) Effect of catalpol on PGC-1 α and TERT protein expression and decreasing p53 protein expression with PGC-1 α and TERT siRNA, n = 10. (b) Effect of catalpol on ROS production with PGC-1 α and TERT siRNA, n = 10. (c) Effect of catalpol on cell apoptosis with PGC-1 α and TERT siRNA, n = 10. (d) Effects of catalpol on cell senescence and DNA damage, n = 10. (e) Telomerase activity, n = 10. (f) oxidative DNA damage, n = 10. ∗∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Activating the PGC-1α/TERT Pathway by Catalpol Ameliorates Atherosclerosis via Modulating ROS Production, DNA Damage, and Telomere Function: Implications on Mitochondria and Telomere Link

    doi: 10.1155/2018/2876350

    Figure Lengend Snippet: Catalpol decreases DNA damage and ROS accumulation and ameliorates telomere function in THP-1 macrophages through the PGC-1 α /TERT pathway. THP-1 cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation and then transfected with small nontargeting RNA for si-control or si-TERT and si-PGC-1 α for 12 h, then treated with ox-LDL or catalpol for an additional 24 h and harvested and analyzed by Western blot analysis. (a) Effect of catalpol on PGC-1 α and TERT protein expression and decreasing p53 protein expression with PGC-1 α and TERT siRNA, n = 10. (b) Effect of catalpol on ROS production with PGC-1 α and TERT siRNA, n = 10. (c) Effect of catalpol on cell apoptosis with PGC-1 α and TERT siRNA, n = 10. (d) Effects of catalpol on cell senescence and DNA damage, n = 10. (e) Telomerase activity, n = 10. (f) oxidative DNA damage, n = 10. ∗∗ p

    Article Snippet: Human THP-1 cells were purchased from ScienCell company (CA, USA) and cultured in RPMI 1640 medium (Gibco, CA, USA) with 10% ( v / v ) fetal bovine serum (Gibco), 20 mg/mL penicillin, and 20 mg/mL streptomycin and maintained at 37°C in a humidified atmosphere of 5% CO2 and then treated with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, Missouri, USA) for 48 h to induce differentiation into macrophages, and then the in vitro model was established by replacing the mediums with a serum-free medium containing oxLDL (100 μ g/mL) for 24 h as previously described.

    Techniques: Pyrolysis Gas Chromatography, Transfection, Western Blot, Expressing, Activity Assay

    Catalpol directly enhances PGC-1 α promoter activity. Luciferase reporter gene vector pGL4 containing the PGC-1 α promoter area was obtained and transfected into THP-1-derived macrophages. Reporter assays were conducted 24 h after administrating with catalpol (0, 5, 20, and 80 μ M) or pioglitazone (5 μ M). The luciferase activity was determined and was normalized for Renilla luciferase activity. (a) The PGC-1 α promoter was inserted into the PGL4 vector. (b) Effect of catalpol on PGC-1 α promoter activity by dual luciferase activity, n = 10. ∗∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Activating the PGC-1α/TERT Pathway by Catalpol Ameliorates Atherosclerosis via Modulating ROS Production, DNA Damage, and Telomere Function: Implications on Mitochondria and Telomere Link

    doi: 10.1155/2018/2876350

    Figure Lengend Snippet: Catalpol directly enhances PGC-1 α promoter activity. Luciferase reporter gene vector pGL4 containing the PGC-1 α promoter area was obtained and transfected into THP-1-derived macrophages. Reporter assays were conducted 24 h after administrating with catalpol (0, 5, 20, and 80 μ M) or pioglitazone (5 μ M). The luciferase activity was determined and was normalized for Renilla luciferase activity. (a) The PGC-1 α promoter was inserted into the PGL4 vector. (b) Effect of catalpol on PGC-1 α promoter activity by dual luciferase activity, n = 10. ∗∗ p

    Article Snippet: Human THP-1 cells were purchased from ScienCell company (CA, USA) and cultured in RPMI 1640 medium (Gibco, CA, USA) with 10% ( v / v ) fetal bovine serum (Gibco), 20 mg/mL penicillin, and 20 mg/mL streptomycin and maintained at 37°C in a humidified atmosphere of 5% CO2 and then treated with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, Missouri, USA) for 48 h to induce differentiation into macrophages, and then the in vitro model was established by replacing the mediums with a serum-free medium containing oxLDL (100 μ g/mL) for 24 h as previously described.

    Techniques: Pyrolysis Gas Chromatography, Activity Assay, Luciferase, Plasmid Preparation, Transfection, Derivative Assay

    Catalpol decreases ROS accumulation and DNA damage and ameliorates telomere function through upregulating PGC-1 α expression. THP-1 cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation and then transfected with small nontargeting RNA for si-control or si-PGC-1 α for 12 h, then treated with oxLDL or catalpol for an additional 24 h and harvested and analyzed by Western blot analysis. (a) Knockdown of PGC-1 α by its specific siRNA, n = 10. (b) Effect of catalpol on increasing PGC-1 α and TERT protein expression and decreasing p53 protein expression was attenuated with PGC-1 α siRNA, n = 10. (c) Effect of catalpol on ROS production with PGC-1 α siRNA, n = 10. (d) Effects of catalpol on cell apoptosis with PGC-1 α siRNA, n = 10. (e) Effects of catalpol on cell senescence and DNA damage, n = 10. (f) Telomerase activity, n = 10. (g) Oxidative DNA damage, n = 10. ∗∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Activating the PGC-1α/TERT Pathway by Catalpol Ameliorates Atherosclerosis via Modulating ROS Production, DNA Damage, and Telomere Function: Implications on Mitochondria and Telomere Link

    doi: 10.1155/2018/2876350

    Figure Lengend Snippet: Catalpol decreases ROS accumulation and DNA damage and ameliorates telomere function through upregulating PGC-1 α expression. THP-1 cells were exposed to PMA (100 ng/mL) for 72 h to induce macrophage formation and then transfected with small nontargeting RNA for si-control or si-PGC-1 α for 12 h, then treated with oxLDL or catalpol for an additional 24 h and harvested and analyzed by Western blot analysis. (a) Knockdown of PGC-1 α by its specific siRNA, n = 10. (b) Effect of catalpol on increasing PGC-1 α and TERT protein expression and decreasing p53 protein expression was attenuated with PGC-1 α siRNA, n = 10. (c) Effect of catalpol on ROS production with PGC-1 α siRNA, n = 10. (d) Effects of catalpol on cell apoptosis with PGC-1 α siRNA, n = 10. (e) Effects of catalpol on cell senescence and DNA damage, n = 10. (f) Telomerase activity, n = 10. (g) Oxidative DNA damage, n = 10. ∗∗ p

    Article Snippet: Human THP-1 cells were purchased from ScienCell company (CA, USA) and cultured in RPMI 1640 medium (Gibco, CA, USA) with 10% ( v / v ) fetal bovine serum (Gibco), 20 mg/mL penicillin, and 20 mg/mL streptomycin and maintained at 37°C in a humidified atmosphere of 5% CO2 and then treated with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, Missouri, USA) for 48 h to induce differentiation into macrophages, and then the in vitro model was established by replacing the mediums with a serum-free medium containing oxLDL (100 μ g/mL) for 24 h as previously described.

    Techniques: Pyrolysis Gas Chromatography, Expressing, Transfection, Western Blot, Activity Assay