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  • 90
    Cell Signaling Technology Inc stat3α
    DHA inhibits TPA-induced PKCδ activation, STAT3 DNA binding activity, β-catenin, and <t>STAT3α</t> expression MCF-7 cells were pretreated with 0, 25, or 100 μM DHA for 24 h followed by incubation with 100 ng/ml of TPA for another 30 min. PKCδ in the plasma membrane and cytosol ( A ) and STAT3α ( B ) and GSK3β ( C ) phosphorylation were determined. ( D ) Total cellular β-catenin and STAT3α and nuclear β-catenin protein levels were determined in cells pretreated with various concentrations of DHA for 24 h followed by incubation with TPA for another 24 h and 4 h, respectively. ( E ) Cells were pretreated with 100 μM DHA for 24 h, 5 μM WP for 4 h, or 5 μM rottlerin (Rot) for 1 h, and then were incubated with TPA for another 6 h. Nuclear extracts (10 μg) were prepared for STAT3 nuclear protein DNA binding activity assay. To confirm the specificity of the nucleotide, 50-fold cold probe and mutant (Mu) were included in the EMSA. One representative experiment out of three independent experiments is shown. ** p
    Stat3α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1531 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology stat3α
    Modulation of <t>STAT3α</t> cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.
    Stat3α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genecopoeia stat3α 3 utr
    Modulation of <t>STAT3α</t> cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.
    Stat3α 3 Utr, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc rabbit anti stat3α
    <t>STAT3α</t> is expressed in the sensory epithelium in the organ of Corti. Immunohistochemical staining of vibratome-cut whole mount cochlea sections for STAT3α (green) showed fluorescent signal in the cytoplasm of inner hair cells (IHC), outer hair cells (OHC) and Deiters cells (DC). Open arrow heads point to OHCs, closed arrow heads point to IHCs, and the arrows point to DCs. Nuclei are stained with Hoescht 33258 (blue). The images are from the upper basal turn of the cochlea and are representative of 3 individual mice.
    Rabbit Anti Stat3α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher human stat3α cdna
    <t>STAT3α</t> is expressed in the sensory epithelium in the organ of Corti. Immunohistochemical staining of vibratome-cut whole mount cochlea sections for STAT3α (green) showed fluorescent signal in the cytoplasm of inner hair cells (IHC), outer hair cells (OHC) and Deiters cells (DC). Open arrow heads point to OHCs, closed arrow heads point to IHCs, and the arrows point to DCs. Nuclei are stained with Hoescht 33258 (blue). The images are from the upper basal turn of the cochlea and are representative of 3 individual mice.
    Human Stat3α Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    OriGene mouse stat3α
    STAT3β is activated in the mammary epithelial cells during involution. ( A ) Western blot analysis of total <t>STAT3α/β</t> in the CDβ cell line and the CD-derived clones. ( B–D ) Western blot analysis of total STAT3α/β
    Mouse Stat3α, supplied by OriGene, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Epitomics anti stat3α
    STAT3β is activated in the mammary epithelial cells during involution. ( A ) Western blot analysis of total <t>STAT3α/β</t> in the CDβ cell line and the CD-derived clones. ( B–D ) Western blot analysis of total STAT3α/β
    Anti Stat3α, supplied by Epitomics, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology anti stat3α carboxy terminal antibody
    Modulation of <t>STAT3α</t> cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.
    Anti Stat3α Carboxy Terminal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cell Signaling Technology Inc anti stat3α β
    Modulation of <t>STAT3α</t> cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.
    Anti Stat3α β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc anti stat3α rabbit igg
    Modulation of <t>STAT3α</t> cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.
    Anti Stat3α Rabbit Igg, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti stat3α rabbit igg/product/Cell Signaling Technology Inc
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    86
    Cell Signaling Technology Inc rabbit anti human stat3α antibody
    Modulation of <t>STAT3α</t> cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.
    Rabbit Anti Human Stat3α Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam anti stat3α
    Rescue experiments using single STAT3 variants. ( a ) Workflow of knockdown/re-expression approach. Different stable cell lines were induced for expression of their individual STAT3 variants or EV control for 3 days before transduction with STAT3 shRNAs with the marker GFP. The shRNA-expressing GFP+ cells were monitored by flow cytometry over 12 days of observation. ( b ) Expression and activation of the single STAT3 variants in ABC DLBCL cells after knockdown of endogenous STAT3 by shRNA#16. OCI-Ly10 cells were manipulated as described in a , and shSTAT3 cells were selected with puromycin. After 5 days of selection, shSTAT3 was induced for expression for 4 days before immunoblotting with STAT3 or phospho-tyrosine 705 (pY705) antibody. Note that this monoclonal antibody did not recognize phospho-tyrosine 704 of STAT3ΔSα and ΔSβ. Expression of pY705 <t>STAT3α</t> or β relative to the uninduced control was quantified by densitometry. ( c ) Insufficient rescue by the individual variants. ABC DLBCL cell lines HBL1 and OCI-Ly10 and control GCB cell line OCI-Ly19 were used following the experimental procedure as described in a . The percentage of GFP+ cells expressing STAT3 shRNAs was normalized to that of the control shRNA at each time point. STAT3Sα-C expressing cells served as positive controls. The experiments were repeated twice with two different STAT3 shRNAs. ( d ) The rescue efficiencies of different STAT3 variant stable cell lines on day 12 were calculated by a formula: (% variant−% EV)/(% STAT3Sα-C−% EV). Error bars represent mean±s.d. ( n =4, * P
    Anti Stat3α, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bachem z vad fmk
    Modulation of STAT3α cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor <t>z-VAD-FMK</t> and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.
    Z Vad Fmk, supplied by Bachem, used in various techniques. Bioz Stars score: 92/100, based on 326 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    z vad fmk - by Bioz Stars, 2020-05
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    99
    Jackson Immuno anti mouse
    Modulation of STAT3α cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor <t>z-VAD-FMK</t> and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.
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    GE Healthcare amersham ecl
    Modulation of STAT3α cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor <t>z-VAD-FMK</t> and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.
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    Active Motif recombinant stat3 protein
    The comparative effect of curcumin and tetrahydrocurcumin on <t>STAT3</t> dimerization. ( A ) Total lysate from H- Ras MCF10A cells treated with curcumin (25 μM) for 12 h was immunoprecipitated with Protein A/G agarose and anti-STAT3 antibody overnight and analyzed by immunobloting with anti-STAT3 antibody. * P
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    SignalChem gst stat3 protein
    The comparative effect of curcumin and tetrahydrocurcumin on <t>STAT3</t> dimerization. ( A ) Total lysate from H- Ras MCF10A cells treated with curcumin (25 μM) for 12 h was immunoprecipitated with Protein A/G agarose and anti-STAT3 antibody overnight and analyzed by immunobloting with anti-STAT3 antibody. * P
    Gst Stat3 Protein, supplied by SignalChem, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Johnson & Johnson stat3 protein signaling
    The comparative effect of curcumin and tetrahydrocurcumin on <t>STAT3</t> dimerization. ( A ) Total lysate from H- Ras MCF10A cells treated with curcumin (25 μM) for 12 h was immunoprecipitated with Protein A/G agarose and anti-STAT3 antibody overnight and analyzed by immunobloting with anti-STAT3 antibody. * P
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    Active Motif transam stat3 transcription factor assay kit
    The comparative effect of curcumin and tetrahydrocurcumin on <t>STAT3</t> dimerization. ( A ) Total lysate from H- Ras MCF10A cells treated with curcumin (25 μM) for 12 h was immunoprecipitated with Protein A/G agarose and anti-STAT3 antibody overnight and analyzed by immunobloting with anti-STAT3 antibody. * P
    Transam Stat3 Transcription Factor Assay Kit, supplied by Active Motif, used in various techniques. Bioz Stars score: 93/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam length gst tagged human stat3 protein
    The comparative effect of curcumin and tetrahydrocurcumin on <t>STAT3</t> dimerization. ( A ) Total lysate from H- Ras MCF10A cells treated with curcumin (25 μM) for 12 h was immunoprecipitated with Protein A/G agarose and anti-STAT3 antibody overnight and analyzed by immunobloting with anti-STAT3 antibody. * P
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    Abcam stat3 protein
    IFN-γ-induced signal transduction, in the presence of FLLL32 and FLLL62. (A) Human ACHN RCC cells or (B) A375 melanoma cells were pre-treated for 16 hours with DMSO (negative control), multiple doses of FLLL32, FLLL62, or curcumin and subsequently stimulated with IFN-γ (10 ng/mL) or PBS (vehicle) for 15 minutes. The expression of pSTAT1 and pSTAT3 were evaluated by immunoblot. Membranes were probed for total <t>STAT3</t> protein, total STAT1 protein and β-actin to control for loading.
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    TaKaRa stat3 transcription factor kit
    LLL12 inhibited <t>STAT3</t> DNA binding activity and expression of downstream targets associated with proliferation and survival of MM tumor cells (A) U266 and ARH-77 MM cells were treated in LLL12 (2.5μM) or DMSO for 24 hours and nuclear extracts were examined for DNA binding activity. LLL12 induced statistically significant (*) reductions in STAT3 DNA binding activity, results are representative of two independent experiments in each cell line. (B) U266 and ARH-77 human MM cell lines were cultured in LLL12 (5 μM) or DMSO for 24 hours. Reverse transcriptase PCR revealed decreased expression of STAT3 target genes in LLL12-treated cells as compared to DMSO control following treatment with LLL12. (C) Downstream STAT3 target proteins, Cyclin D1, Survivin, Bcl-2, and DNMT1 were downregulated by LLL12 as shown by western blot analysis. (D) MM.1S cells were stimulated with IL-6 (2.5–10 ng/ml) for 48 hours in the presence or absence of LLL12 (2.5μM) for the last 24 hours. Reverse transcriptase PCR showed IL-6 enhanced expression of STAT3 target genes, which was blocked by LLL12.
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    Image Search Results


    DHA inhibits TPA-induced PKCδ activation, STAT3 DNA binding activity, β-catenin, and STAT3α expression MCF-7 cells were pretreated with 0, 25, or 100 μM DHA for 24 h followed by incubation with 100 ng/ml of TPA for another 30 min. PKCδ in the plasma membrane and cytosol ( A ) and STAT3α ( B ) and GSK3β ( C ) phosphorylation were determined. ( D ) Total cellular β-catenin and STAT3α and nuclear β-catenin protein levels were determined in cells pretreated with various concentrations of DHA for 24 h followed by incubation with TPA for another 24 h and 4 h, respectively. ( E ) Cells were pretreated with 100 μM DHA for 24 h, 5 μM WP for 4 h, or 5 μM rottlerin (Rot) for 1 h, and then were incubated with TPA for another 6 h. Nuclear extracts (10 μg) were prepared for STAT3 nuclear protein DNA binding activity assay. To confirm the specificity of the nucleotide, 50-fold cold probe and mutant (Mu) were included in the EMSA. One representative experiment out of three independent experiments is shown. ** p

    Journal: Oncotarget

    Article Title: Docosahexaenoic acid inhibits 12-O-tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

    doi: 10.18632/oncotarget.7301

    Figure Lengend Snippet: DHA inhibits TPA-induced PKCδ activation, STAT3 DNA binding activity, β-catenin, and STAT3α expression MCF-7 cells were pretreated with 0, 25, or 100 μM DHA for 24 h followed by incubation with 100 ng/ml of TPA for another 30 min. PKCδ in the plasma membrane and cytosol ( A ) and STAT3α ( B ) and GSK3β ( C ) phosphorylation were determined. ( D ) Total cellular β-catenin and STAT3α and nuclear β-catenin protein levels were determined in cells pretreated with various concentrations of DHA for 24 h followed by incubation with TPA for another 24 h and 4 h, respectively. ( E ) Cells were pretreated with 100 μM DHA for 24 h, 5 μM WP for 4 h, or 5 μM rottlerin (Rot) for 1 h, and then were incubated with TPA for another 6 h. Nuclear extracts (10 μg) were prepared for STAT3 nuclear protein DNA binding activity assay. To confirm the specificity of the nucleotide, 50-fold cold probe and mutant (Mu) were included in the EMSA. One representative experiment out of three independent experiments is shown. ** p

    Article Snippet: Reagents DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

    Techniques: Activation Assay, Binding Assay, Activity Assay, Expressing, Incubation, Mutagenesis

    TPA induces cellular β-catenin and STAT3α protein expression and nuclear translocation and β-catenin siRNA abolishes TPA-induced STAT3α and fascin-1 expression in MCF-7 cells After treatment with various concentrations of TPA for 24 h ( A ) or with 100 ng/ml of TPA for 0–24 h ( B ), cellular β-catenin, STAT3α, and fascin-1 expression as well as nuclear (N) β-catenin and STAT3α expression were determined by Western blotting. ( C ) Cells were transfected with β-catenin siRNA or nontargeting control (NTC) and were then treated with 100 ng/ml of TPA for an additional 24 h. β-catenin, STAT3α, and fascin-1 proteins were determined. ( D ) MCF-7 cells were treated with 100 ng/ml of TPA for 6 h, and cell lysate was prepared for ChIP-PCR assay for STAT3 binding in fascin-1 gene in MCF-7 cells. “Input”, total input DNA; “H3”, DNA-protein complex pulled down by acetyl Histone H3; “STAT3”, DNA-protein complex pulled down by anti-STAT3; and “IgG”, DNA-protein complex pulled down by rabbit IgG antibody. H3 served as a positive control for STAT3 binding. Values are mean ± SD, n = 3. * p

    Journal: Oncotarget

    Article Title: Docosahexaenoic acid inhibits 12-O-tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

    doi: 10.18632/oncotarget.7301

    Figure Lengend Snippet: TPA induces cellular β-catenin and STAT3α protein expression and nuclear translocation and β-catenin siRNA abolishes TPA-induced STAT3α and fascin-1 expression in MCF-7 cells After treatment with various concentrations of TPA for 24 h ( A ) or with 100 ng/ml of TPA for 0–24 h ( B ), cellular β-catenin, STAT3α, and fascin-1 expression as well as nuclear (N) β-catenin and STAT3α expression were determined by Western blotting. ( C ) Cells were transfected with β-catenin siRNA or nontargeting control (NTC) and were then treated with 100 ng/ml of TPA for an additional 24 h. β-catenin, STAT3α, and fascin-1 proteins were determined. ( D ) MCF-7 cells were treated with 100 ng/ml of TPA for 6 h, and cell lysate was prepared for ChIP-PCR assay for STAT3 binding in fascin-1 gene in MCF-7 cells. “Input”, total input DNA; “H3”, DNA-protein complex pulled down by acetyl Histone H3; “STAT3”, DNA-protein complex pulled down by anti-STAT3; and “IgG”, DNA-protein complex pulled down by rabbit IgG antibody. H3 served as a positive control for STAT3 binding. Values are mean ± SD, n = 3. * p

    Article Snippet: Reagents DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

    Techniques: Expressing, Translocation Assay, Western Blot, Transfection, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Positive Control

    TPA increase in STAT3α phosphorylation up-regulates β-catenin and fascin-1 expression MCF-7 cells were treated with various concentrations of TPA for 30 min ( A ), were treated with 100 ng/ml of TPA for different time periods ( B ), or were pretreated with 1 or 5 μM WP1066 for 4 h followed by incubation with 100 ng/ml of TPA for another 30 min ( C ). STAT3α phosphorylation at Tyr705 was measured. Cells were pretreated with 1 or 5 μM WP1066 for 4 h followed by incubation with 100 ng/ml of TPA for another 24 h. The amounts of cellular β-catenin protein ( D ) were determined. Changes in β-catenin ( E ) and fascin-1 mRNA ( F ) and protein ( G ) were measured in cells pretreated with or without 5 μM WP1066 for 4 h, followed by incubation with TPA for another 18 h and 24 h, respectively. ( H ) Cells were transfected with STAT3 expression vector or pcDNA3.1 ( − ) control vector and were then treated with 100 ng/ml of TPA for an additional 24 h. STAT3α, β-catenin, and fascin-1 proteins were determined. One representative experiment out of three independent experiments is shown. Values are presented as mean ± SD, n = 3. * p

    Journal: Oncotarget

    Article Title: Docosahexaenoic acid inhibits 12-O-tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

    doi: 10.18632/oncotarget.7301

    Figure Lengend Snippet: TPA increase in STAT3α phosphorylation up-regulates β-catenin and fascin-1 expression MCF-7 cells were treated with various concentrations of TPA for 30 min ( A ), were treated with 100 ng/ml of TPA for different time periods ( B ), or were pretreated with 1 or 5 μM WP1066 for 4 h followed by incubation with 100 ng/ml of TPA for another 30 min ( C ). STAT3α phosphorylation at Tyr705 was measured. Cells were pretreated with 1 or 5 μM WP1066 for 4 h followed by incubation with 100 ng/ml of TPA for another 24 h. The amounts of cellular β-catenin protein ( D ) were determined. Changes in β-catenin ( E ) and fascin-1 mRNA ( F ) and protein ( G ) were measured in cells pretreated with or without 5 μM WP1066 for 4 h, followed by incubation with TPA for another 18 h and 24 h, respectively. ( H ) Cells were transfected with STAT3 expression vector or pcDNA3.1 ( − ) control vector and were then treated with 100 ng/ml of TPA for an additional 24 h. STAT3α, β-catenin, and fascin-1 proteins were determined. One representative experiment out of three independent experiments is shown. Values are presented as mean ± SD, n = 3. * p

    Article Snippet: Reagents DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

    Techniques: Expressing, Incubation, Transfection, Plasmid Preparation

    PKC is an upstream mediator of STAT3α and GSK3β phosphorylation induced by TPA MCF-7 cells were treated with various concentrations of TPA for 30 min and PKCδ in the plasma membrane and cytosolic fractions were determined ( A ). Cells were pretreated with or without 0.5 μM nonselective PKC inhibitor GF109203X (GF) or 5 μM PKCδ-specific inhibitor rottlerin (Rot) for 1 h followed by incubation with 100 ng/ml of TPA for another 30 min. STAT3α and GSK3β phosphorylation were measured ( B ). β-Catenin, STAT3α, and fascin-1 mRNA ( C ) and protein ( D ) levels were determined after 18 h and 24 h with TPA treatment, respectively. One representative experiment out of three independent experiments is shown. Mean ± SD, n = 3. * p

    Journal: Oncotarget

    Article Title: Docosahexaenoic acid inhibits 12-O-tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

    doi: 10.18632/oncotarget.7301

    Figure Lengend Snippet: PKC is an upstream mediator of STAT3α and GSK3β phosphorylation induced by TPA MCF-7 cells were treated with various concentrations of TPA for 30 min and PKCδ in the plasma membrane and cytosolic fractions were determined ( A ). Cells were pretreated with or without 0.5 μM nonselective PKC inhibitor GF109203X (GF) or 5 μM PKCδ-specific inhibitor rottlerin (Rot) for 1 h followed by incubation with 100 ng/ml of TPA for another 30 min. STAT3α and GSK3β phosphorylation were measured ( B ). β-Catenin, STAT3α, and fascin-1 mRNA ( C ) and protein ( D ) levels were determined after 18 h and 24 h with TPA treatment, respectively. One representative experiment out of three independent experiments is shown. Mean ± SD, n = 3. * p

    Article Snippet: Reagents DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

    Techniques: Incubation

    DHA and silencing of β-catenin, STAT3α, and fascin-1 suppress Hs578T cell migration ( A ) β-catenin, STAT3α, and fascin-1 expression in MCF-7 and Hs578T cells were determined by Western blotting. ( B ) Hs578T cells were treated with various concentrations of DHA for 24 h. ( C ) Cells were transiently transfected with β-catenin, STAT3, and fascin-1 siRNA or with nontargeting control (NTC) followed by treated with or without 100 μM DHA, 5 μM Rot or 5 μM WP1066 for an additional 24 h. Protein levels of β-catenin, STAT3α, and fascin-1 (C) as well as cell migration ( D ) were determined. One representative experiment out of three independent experiments is shown.

    Journal: Oncotarget

    Article Title: Docosahexaenoic acid inhibits 12-O-tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

    doi: 10.18632/oncotarget.7301

    Figure Lengend Snippet: DHA and silencing of β-catenin, STAT3α, and fascin-1 suppress Hs578T cell migration ( A ) β-catenin, STAT3α, and fascin-1 expression in MCF-7 and Hs578T cells were determined by Western blotting. ( B ) Hs578T cells were treated with various concentrations of DHA for 24 h. ( C ) Cells were transiently transfected with β-catenin, STAT3, and fascin-1 siRNA or with nontargeting control (NTC) followed by treated with or without 100 μM DHA, 5 μM Rot or 5 μM WP1066 for an additional 24 h. Protein levels of β-catenin, STAT3α, and fascin-1 (C) as well as cell migration ( D ) were determined. One representative experiment out of three independent experiments is shown.

    Article Snippet: Reagents DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

    Techniques: Migration, Expressing, Western Blot, Transfection

    The JAK/STAT3 pathway is activated in reactive astrocytes in the mouse model of HD. A , Images of brain sections showing double staining for GFAP (red) and STAT3 (green) on mouse brain sections, 6 weeks after the infection of striatal neurons with lenti-Htt18Q or lenti-Htt82Q. Astrocytes in the Htt82Q striatum are hypertrophic and express higher levels of STAT3 in their nucleus relative to resting astrocytes in the Htt18Q striatum. B , C , The number of nSTAT3 + /GFAP + cells ( B ) and the percentage of cells displaying strong staining for STAT3 ( C ) are significantly higher in the Htt82Q striatum than in the Htt18Q striatum. n = 6. ** p

    Journal: The Journal of Neuroscience

    Article Title: The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases

    doi: 10.1523/JNEUROSCI.3516-14.2015

    Figure Lengend Snippet: The JAK/STAT3 pathway is activated in reactive astrocytes in the mouse model of HD. A , Images of brain sections showing double staining for GFAP (red) and STAT3 (green) on mouse brain sections, 6 weeks after the infection of striatal neurons with lenti-Htt18Q or lenti-Htt82Q. Astrocytes in the Htt82Q striatum are hypertrophic and express higher levels of STAT3 in their nucleus relative to resting astrocytes in the Htt18Q striatum. B , C , The number of nSTAT3 + /GFAP + cells ( B ) and the percentage of cells displaying strong staining for STAT3 ( C ) are significantly higher in the Htt82Q striatum than in the Htt18Q striatum. n = 6. ** p

    Article Snippet: Sections were then blocked with 4.5% normal goat serum (Sigma) and incubated with primary antibodies directed against the following proteins: 4G8 (1:500, mouse; Signet Covance), EM48 (1:200, mouse; Millipore Bioscience Research Reagents), GFAP–Cy3 (1:1000, mouse; Invitrogen), IBA1 (1:1000, rabbit; Wako), S100β (1:500, mouse; Invitrogen), STAT3 (STAT3α, 1:200, rabbit; Cell Signaling Technology), and vimentin (1:1000, chicken; Abcam).

    Techniques: Double Staining, Infection, Staining

    The JAK/STAT3 pathway is activated in reactive astrocytes in the primate model of HD. A , Images of brain sections from macaques injected with lenti-Htt82Q in the putamen showing triple staining for STAT3 (green), GFAP (red), and EM48 (magenta). Seventeen months after infection with lenti-Htt82Q in the putamen, EM48 + aggregates of mHtt are observed, as well as prominent astrocyte reactivity. The immunoreactivity for STAT3 is much stronger in GFAP + reactive astrocytes than in resting astrocytes found outside the injected area in the same animal. Images are representative of all three macaques. Scale bars: 40 μm; enlargement, 10 μm.

    Journal: The Journal of Neuroscience

    Article Title: The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases

    doi: 10.1523/JNEUROSCI.3516-14.2015

    Figure Lengend Snippet: The JAK/STAT3 pathway is activated in reactive astrocytes in the primate model of HD. A , Images of brain sections from macaques injected with lenti-Htt82Q in the putamen showing triple staining for STAT3 (green), GFAP (red), and EM48 (magenta). Seventeen months after infection with lenti-Htt82Q in the putamen, EM48 + aggregates of mHtt are observed, as well as prominent astrocyte reactivity. The immunoreactivity for STAT3 is much stronger in GFAP + reactive astrocytes than in resting astrocytes found outside the injected area in the same animal. Images are representative of all three macaques. Scale bars: 40 μm; enlargement, 10 μm.

    Article Snippet: Sections were then blocked with 4.5% normal goat serum (Sigma) and incubated with primary antibodies directed against the following proteins: 4G8 (1:500, mouse; Signet Covance), EM48 (1:200, mouse; Millipore Bioscience Research Reagents), GFAP–Cy3 (1:1000, mouse; Invitrogen), IBA1 (1:1000, rabbit; Wako), S100β (1:500, mouse; Invitrogen), STAT3 (STAT3α, 1:200, rabbit; Cell Signaling Technology), and vimentin (1:1000, chicken; Abcam).

    Techniques: Injection, Staining, Infection

    The JAK/STAT3 pathway is activated in reactive astrocytes in 3xTg-AD mice. A , B , Images of brain sections from the subiculum of 12-month-old 3xTg-AD mice. STAT3 (green) accumulates in the nucleus of reactive astrocytes labeled with GFAP ( A , red) or vimentin ( B , red), especially around amyloid plaques (arrowheads). C , The number of nSTAT3 + /GFAP + cells is significantly higher in 3xTg-AD mice than in age-matched WT controls. n = 3–5 per group. *** p

    Journal: The Journal of Neuroscience

    Article Title: The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases

    doi: 10.1523/JNEUROSCI.3516-14.2015

    Figure Lengend Snippet: The JAK/STAT3 pathway is activated in reactive astrocytes in 3xTg-AD mice. A , B , Images of brain sections from the subiculum of 12-month-old 3xTg-AD mice. STAT3 (green) accumulates in the nucleus of reactive astrocytes labeled with GFAP ( A , red) or vimentin ( B , red), especially around amyloid plaques (arrowheads). C , The number of nSTAT3 + /GFAP + cells is significantly higher in 3xTg-AD mice than in age-matched WT controls. n = 3–5 per group. *** p

    Article Snippet: Sections were then blocked with 4.5% normal goat serum (Sigma) and incubated with primary antibodies directed against the following proteins: 4G8 (1:500, mouse; Signet Covance), EM48 (1:200, mouse; Millipore Bioscience Research Reagents), GFAP–Cy3 (1:1000, mouse; Invitrogen), IBA1 (1:1000, rabbit; Wako), S100β (1:500, mouse; Invitrogen), STAT3 (STAT3α, 1:200, rabbit; Cell Signaling Technology), and vimentin (1:1000, chicken; Abcam).

    Techniques: Mouse Assay, Labeling

    The JAK/STAT3 pathway is activated in reactive astrocytes in APP/PS1dE9 mice. A , B , Images of brain sections from 8-month-old APP/PS1dE9 mice showing double staining for STAT3 (green) and reactive astrocyte markers ( A , GFAP; B , vimentin). APP/PS1dE9 mice display reactive astrocytes that overexpress GFAP, vimentin, and STAT3 around amyloid plaques (arrowhead) in the hippocampus. STAT3 accumulates in the nucleus of reactive astrocytes (see enlargement). C , The number of GFAP + astrocytes coexpressing STAT3 in the nucleus (nSTAT3 + /GFAP + cells) is significantly higher in APP/PS1dE9 mice than in WT mice. D , The percentage of cells showing strong staining for STAT3 is higher in APP/PS1dE9 mice than in WT mice. n = 3–4 per group. * p

    Journal: The Journal of Neuroscience

    Article Title: The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases

    doi: 10.1523/JNEUROSCI.3516-14.2015

    Figure Lengend Snippet: The JAK/STAT3 pathway is activated in reactive astrocytes in APP/PS1dE9 mice. A , B , Images of brain sections from 8-month-old APP/PS1dE9 mice showing double staining for STAT3 (green) and reactive astrocyte markers ( A , GFAP; B , vimentin). APP/PS1dE9 mice display reactive astrocytes that overexpress GFAP, vimentin, and STAT3 around amyloid plaques (arrowhead) in the hippocampus. STAT3 accumulates in the nucleus of reactive astrocytes (see enlargement). C , The number of GFAP + astrocytes coexpressing STAT3 in the nucleus (nSTAT3 + /GFAP + cells) is significantly higher in APP/PS1dE9 mice than in WT mice. D , The percentage of cells showing strong staining for STAT3 is higher in APP/PS1dE9 mice than in WT mice. n = 3–4 per group. * p

    Article Snippet: Sections were then blocked with 4.5% normal goat serum (Sigma) and incubated with primary antibodies directed against the following proteins: 4G8 (1:500, mouse; Signet Covance), EM48 (1:200, mouse; Millipore Bioscience Research Reagents), GFAP–Cy3 (1:1000, mouse; Invitrogen), IBA1 (1:1000, rabbit; Wako), S100β (1:500, mouse; Invitrogen), STAT3 (STAT3α, 1:200, rabbit; Cell Signaling Technology), and vimentin (1:1000, chicken; Abcam).

    Techniques: Mouse Assay, Double Staining, Staining

    The JAK/STAT3 pathway is responsible for astrocyte reactivity in 3xTg-AD mice. A , Images of double staining for GFP (green) and STAT3 (red) in 7- to 8-month-old 3xTg-AD mice injected in the subiculum with lenti-GFP or lenti-SOCS3 plus lenti-GFP (same total virus load). STAT3 expression becomes undetectable in astrocytes infected with lenti-SOCS3. B , The number of GFP + astrocytes coexpressing STAT3 in the nucleus (GFP + /nSTAT3 + cells) is significantly lower in the SOCS3 group than in the GFP control group. C , Images of GFP (green) and GFAP (red) staining in brain sections from 3xTg-AD mice injected with lenti-GFP or lenti-SOCS3 plus lenti-GFP in the subiculum. Lenti-SOCS3 injection strongly reduces GFAP expression in the injected area (delimited by white dots) in 3xTg-AD mice. Note that infected astrocytes in the SOCS3 group have a bushy morphology typical of resting astrocytes, whereas cells in the GFP group are hypertrophic with enlarged primary processes. D , Quantification of the GFAP + area in 3xTg-AD mice injected with lenti-SOCS3 plus lenti-GFP or lenti-GFP alone. E , The number of GFP + astrocytes coexpressing GFAP is significantly lower in the SOCS3 group than in the GFP control group. F , Immunofluorescent labeling for the astrocyte marker S100β. G , Quantification of the number of infected astrocytes coexpressing S100β (GFP + /S100β + cells) shows that its expression is not altered by SOCS3. n = 3–5 per group. * p

    Journal: The Journal of Neuroscience

    Article Title: The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases

    doi: 10.1523/JNEUROSCI.3516-14.2015

    Figure Lengend Snippet: The JAK/STAT3 pathway is responsible for astrocyte reactivity in 3xTg-AD mice. A , Images of double staining for GFP (green) and STAT3 (red) in 7- to 8-month-old 3xTg-AD mice injected in the subiculum with lenti-GFP or lenti-SOCS3 plus lenti-GFP (same total virus load). STAT3 expression becomes undetectable in astrocytes infected with lenti-SOCS3. B , The number of GFP + astrocytes coexpressing STAT3 in the nucleus (GFP + /nSTAT3 + cells) is significantly lower in the SOCS3 group than in the GFP control group. C , Images of GFP (green) and GFAP (red) staining in brain sections from 3xTg-AD mice injected with lenti-GFP or lenti-SOCS3 plus lenti-GFP in the subiculum. Lenti-SOCS3 injection strongly reduces GFAP expression in the injected area (delimited by white dots) in 3xTg-AD mice. Note that infected astrocytes in the SOCS3 group have a bushy morphology typical of resting astrocytes, whereas cells in the GFP group are hypertrophic with enlarged primary processes. D , Quantification of the GFAP + area in 3xTg-AD mice injected with lenti-SOCS3 plus lenti-GFP or lenti-GFP alone. E , The number of GFP + astrocytes coexpressing GFAP is significantly lower in the SOCS3 group than in the GFP control group. F , Immunofluorescent labeling for the astrocyte marker S100β. G , Quantification of the number of infected astrocytes coexpressing S100β (GFP + /S100β + cells) shows that its expression is not altered by SOCS3. n = 3–5 per group. * p

    Article Snippet: Sections were then blocked with 4.5% normal goat serum (Sigma) and incubated with primary antibodies directed against the following proteins: 4G8 (1:500, mouse; Signet Covance), EM48 (1:200, mouse; Millipore Bioscience Research Reagents), GFAP–Cy3 (1:1000, mouse; Invitrogen), IBA1 (1:1000, rabbit; Wako), S100β (1:500, mouse; Invitrogen), STAT3 (STAT3α, 1:200, rabbit; Cell Signaling Technology), and vimentin (1:1000, chicken; Abcam).

    Techniques: Mouse Assay, Double Staining, Injection, Expressing, Infection, Staining, Labeling, Marker

    The JAK/STAT3 pathway is responsible for astrocyte reactivity in the lentiviral-based mouse model of HD. A , Immunofluorescent staining of GFP (green) and STAT3 (red) in mice injected in the left striatum with lenti-Htt82Q plus lenti-GFP and the right striatum with lenti-Htt82Q plus lenti-SOCS3 plus lenti-GFP. STAT3 expression becomes undetectable in astrocytes infected with lenti-SOCS3. B , C , The number of GFP + /nSTAT3 + astrocytes ( B ) and the percentage of cells showing strong staining for STAT3 ( C ) is significantly lower in the right striatum injected with lenti-SOCS3 than in the control striatum. D , SOCS3 expression strongly reduces GFAP expression (red) in the injected area (GFP + , green). Infected astrocytes in the SOCS3 group display thin processes and complex ramifications, unlike hypertrophic reactive astrocytes in the control group. E , Quantification confirms that the GFAP + area is significantly smaller in the striatum injected with lenti-SOCS3 than in the control striatum. F , The number of GFP + astrocytes coexpressing GFAP is significantly lower in the striatum expressing SOCS3 than in the control striatum. G , Immunofluorescent staining for the astrocyte marker S100β (red). H , The number of GFP + /S100β + cells is not different between the two groups. I–K , qRT-PCR analysis on mice injected in the striatum with lenti-Htt18Q plus lenti-GFP, lenti-Htt18Q plus lenti-SOCS3 plus lenti-GFP, lenti-Htt82Q plus lenti-GFP, or lenti-Htt82Q plus lenti-SOCS3 plus lenti-GFP (same total virus load). I , Socs3 mRNA is overexpressed > 100 times after lenti-SOCS3 injection. The expression of gfap ( J ) and vimentin ( K ) is induced by lenti-Htt82Q and is restored to levels observed in the Htt18Q controls by the expression of SOCS3. Note that lenti-SOCS3 has no effect in resting astrocytes. n = 4–11 per group. * p

    Journal: The Journal of Neuroscience

    Article Title: The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases

    doi: 10.1523/JNEUROSCI.3516-14.2015

    Figure Lengend Snippet: The JAK/STAT3 pathway is responsible for astrocyte reactivity in the lentiviral-based mouse model of HD. A , Immunofluorescent staining of GFP (green) and STAT3 (red) in mice injected in the left striatum with lenti-Htt82Q plus lenti-GFP and the right striatum with lenti-Htt82Q plus lenti-SOCS3 plus lenti-GFP. STAT3 expression becomes undetectable in astrocytes infected with lenti-SOCS3. B , C , The number of GFP + /nSTAT3 + astrocytes ( B ) and the percentage of cells showing strong staining for STAT3 ( C ) is significantly lower in the right striatum injected with lenti-SOCS3 than in the control striatum. D , SOCS3 expression strongly reduces GFAP expression (red) in the injected area (GFP + , green). Infected astrocytes in the SOCS3 group display thin processes and complex ramifications, unlike hypertrophic reactive astrocytes in the control group. E , Quantification confirms that the GFAP + area is significantly smaller in the striatum injected with lenti-SOCS3 than in the control striatum. F , The number of GFP + astrocytes coexpressing GFAP is significantly lower in the striatum expressing SOCS3 than in the control striatum. G , Immunofluorescent staining for the astrocyte marker S100β (red). H , The number of GFP + /S100β + cells is not different between the two groups. I–K , qRT-PCR analysis on mice injected in the striatum with lenti-Htt18Q plus lenti-GFP, lenti-Htt18Q plus lenti-SOCS3 plus lenti-GFP, lenti-Htt82Q plus lenti-GFP, or lenti-Htt82Q plus lenti-SOCS3 plus lenti-GFP (same total virus load). I , Socs3 mRNA is overexpressed > 100 times after lenti-SOCS3 injection. The expression of gfap ( J ) and vimentin ( K ) is induced by lenti-Htt82Q and is restored to levels observed in the Htt18Q controls by the expression of SOCS3. Note that lenti-SOCS3 has no effect in resting astrocytes. n = 4–11 per group. * p

    Article Snippet: Sections were then blocked with 4.5% normal goat serum (Sigma) and incubated with primary antibodies directed against the following proteins: 4G8 (1:500, mouse; Signet Covance), EM48 (1:200, mouse; Millipore Bioscience Research Reagents), GFAP–Cy3 (1:1000, mouse; Invitrogen), IBA1 (1:1000, rabbit; Wako), S100β (1:500, mouse; Invitrogen), STAT3 (STAT3α, 1:200, rabbit; Cell Signaling Technology), and vimentin (1:1000, chicken; Abcam).

    Techniques: Staining, Mouse Assay, Injection, Expressing, Infection, Marker, Quantitative RT-PCR

    Modulation of STAT3α cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.

    Journal: BMC Cancer

    Article Title: Caspase-dependent proteolytic cleavage of STAT3? in ES cells, in mammary glands undergoing forced involution and in breast cancer cell lines

    doi: 10.1186/1471-2407-7-29

    Figure Lengend Snippet: Modulation of STAT3α cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.

    Article Snippet: STAT3α is cleaved into two fragments and cleavage is inhibited by the nitric oxide donor sodium nitroprusside Immunoblots of HM-1 murine ES cell cytoplasmic extracts with a polyclonal antibody (Santa Cruz, sc-482) which recognises an epitope at the carboxy terminus of the mouse STAT3α protein, revealed both the expected 92 kDa STAT3α species and another immunoreactive species of approximately 43 kDa (Fig. ).

    Techniques: Activity Assay, Irradiation, Inhibition, Cell Culture, Cell Harvesting, Western Blot, Knock-Out, Transgenic Assay

    Proteolytic cleavage of STAT3α in HM-1 murine ES cells to 48 kDa and 43 kDa fragments can be inhibited by the NO-donor SNP. A. An antibody (Santa Cruz sc-482) directed against amino acids 750–769 at the carboxy terminus of STAT3α revealed full-length STAT3α and a 43 kDa carboxy terminal cleavage product. 90 μg of protein were loaded per lane. B. Blot stripped and re-probed with an antibody (Cell Signaling Technology #9132) that recognises an epitope around the STAT3 Y705 residue within the carboxy terminal region, again detecting the 43 kDa cleavage fragment. C. Blot stripped and re-probed with an antibody that recognises an epitope within the STAT3 amino terminal amino acid 1–175 region (BD/Transduction Laboratories S21320/610190), detecting full-length STAT3α and a STAT3-derived 48 kDa amino terminal cleavage fragment. Cell culture treatment with the NO-donor (and caspase inhibitor) SNP for 2 hours reduced the level of the 43 kDa and 48 kDa fragments in a dose-dependent manner (lanes 1–3, 6–8 in panels A - C ) in HM-1 ES cells cultured in either the presence (+LIF, lanes 1–5) or absence (-LIF, lanes 6y10) of added LIF in the medium. In contrast, the proteasome inhibitor MG132 added to 10 μM in the medium for 2 hours (with DMSO vehicle to a final concentration of 0.2% vol/vol) could not inhibit cleavage (lanes 4, 9 in A - C ) relative to untreated cells, or DMSO vehicle alone (D). D . Blot stripped and re-probed with an anti β-Actin monoclonal antibody (Sigma, AC-74) to confirm equal amounts of protein loaded per lane.

    Journal: BMC Cancer

    Article Title: Caspase-dependent proteolytic cleavage of STAT3? in ES cells, in mammary glands undergoing forced involution and in breast cancer cell lines

    doi: 10.1186/1471-2407-7-29

    Figure Lengend Snippet: Proteolytic cleavage of STAT3α in HM-1 murine ES cells to 48 kDa and 43 kDa fragments can be inhibited by the NO-donor SNP. A. An antibody (Santa Cruz sc-482) directed against amino acids 750–769 at the carboxy terminus of STAT3α revealed full-length STAT3α and a 43 kDa carboxy terminal cleavage product. 90 μg of protein were loaded per lane. B. Blot stripped and re-probed with an antibody (Cell Signaling Technology #9132) that recognises an epitope around the STAT3 Y705 residue within the carboxy terminal region, again detecting the 43 kDa cleavage fragment. C. Blot stripped and re-probed with an antibody that recognises an epitope within the STAT3 amino terminal amino acid 1–175 region (BD/Transduction Laboratories S21320/610190), detecting full-length STAT3α and a STAT3-derived 48 kDa amino terminal cleavage fragment. Cell culture treatment with the NO-donor (and caspase inhibitor) SNP for 2 hours reduced the level of the 43 kDa and 48 kDa fragments in a dose-dependent manner (lanes 1–3, 6–8 in panels A - C ) in HM-1 ES cells cultured in either the presence (+LIF, lanes 1–5) or absence (-LIF, lanes 6y10) of added LIF in the medium. In contrast, the proteasome inhibitor MG132 added to 10 μM in the medium for 2 hours (with DMSO vehicle to a final concentration of 0.2% vol/vol) could not inhibit cleavage (lanes 4, 9 in A - C ) relative to untreated cells, or DMSO vehicle alone (D). D . Blot stripped and re-probed with an anti β-Actin monoclonal antibody (Sigma, AC-74) to confirm equal amounts of protein loaded per lane.

    Article Snippet: STAT3α is cleaved into two fragments and cleavage is inhibited by the nitric oxide donor sodium nitroprusside Immunoblots of HM-1 murine ES cell cytoplasmic extracts with a polyclonal antibody (Santa Cruz, sc-482) which recognises an epitope at the carboxy terminus of the mouse STAT3α protein, revealed both the expected 92 kDa STAT3α species and another immunoreactive species of approximately 43 kDa (Fig. ).

    Techniques: Transduction, Derivative Assay, Cell Culture, Concentration Assay

    Cleavage of STAT3α in human breast cancer cell lines. Whole cell extracts from one normal human breast cell line (HB4A) and 7 breast cancer cell lines with varying levels of activated, tyrosine phosphorylated, STAT3α were immunoblotted with either the anti-STAT3 amino terminal antibody (upper panel, 48 kDa cleaved fragment) or the anti-STAT3 PY705 antibody (lower panel, 43 kDa cleaved fragment). Over exposure of the upper blot revealed an additional cleavage product of approximately 25 kDa that was most apparent in the GI-101 cell line which has the highest level of tyrosine phosphorylated STAT3α in the breast cancer cell lines examined. Extracts from C57Bl/6 mouse mammary glands were loaded as controls: 5d Gest = day 5 pregnancy; 10d Lac = 10 days of lactation; 96 Hr In = 96 hours following forced weaning at day 10 lactation. Arrows indicate cleaved fragments.

    Journal: BMC Cancer

    Article Title: Caspase-dependent proteolytic cleavage of STAT3? in ES cells, in mammary glands undergoing forced involution and in breast cancer cell lines

    doi: 10.1186/1471-2407-7-29

    Figure Lengend Snippet: Cleavage of STAT3α in human breast cancer cell lines. Whole cell extracts from one normal human breast cell line (HB4A) and 7 breast cancer cell lines with varying levels of activated, tyrosine phosphorylated, STAT3α were immunoblotted with either the anti-STAT3 amino terminal antibody (upper panel, 48 kDa cleaved fragment) or the anti-STAT3 PY705 antibody (lower panel, 43 kDa cleaved fragment). Over exposure of the upper blot revealed an additional cleavage product of approximately 25 kDa that was most apparent in the GI-101 cell line which has the highest level of tyrosine phosphorylated STAT3α in the breast cancer cell lines examined. Extracts from C57Bl/6 mouse mammary glands were loaded as controls: 5d Gest = day 5 pregnancy; 10d Lac = 10 days of lactation; 96 Hr In = 96 hours following forced weaning at day 10 lactation. Arrows indicate cleaved fragments.

    Article Snippet: STAT3α is cleaved into two fragments and cleavage is inhibited by the nitric oxide donor sodium nitroprusside Immunoblots of HM-1 murine ES cell cytoplasmic extracts with a polyclonal antibody (Santa Cruz, sc-482) which recognises an epitope at the carboxy terminus of the mouse STAT3α protein, revealed both the expected 92 kDa STAT3α species and another immunoreactive species of approximately 43 kDa (Fig. ).

    Techniques:

    STAT3α is expressed in the sensory epithelium in the organ of Corti. Immunohistochemical staining of vibratome-cut whole mount cochlea sections for STAT3α (green) showed fluorescent signal in the cytoplasm of inner hair cells (IHC), outer hair cells (OHC) and Deiters cells (DC). Open arrow heads point to OHCs, closed arrow heads point to IHCs, and the arrows point to DCs. Nuclei are stained with Hoescht 33258 (blue). The images are from the upper basal turn of the cochlea and are representative of 3 individual mice.

    Journal: PLoS ONE

    Article Title: JAK2/STAT3 Inhibition Attenuates Noise-Induced Hearing Loss

    doi: 10.1371/journal.pone.0108276

    Figure Lengend Snippet: STAT3α is expressed in the sensory epithelium in the organ of Corti. Immunohistochemical staining of vibratome-cut whole mount cochlea sections for STAT3α (green) showed fluorescent signal in the cytoplasm of inner hair cells (IHC), outer hair cells (OHC) and Deiters cells (DC). Open arrow heads point to OHCs, closed arrow heads point to IHCs, and the arrows point to DCs. Nuclei are stained with Hoescht 33258 (blue). The images are from the upper basal turn of the cochlea and are representative of 3 individual mice.

    Article Snippet: The tissue sections were immunolabeled with rabbit anti-STAT3α (1∶100, #8768, Cell Signaling Technology, Danvers, MA) or anti-pSTAT3 (Y705) antibody (#9145) at 1∶100 dilution overnight at 4°C followed by incubation with donkey anti-rabbit secondary antibody (Alexafluor 488, Life Technologies, Grand Island, NY).

    Techniques: Immunohistochemistry, Staining, Mouse Assay

    STAT3β is activated in the mammary epithelial cells during involution. ( A ) Western blot analysis of total STAT3α/β in the CDβ cell line and the CD-derived clones. ( B–D ) Western blot analysis of total STAT3α/β

    Journal: Journal of Cell Science

    Article Title: A novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance

    doi: 10.1242/jcs.119214

    Figure Lengend Snippet: STAT3β is activated in the mammary epithelial cells during involution. ( A ) Western blot analysis of total STAT3α/β in the CDβ cell line and the CD-derived clones. ( B–D ) Western blot analysis of total STAT3α/β

    Article Snippet: 293T cells were transiently transfected with cDNA encoding mouse STAT3β (OriGene Technologies, cat. no. MC221089), mouse STAT3α (OriGene Technologies, cat. no. MC221487) and control (OriGene Technologies, cat. no. PS100001), according to the manufacturer's protocol.

    Techniques: Western Blot, Derivative Assay, Clone Assay

    Effect of knockdown on miR146b known targets as well as on STAT3α/β. ( A ) Western analysis of Smad4, IRAK1, STAT5a, NFκB, TRAF6, ELF5 and β-actin in the alveolar progenitor cell line 48 hours post-transfection in

    Journal: Journal of Cell Science

    Article Title: A novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance

    doi: 10.1242/jcs.119214

    Figure Lengend Snippet: Effect of knockdown on miR146b known targets as well as on STAT3α/β. ( A ) Western analysis of Smad4, IRAK1, STAT5a, NFκB, TRAF6, ELF5 and β-actin in the alveolar progenitor cell line 48 hours post-transfection in

    Article Snippet: 293T cells were transiently transfected with cDNA encoding mouse STAT3β (OriGene Technologies, cat. no. MC221089), mouse STAT3α (OriGene Technologies, cat. no. MC221487) and control (OriGene Technologies, cat. no. PS100001), according to the manufacturer's protocol.

    Techniques: Western Blot, Transfection

    MiR-146b targets the 3′UTR of STAT3α/β. ( A ) A diagram of the 3′UTRs of STAT3α and β computationally predicted binding sites for miR-146b. ( B ) Vector backbone of STAT3α/β 3′UTR reporter

    Journal: Journal of Cell Science

    Article Title: A novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance

    doi: 10.1242/jcs.119214

    Figure Lengend Snippet: MiR-146b targets the 3′UTR of STAT3α/β. ( A ) A diagram of the 3′UTRs of STAT3α and β computationally predicted binding sites for miR-146b. ( B ) Vector backbone of STAT3α/β 3′UTR reporter

    Article Snippet: 293T cells were transiently transfected with cDNA encoding mouse STAT3β (OriGene Technologies, cat. no. MC221089), mouse STAT3α (OriGene Technologies, cat. no. MC221487) and control (OriGene Technologies, cat. no. PS100001), according to the manufacturer's protocol.

    Techniques: Binding Assay, Plasmid Preparation

    Modulation of STAT3α cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.

    Journal: BMC Cancer

    Article Title: Caspase-dependent proteolytic cleavage of STAT3? in ES cells, in mammary glands undergoing forced involution and in breast cancer cell lines

    doi: 10.1186/1471-2407-7-29

    Figure Lengend Snippet: Modulation of STAT3α cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.

    Article Snippet: UV irradiation of HM-1 ES cells (254 nm, 1 mJ/cm2 ) and harvesting 13 hours later revealed the cleavage of approximately 50% of the STAT3α to yield the same size product in whole cell extracts as detected previously with the anti-STAT3α carboxy terminal antibody (Santa Cruz, sc-482) (Fig. , lanes 3, 4).

    Techniques: Activity Assay, Irradiation, Inhibition, Cell Culture, Cell Harvesting, Western Blot, Knock-Out, Transgenic Assay

    Proteolytic cleavage of STAT3α in HM-1 murine ES cells to 48 kDa and 43 kDa fragments can be inhibited by the NO-donor SNP. A. An antibody (Santa Cruz sc-482) directed against amino acids 750–769 at the carboxy terminus of STAT3α revealed full-length STAT3α and a 43 kDa carboxy terminal cleavage product. 90 μg of protein were loaded per lane. B. Blot stripped and re-probed with an antibody (Cell Signaling Technology #9132) that recognises an epitope around the STAT3 Y705 residue within the carboxy terminal region, again detecting the 43 kDa cleavage fragment. C. Blot stripped and re-probed with an antibody that recognises an epitope within the STAT3 amino terminal amino acid 1–175 region (BD/Transduction Laboratories S21320/610190), detecting full-length STAT3α and a STAT3-derived 48 kDa amino terminal cleavage fragment. Cell culture treatment with the NO-donor (and caspase inhibitor) SNP for 2 hours reduced the level of the 43 kDa and 48 kDa fragments in a dose-dependent manner (lanes 1–3, 6–8 in panels A - C ) in HM-1 ES cells cultured in either the presence (+LIF, lanes 1–5) or absence (-LIF, lanes 6y10) of added LIF in the medium. In contrast, the proteasome inhibitor MG132 added to 10 μM in the medium for 2 hours (with DMSO vehicle to a final concentration of 0.2% vol/vol) could not inhibit cleavage (lanes 4, 9 in A - C ) relative to untreated cells, or DMSO vehicle alone (D). D . Blot stripped and re-probed with an anti β-Actin monoclonal antibody (Sigma, AC-74) to confirm equal amounts of protein loaded per lane.

    Journal: BMC Cancer

    Article Title: Caspase-dependent proteolytic cleavage of STAT3? in ES cells, in mammary glands undergoing forced involution and in breast cancer cell lines

    doi: 10.1186/1471-2407-7-29

    Figure Lengend Snippet: Proteolytic cleavage of STAT3α in HM-1 murine ES cells to 48 kDa and 43 kDa fragments can be inhibited by the NO-donor SNP. A. An antibody (Santa Cruz sc-482) directed against amino acids 750–769 at the carboxy terminus of STAT3α revealed full-length STAT3α and a 43 kDa carboxy terminal cleavage product. 90 μg of protein were loaded per lane. B. Blot stripped and re-probed with an antibody (Cell Signaling Technology #9132) that recognises an epitope around the STAT3 Y705 residue within the carboxy terminal region, again detecting the 43 kDa cleavage fragment. C. Blot stripped and re-probed with an antibody that recognises an epitope within the STAT3 amino terminal amino acid 1–175 region (BD/Transduction Laboratories S21320/610190), detecting full-length STAT3α and a STAT3-derived 48 kDa amino terminal cleavage fragment. Cell culture treatment with the NO-donor (and caspase inhibitor) SNP for 2 hours reduced the level of the 43 kDa and 48 kDa fragments in a dose-dependent manner (lanes 1–3, 6–8 in panels A - C ) in HM-1 ES cells cultured in either the presence (+LIF, lanes 1–5) or absence (-LIF, lanes 6y10) of added LIF in the medium. In contrast, the proteasome inhibitor MG132 added to 10 μM in the medium for 2 hours (with DMSO vehicle to a final concentration of 0.2% vol/vol) could not inhibit cleavage (lanes 4, 9 in A - C ) relative to untreated cells, or DMSO vehicle alone (D). D . Blot stripped and re-probed with an anti β-Actin monoclonal antibody (Sigma, AC-74) to confirm equal amounts of protein loaded per lane.

    Article Snippet: UV irradiation of HM-1 ES cells (254 nm, 1 mJ/cm2 ) and harvesting 13 hours later revealed the cleavage of approximately 50% of the STAT3α to yield the same size product in whole cell extracts as detected previously with the anti-STAT3α carboxy terminal antibody (Santa Cruz, sc-482) (Fig. , lanes 3, 4).

    Techniques: Transduction, Derivative Assay, Cell Culture, Concentration Assay

    Cleavage of STAT3α in human breast cancer cell lines. Whole cell extracts from one normal human breast cell line (HB4A) and 7 breast cancer cell lines with varying levels of activated, tyrosine phosphorylated, STAT3α were immunoblotted with either the anti-STAT3 amino terminal antibody (upper panel, 48 kDa cleaved fragment) or the anti-STAT3 PY705 antibody (lower panel, 43 kDa cleaved fragment). Over exposure of the upper blot revealed an additional cleavage product of approximately 25 kDa that was most apparent in the GI-101 cell line which has the highest level of tyrosine phosphorylated STAT3α in the breast cancer cell lines examined. Extracts from C57Bl/6 mouse mammary glands were loaded as controls: 5d Gest = day 5 pregnancy; 10d Lac = 10 days of lactation; 96 Hr In = 96 hours following forced weaning at day 10 lactation. Arrows indicate cleaved fragments.

    Journal: BMC Cancer

    Article Title: Caspase-dependent proteolytic cleavage of STAT3? in ES cells, in mammary glands undergoing forced involution and in breast cancer cell lines

    doi: 10.1186/1471-2407-7-29

    Figure Lengend Snippet: Cleavage of STAT3α in human breast cancer cell lines. Whole cell extracts from one normal human breast cell line (HB4A) and 7 breast cancer cell lines with varying levels of activated, tyrosine phosphorylated, STAT3α were immunoblotted with either the anti-STAT3 amino terminal antibody (upper panel, 48 kDa cleaved fragment) or the anti-STAT3 PY705 antibody (lower panel, 43 kDa cleaved fragment). Over exposure of the upper blot revealed an additional cleavage product of approximately 25 kDa that was most apparent in the GI-101 cell line which has the highest level of tyrosine phosphorylated STAT3α in the breast cancer cell lines examined. Extracts from C57Bl/6 mouse mammary glands were loaded as controls: 5d Gest = day 5 pregnancy; 10d Lac = 10 days of lactation; 96 Hr In = 96 hours following forced weaning at day 10 lactation. Arrows indicate cleaved fragments.

    Article Snippet: UV irradiation of HM-1 ES cells (254 nm, 1 mJ/cm2 ) and harvesting 13 hours later revealed the cleavage of approximately 50% of the STAT3α to yield the same size product in whole cell extracts as detected previously with the anti-STAT3α carboxy terminal antibody (Santa Cruz, sc-482) (Fig. , lanes 3, 4).

    Techniques:

    Rescue experiments using single STAT3 variants. ( a ) Workflow of knockdown/re-expression approach. Different stable cell lines were induced for expression of their individual STAT3 variants or EV control for 3 days before transduction with STAT3 shRNAs with the marker GFP. The shRNA-expressing GFP+ cells were monitored by flow cytometry over 12 days of observation. ( b ) Expression and activation of the single STAT3 variants in ABC DLBCL cells after knockdown of endogenous STAT3 by shRNA#16. OCI-Ly10 cells were manipulated as described in a , and shSTAT3 cells were selected with puromycin. After 5 days of selection, shSTAT3 was induced for expression for 4 days before immunoblotting with STAT3 or phospho-tyrosine 705 (pY705) antibody. Note that this monoclonal antibody did not recognize phospho-tyrosine 704 of STAT3ΔSα and ΔSβ. Expression of pY705 STAT3α or β relative to the uninduced control was quantified by densitometry. ( c ) Insufficient rescue by the individual variants. ABC DLBCL cell lines HBL1 and OCI-Ly10 and control GCB cell line OCI-Ly19 were used following the experimental procedure as described in a . The percentage of GFP+ cells expressing STAT3 shRNAs was normalized to that of the control shRNA at each time point. STAT3Sα-C expressing cells served as positive controls. The experiments were repeated twice with two different STAT3 shRNAs. ( d ) The rescue efficiencies of different STAT3 variant stable cell lines on day 12 were calculated by a formula: (% variant−% EV)/(% STAT3Sα-C−% EV). Error bars represent mean±s.d. ( n =4, * P

    Journal: Oncogenesis

    Article Title: A mix of S and ΔS variants of STAT3 enable survival of activated B-cell-like diffuse large B-cell lymphoma cells in culture

    doi: 10.1038/oncsis.2015.44

    Figure Lengend Snippet: Rescue experiments using single STAT3 variants. ( a ) Workflow of knockdown/re-expression approach. Different stable cell lines were induced for expression of their individual STAT3 variants or EV control for 3 days before transduction with STAT3 shRNAs with the marker GFP. The shRNA-expressing GFP+ cells were monitored by flow cytometry over 12 days of observation. ( b ) Expression and activation of the single STAT3 variants in ABC DLBCL cells after knockdown of endogenous STAT3 by shRNA#16. OCI-Ly10 cells were manipulated as described in a , and shSTAT3 cells were selected with puromycin. After 5 days of selection, shSTAT3 was induced for expression for 4 days before immunoblotting with STAT3 or phospho-tyrosine 705 (pY705) antibody. Note that this monoclonal antibody did not recognize phospho-tyrosine 704 of STAT3ΔSα and ΔSβ. Expression of pY705 STAT3α or β relative to the uninduced control was quantified by densitometry. ( c ) Insufficient rescue by the individual variants. ABC DLBCL cell lines HBL1 and OCI-Ly10 and control GCB cell line OCI-Ly19 were used following the experimental procedure as described in a . The percentage of GFP+ cells expressing STAT3 shRNAs was normalized to that of the control shRNA at each time point. STAT3Sα-C expressing cells served as positive controls. The experiments were repeated twice with two different STAT3 shRNAs. ( d ) The rescue efficiencies of different STAT3 variant stable cell lines on day 12 were calculated by a formula: (% variant−% EV)/(% STAT3Sα-C−% EV). Error bars represent mean±s.d. ( n =4, * P

    Article Snippet: The primary antibodies included anti-STAT3pan (124H6, from Cell Signaling Technology, Danvers, MA, USA), anti-STAT3α (9D8, ab119352, from Abcam, Cambridge, MA, USA), anti-STAT3β, anti-phospho-STAT3 (Tyr-705) (D3A7) (#9145, from Cell Signaling Technology), anti-NFKBIA (L35A5, from Cell Signaling Technology), anti-NFKBIZ (TA502768S, from OriGene Technologies, Rockville, MD, USA), anti-α-tubulin (sc-8035, from Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-actin (#4967, from Cell Signaling Technology) and anti-histone-H3 (Abcam, #ab1791).

    Techniques: Expressing, Stable Transfection, Transduction, Marker, shRNA, Flow Cytometry, Cytometry, Activation Assay, Selection, Variant Assay

    Rescue experiments using combinations of STAT3 variants. ( a ) Verification of expression of combinatorial STAT3 variants in OCI-Ly10 cells after 3 days of induction by immunoblotting with specific antibodies to STAT3α and β (left panel) or qPCR (right panel) (see Materials and Methods for details). It should be stressed that neither method distinguishes between exogenously and endogenously expressed STAT3 variants. ( b , c ) Verification of expression of combinatorial STAT3 variants in OCI-Ly10 cells after 3 days of induction by immunoblotting ( b ) or qPCR ( c ) when endogenous STAT3 variants were knocked down by STAT3 shRNA. ( d ) Partial rescue by re-expression of Sα and ΔSα or Sβ and ΔSβ or together all four variants. ABC DLBCL cell lines HBL1 and OCI-Ly10 were used following the experimental procedure as described in a . The percentage of GFP+ cells expressing STAT3 shRNAs was normalized to that of the control shRNA at each time point. STAT3Sα-C expressing cells served as positive controls. The experiments were repeated twice with two different STAT3 shRNAs. ( e ) The rescue efficiencies of different combinatorial STAT3 variant stable cell lines on day 12 were calculated by a formula: (% variant−% EV)/(% STAT3Sα-C−% EV). Error bars represent mean±s.d. ( n =4, * P

    Journal: Oncogenesis

    Article Title: A mix of S and ΔS variants of STAT3 enable survival of activated B-cell-like diffuse large B-cell lymphoma cells in culture

    doi: 10.1038/oncsis.2015.44

    Figure Lengend Snippet: Rescue experiments using combinations of STAT3 variants. ( a ) Verification of expression of combinatorial STAT3 variants in OCI-Ly10 cells after 3 days of induction by immunoblotting with specific antibodies to STAT3α and β (left panel) or qPCR (right panel) (see Materials and Methods for details). It should be stressed that neither method distinguishes between exogenously and endogenously expressed STAT3 variants. ( b , c ) Verification of expression of combinatorial STAT3 variants in OCI-Ly10 cells after 3 days of induction by immunoblotting ( b ) or qPCR ( c ) when endogenous STAT3 variants were knocked down by STAT3 shRNA. ( d ) Partial rescue by re-expression of Sα and ΔSα or Sβ and ΔSβ or together all four variants. ABC DLBCL cell lines HBL1 and OCI-Ly10 were used following the experimental procedure as described in a . The percentage of GFP+ cells expressing STAT3 shRNAs was normalized to that of the control shRNA at each time point. STAT3Sα-C expressing cells served as positive controls. The experiments were repeated twice with two different STAT3 shRNAs. ( e ) The rescue efficiencies of different combinatorial STAT3 variant stable cell lines on day 12 were calculated by a formula: (% variant−% EV)/(% STAT3Sα-C−% EV). Error bars represent mean±s.d. ( n =4, * P

    Article Snippet: The primary antibodies included anti-STAT3pan (124H6, from Cell Signaling Technology, Danvers, MA, USA), anti-STAT3α (9D8, ab119352, from Abcam, Cambridge, MA, USA), anti-STAT3β, anti-phospho-STAT3 (Tyr-705) (D3A7) (#9145, from Cell Signaling Technology), anti-NFKBIA (L35A5, from Cell Signaling Technology), anti-NFKBIZ (TA502768S, from OriGene Technologies, Rockville, MD, USA), anti-α-tubulin (sc-8035, from Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-actin (#4967, from Cell Signaling Technology) and anti-histone-H3 (Abcam, #ab1791).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, shRNA, Variant Assay, Stable Transfection

    Modulation of STAT3α cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.

    Journal: BMC Cancer

    Article Title: Caspase-dependent proteolytic cleavage of STAT3? in ES cells, in mammary glands undergoing forced involution and in breast cancer cell lines

    doi: 10.1186/1471-2407-7-29

    Figure Lengend Snippet: Modulation of STAT3α cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm 2 , with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C . Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout ( JAK2 +/- ) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E . Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.

    Article Snippet: Immunoblotting analysis of whole cell extracts showed that z-VAD-FMK could inhibit STAT3α cleavage in a dose-dependent manner, with the generation of the 43 kDa and 48 kDa carboxyl- and amino-terminal fragments essentially abolished in the presence of 60 μM z-VAD-FMK (Fig. , lanes 1, 2, 6–8, C-terminal STAT3α and N-terminal STAT3 panels).

    Techniques: Activity Assay, Irradiation, Inhibition, Cell Culture, Cell Harvesting, Western Blot, Knock-Out, Transgenic Assay

    The comparative effect of curcumin and tetrahydrocurcumin on STAT3 dimerization. ( A ) Total lysate from H- Ras MCF10A cells treated with curcumin (25 μM) for 12 h was immunoprecipitated with Protein A/G agarose and anti-STAT3 antibody overnight and analyzed by immunobloting with anti-STAT3 antibody. * P

    Journal: Scientific Reports

    Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells

    doi: 10.1038/s41598-018-23840-2

    Figure Lengend Snippet: The comparative effect of curcumin and tetrahydrocurcumin on STAT3 dimerization. ( A ) Total lysate from H- Ras MCF10A cells treated with curcumin (25 μM) for 12 h was immunoprecipitated with Protein A/G agarose and anti-STAT3 antibody overnight and analyzed by immunobloting with anti-STAT3 antibody. * P

    Article Snippet: The recombinant STAT3 protein (Active Motif) was treated with curcumin, and the solution mixture was added to the urea buffer (1 M urea, 50 mM Tris-HCl, pH 7.8).

    Techniques: Immunoprecipitation, Western Blot

    Curcumin-induced apoptosis of H- Ras MCF10A cells through inhibition of STAT3 signaling. ( A ) H- Ras MCF10A cells were treated with indicated concentrations of curcumin for 24 h. The expression of phosphorylated STAT3 was assessed by Western blot analysis. Actin was used as an equal loading control. * P

    Journal: Scientific Reports

    Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells

    doi: 10.1038/s41598-018-23840-2

    Figure Lengend Snippet: Curcumin-induced apoptosis of H- Ras MCF10A cells through inhibition of STAT3 signaling. ( A ) H- Ras MCF10A cells were treated with indicated concentrations of curcumin for 24 h. The expression of phosphorylated STAT3 was assessed by Western blot analysis. Actin was used as an equal loading control. * P

    Article Snippet: The recombinant STAT3 protein (Active Motif) was treated with curcumin, and the solution mixture was added to the urea buffer (1 M urea, 50 mM Tris-HCl, pH 7.8).

    Techniques: Inhibition, Expressing, Western Blot

    A proposed mechanism underlying the inactivation of STAT3 by curcumin. Covalent modification of STAT3 at its cysteine 259 residue by curcumin interrupts phosphorylation, dimerization and nuclear translocation of STAT3 and consequently induces apoptotic death in H- Ras MCF10A cells.

    Journal: Scientific Reports

    Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells

    doi: 10.1038/s41598-018-23840-2

    Figure Lengend Snippet: A proposed mechanism underlying the inactivation of STAT3 by curcumin. Covalent modification of STAT3 at its cysteine 259 residue by curcumin interrupts phosphorylation, dimerization and nuclear translocation of STAT3 and consequently induces apoptotic death in H- Ras MCF10A cells.

    Article Snippet: The recombinant STAT3 protein (Active Motif) was treated with curcumin, and the solution mixture was added to the urea buffer (1 M urea, 50 mM Tris-HCl, pH 7.8).

    Techniques: Modification, Translocation Assay

    Identification of curcumin binding sites in recombinant STAT3 by LC-MS/MS. Annotated mass spectrum illustrating the binding of curcumin to STAT3 at cysteine 251 and 259 residues was observed. MS/MS spectrum of precursor ion at m/z 1033.49304 [M + 2 H] +2 corresponds to the amino acid sequence of STAT3. The elucidation of fragment ion at m/z 177.05501 from the structure was given in the inset of the spectra. The sample preparation and other experimental details for mass spectral analysis are described in Methods.

    Journal: Scientific Reports

    Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells

    doi: 10.1038/s41598-018-23840-2

    Figure Lengend Snippet: Identification of curcumin binding sites in recombinant STAT3 by LC-MS/MS. Annotated mass spectrum illustrating the binding of curcumin to STAT3 at cysteine 251 and 259 residues was observed. MS/MS spectrum of precursor ion at m/z 1033.49304 [M + 2 H] +2 corresponds to the amino acid sequence of STAT3. The elucidation of fragment ion at m/z 177.05501 from the structure was given in the inset of the spectra. The sample preparation and other experimental details for mass spectral analysis are described in Methods.

    Article Snippet: The recombinant STAT3 protein (Active Motif) was treated with curcumin, and the solution mixture was added to the urea buffer (1 M urea, 50 mM Tris-HCl, pH 7.8).

    Techniques: Binding Assay, Recombinant, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Sample Prep

    Direct interaction of curcumin with endogenous STAT3 and the cysteine 259 residue of STAT3 as a putative binding site. ( A ) Curcumin-conjugated Sepharose-4B and tetrahydrocurcumin-conjugated Sepharose-4B were incubated with H- Ras MCF10A cell lysates overnight. The STAT3 bound to the curcumin- and tetrahydrocurcumin-conjugated Sepharose 4B beads was pulled down by centrifugation and detected by Western blot analysis. ( B ) PC-3 cells transiently transfected with GFP-tagged WT STAT3, GFP-tagged C251A or GFP-tagged C259A were stimulated with IL-60 (50 ng/ml) for 12 h. The cell lysates were incubated with curcumin-conjugated Sepharose-4B. WT STAT3 and STAT3 mutants were detected by anti-STAT3 antibody. * P

    Journal: Scientific Reports

    Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells

    doi: 10.1038/s41598-018-23840-2

    Figure Lengend Snippet: Direct interaction of curcumin with endogenous STAT3 and the cysteine 259 residue of STAT3 as a putative binding site. ( A ) Curcumin-conjugated Sepharose-4B and tetrahydrocurcumin-conjugated Sepharose-4B were incubated with H- Ras MCF10A cell lysates overnight. The STAT3 bound to the curcumin- and tetrahydrocurcumin-conjugated Sepharose 4B beads was pulled down by centrifugation and detected by Western blot analysis. ( B ) PC-3 cells transiently transfected with GFP-tagged WT STAT3, GFP-tagged C251A or GFP-tagged C259A were stimulated with IL-60 (50 ng/ml) for 12 h. The cell lysates were incubated with curcumin-conjugated Sepharose-4B. WT STAT3 and STAT3 mutants were detected by anti-STAT3 antibody. * P

    Article Snippet: The recombinant STAT3 protein (Active Motif) was treated with curcumin, and the solution mixture was added to the urea buffer (1 M urea, 50 mM Tris-HCl, pH 7.8).

    Techniques: Binding Assay, Incubation, Centrifugation, Western Blot, Transfection

    Involvement of the α,β unsaturated carbonyl group of curcumin in its inhibition of STAT3 activation and induction of apoptosis. ( A ) Curcumin has the two α, β unsaturated carbonyl groups which are eliminated in tetrahydrocurcumin by hydrogenating the double bonds. ( B ) H- Ras MCF10A cells were incubated with vehicle, curcumin (25 μM) or tetrahydrocurcumin (25 μM). The expression of phosphorylated STAT3, STAT3 and related kinase was determind by Western blot analysis. ( C ) H- Ras MCF10A cells were treated with curcumin or tetrahydrocurcumin (25 μM each) for 12 h and then incubated with Annexin V and PI for 15 min. The population of apoptotic cells was measured by FACS analysis. ( D ) H- Ras MCF10A cells were cultured in soft agar for 10 days. Curcumin or tetrahydrocurcumin was added to the medium every other day for another 10 days. The colony formation was visualized under the microscope. ** P

    Journal: Scientific Reports

    Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells

    doi: 10.1038/s41598-018-23840-2

    Figure Lengend Snippet: Involvement of the α,β unsaturated carbonyl group of curcumin in its inhibition of STAT3 activation and induction of apoptosis. ( A ) Curcumin has the two α, β unsaturated carbonyl groups which are eliminated in tetrahydrocurcumin by hydrogenating the double bonds. ( B ) H- Ras MCF10A cells were incubated with vehicle, curcumin (25 μM) or tetrahydrocurcumin (25 μM). The expression of phosphorylated STAT3, STAT3 and related kinase was determind by Western blot analysis. ( C ) H- Ras MCF10A cells were treated with curcumin or tetrahydrocurcumin (25 μM each) for 12 h and then incubated with Annexin V and PI for 15 min. The population of apoptotic cells was measured by FACS analysis. ( D ) H- Ras MCF10A cells were cultured in soft agar for 10 days. Curcumin or tetrahydrocurcumin was added to the medium every other day for another 10 days. The colony formation was visualized under the microscope. ** P

    Article Snippet: The recombinant STAT3 protein (Active Motif) was treated with curcumin, and the solution mixture was added to the urea buffer (1 M urea, 50 mM Tris-HCl, pH 7.8).

    Techniques: Inhibition, Activation Assay, Incubation, Expressing, Western Blot, FACS, Cell Culture, Microscopy

    The effect of curcumin and tetrahydrocurcumin on phosphorylation, nuclear translocation, and transcriptional activity of STAT3. ( A ) H- Ras MCF10A cells were treated with curcumin and tetrahydrocurcumin (25 μM each) for 12 h. Cytosolic and nuclear fractions were extracted and subjected to Western blot analysis for measuring total and phosphorylated STAT3 levels. ( B ) Luciferase activity was measured in HeLa/P-STAT3-luc cells preincubated with curcumin or tetrahydrocurcumin (25 μM each) for 12 h and then stimulated with OSM for additional 6 h. * P

    Journal: Scientific Reports

    Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells

    doi: 10.1038/s41598-018-23840-2

    Figure Lengend Snippet: The effect of curcumin and tetrahydrocurcumin on phosphorylation, nuclear translocation, and transcriptional activity of STAT3. ( A ) H- Ras MCF10A cells were treated with curcumin and tetrahydrocurcumin (25 μM each) for 12 h. Cytosolic and nuclear fractions were extracted and subjected to Western blot analysis for measuring total and phosphorylated STAT3 levels. ( B ) Luciferase activity was measured in HeLa/P-STAT3-luc cells preincubated with curcumin or tetrahydrocurcumin (25 μM each) for 12 h and then stimulated with OSM for additional 6 h. * P

    Article Snippet: The recombinant STAT3 protein (Active Motif) was treated with curcumin, and the solution mixture was added to the urea buffer (1 M urea, 50 mM Tris-HCl, pH 7.8).

    Techniques: Translocation Assay, Activity Assay, Western Blot, Luciferase

    Comparison between MCF10A and H- Ras MCF10A cells and the effect of curcumin on induction of apoptosis. ( A ) Total cell lysates of MCF10A and H- Ras MCF10A cells were prepared, and the expression of phosphorylated STAT3 was assessed by Western blot analysis. ( B ) The same number of MCF10A and H- Ras MCF10A cells (2 × 10 3 ) was seeded onto Lipidure®-Coat Plate A-U96 and incubated for 5 days. The spheroid formations were observed under the microscope. scale bar, 500 μm. Data are means ± standard deviation (SD). ns: not significant; * P

    Journal: Scientific Reports

    Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells

    doi: 10.1038/s41598-018-23840-2

    Figure Lengend Snippet: Comparison between MCF10A and H- Ras MCF10A cells and the effect of curcumin on induction of apoptosis. ( A ) Total cell lysates of MCF10A and H- Ras MCF10A cells were prepared, and the expression of phosphorylated STAT3 was assessed by Western blot analysis. ( B ) The same number of MCF10A and H- Ras MCF10A cells (2 × 10 3 ) was seeded onto Lipidure®-Coat Plate A-U96 and incubated for 5 days. The spheroid formations were observed under the microscope. scale bar, 500 μm. Data are means ± standard deviation (SD). ns: not significant; * P

    Article Snippet: The recombinant STAT3 protein (Active Motif) was treated with curcumin, and the solution mixture was added to the urea buffer (1 M urea, 50 mM Tris-HCl, pH 7.8).

    Techniques: Expressing, Western Blot, Incubation, Microscopy, Standard Deviation

    STAT3 thiol modification by curcumin. ( A ) H- Ras MCF10A cells were treated with curcumin (25 μM) in the presence or absence of NAC (10 mM) or DTT (0.5 mM). ( B ) Luciferase activity was measured with HeLa/P-STAT3-luc cells preincubated with curcumin (25 µM) in the presence or absence of NAC (10 mM) or DTT (0.5 mM) for 12 h and then stimulated with OSM for another 6 h. ** P

    Journal: Scientific Reports

    Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells

    doi: 10.1038/s41598-018-23840-2

    Figure Lengend Snippet: STAT3 thiol modification by curcumin. ( A ) H- Ras MCF10A cells were treated with curcumin (25 μM) in the presence or absence of NAC (10 mM) or DTT (0.5 mM). ( B ) Luciferase activity was measured with HeLa/P-STAT3-luc cells preincubated with curcumin (25 µM) in the presence or absence of NAC (10 mM) or DTT (0.5 mM) for 12 h and then stimulated with OSM for another 6 h. ** P

    Article Snippet: The recombinant STAT3 protein (Active Motif) was treated with curcumin, and the solution mixture was added to the urea buffer (1 M urea, 50 mM Tris-HCl, pH 7.8).

    Techniques: Modification, Luciferase, Activity Assay

    IFN-γ-induced signal transduction, in the presence of FLLL32 and FLLL62. (A) Human ACHN RCC cells or (B) A375 melanoma cells were pre-treated for 16 hours with DMSO (negative control), multiple doses of FLLL32, FLLL62, or curcumin and subsequently stimulated with IFN-γ (10 ng/mL) or PBS (vehicle) for 15 minutes. The expression of pSTAT1 and pSTAT3 were evaluated by immunoblot. Membranes were probed for total STAT3 protein, total STAT1 protein and β-actin to control for loading.

    Journal: PLoS ONE

    Article Title: Structurally Modified Curcumin Analogs Inhibit STAT3 Phosphorylation and Promote Apoptosis of Human Renal Cell Carcinoma and Melanoma Cell Lines

    doi: 10.1371/journal.pone.0040724

    Figure Lengend Snippet: IFN-γ-induced signal transduction, in the presence of FLLL32 and FLLL62. (A) Human ACHN RCC cells or (B) A375 melanoma cells were pre-treated for 16 hours with DMSO (negative control), multiple doses of FLLL32, FLLL62, or curcumin and subsequently stimulated with IFN-γ (10 ng/mL) or PBS (vehicle) for 15 minutes. The expression of pSTAT1 and pSTAT3 were evaluated by immunoblot. Membranes were probed for total STAT3 protein, total STAT1 protein and β-actin to control for loading.

    Article Snippet: For fluorescence polarization assays, STAT3 protein ( > 90% purity) was purchased from Abcam (Cambridge, MA).

    Techniques: Transduction, Negative Control, Expressing

    FLLL32 and FLLL62 inhibit STAT3 dimerization in vitro . (A) Saturation curves for fluorescence polarization assay. Data represent the change in specific binding observed when 4 nM 5-FAM-SpYLPQTV was incubated with increasing concentrations of STAT3 in the presence and absence of 50 µM FLLL32. (B) Scatchard Plot analysis of the binding of 4 nM 5-FAM-SpYLPQTV in the presence and absence of 50 µM FLLL32 over increasing concentrations of STAT3. Specific binding was plotted along the x-axis while the quotient of the specific binding divided by the corresponding concentration of STAT3 was plotted along the y-axis. The slope of each data set is equal to −1/K d while the x-intercept is equal to B max . (C) Hill Plot analysis of the binding of 4 nM 5-FAM-SpYLPQTV in the presence and absence of 50 µM FLLL32 and FLLL62 over increasing concentrations of STAT3. The log of the quotient of the specific binding divided by the difference between the B max and specific binding is plotted along the y-axis while the log of the corresponding concentration of STAT3 is plotted along the x-axis. A slope of 1 indicates noncooperativity in binding. A right-handed shift of the plot shows an increase in the K d .

    Journal: PLoS ONE

    Article Title: Structurally Modified Curcumin Analogs Inhibit STAT3 Phosphorylation and Promote Apoptosis of Human Renal Cell Carcinoma and Melanoma Cell Lines

    doi: 10.1371/journal.pone.0040724

    Figure Lengend Snippet: FLLL32 and FLLL62 inhibit STAT3 dimerization in vitro . (A) Saturation curves for fluorescence polarization assay. Data represent the change in specific binding observed when 4 nM 5-FAM-SpYLPQTV was incubated with increasing concentrations of STAT3 in the presence and absence of 50 µM FLLL32. (B) Scatchard Plot analysis of the binding of 4 nM 5-FAM-SpYLPQTV in the presence and absence of 50 µM FLLL32 over increasing concentrations of STAT3. Specific binding was plotted along the x-axis while the quotient of the specific binding divided by the corresponding concentration of STAT3 was plotted along the y-axis. The slope of each data set is equal to −1/K d while the x-intercept is equal to B max . (C) Hill Plot analysis of the binding of 4 nM 5-FAM-SpYLPQTV in the presence and absence of 50 µM FLLL32 and FLLL62 over increasing concentrations of STAT3. The log of the quotient of the specific binding divided by the difference between the B max and specific binding is plotted along the y-axis while the log of the corresponding concentration of STAT3 is plotted along the x-axis. A slope of 1 indicates noncooperativity in binding. A right-handed shift of the plot shows an increase in the K d .

    Article Snippet: For fluorescence polarization assays, STAT3 protein ( > 90% purity) was purchased from Abcam (Cambridge, MA).

    Techniques: In Vitro, Fluorescence, Binding Assay, Incubation, Concentration Assay

    FLLL32 and FLLL62 induce apoptosis in human RCC cell lines. Annexin V/propidium iodide (Ann V/PI) staining of human RCC lines (Caki, ACHN, SK-RC-45, SK-RC-54) following a continuous 48 hour treatment with various doses of (A) FLLL32 or (B) FLLL62. Data were derived from at least three independent experiments. 95% confidence bounds are shown for the absolute IC 50 estimates. (C) Representative flow cytometric analysis of apoptosis in human ACHN cells following a 48 hour treatment with DMSO (vehicle), FLLL32 or FLLL62. Inhibition of STAT3 phosphorylation (at Tyr 705 ) was confirmed via immunoblot following a 24 hour treatment with (D) FLLL32 or FLLL62. Processing of PARP from its native to its cleaved form was also assessed as a marker of apoptosis. The STAT3 regulated gene, Cyclin D1 was also reduced by FLLL32. Membranes were probed for total STAT3 protein and β-actin to control for loading. (E) Immunoblot analysis of ERK, p38 MAPK and AKT phosphorylation in human RCC lines after a 48 hour treatment with FLLL32 or FLLL62. (F) Reduced STAT3-phosphorylation and PARP cleavage in human metastatic A375 and Hs294T melanoma cell lines following a 24 hour treatment with FLLL62.

    Journal: PLoS ONE

    Article Title: Structurally Modified Curcumin Analogs Inhibit STAT3 Phosphorylation and Promote Apoptosis of Human Renal Cell Carcinoma and Melanoma Cell Lines

    doi: 10.1371/journal.pone.0040724

    Figure Lengend Snippet: FLLL32 and FLLL62 induce apoptosis in human RCC cell lines. Annexin V/propidium iodide (Ann V/PI) staining of human RCC lines (Caki, ACHN, SK-RC-45, SK-RC-54) following a continuous 48 hour treatment with various doses of (A) FLLL32 or (B) FLLL62. Data were derived from at least three independent experiments. 95% confidence bounds are shown for the absolute IC 50 estimates. (C) Representative flow cytometric analysis of apoptosis in human ACHN cells following a 48 hour treatment with DMSO (vehicle), FLLL32 or FLLL62. Inhibition of STAT3 phosphorylation (at Tyr 705 ) was confirmed via immunoblot following a 24 hour treatment with (D) FLLL32 or FLLL62. Processing of PARP from its native to its cleaved form was also assessed as a marker of apoptosis. The STAT3 regulated gene, Cyclin D1 was also reduced by FLLL32. Membranes were probed for total STAT3 protein and β-actin to control for loading. (E) Immunoblot analysis of ERK, p38 MAPK and AKT phosphorylation in human RCC lines after a 48 hour treatment with FLLL32 or FLLL62. (F) Reduced STAT3-phosphorylation and PARP cleavage in human metastatic A375 and Hs294T melanoma cell lines following a 24 hour treatment with FLLL62.

    Article Snippet: For fluorescence polarization assays, STAT3 protein ( > 90% purity) was purchased from Abcam (Cambridge, MA).

    Techniques: Staining, Derivative Assay, Flow Cytometry, Inhibition, Marker

    Molecular structure of FLLL32 and FLLL62 curcumin analogs. (A) Structural representation of curcumin and the FLLL32 and FLLL62 diketone analogs. (B) Computational representation of FLLL32 and FLLL62 binding to the STAT3 SH2 domain. The SH2 surface was shown with electrostatic potential, with blue representing positive charged surface and red representing negative charges. Both compounds bind to the pTyr705 site identically with a slight difference occurring at the Leu706 site.

    Journal: PLoS ONE

    Article Title: Structurally Modified Curcumin Analogs Inhibit STAT3 Phosphorylation and Promote Apoptosis of Human Renal Cell Carcinoma and Melanoma Cell Lines

    doi: 10.1371/journal.pone.0040724

    Figure Lengend Snippet: Molecular structure of FLLL32 and FLLL62 curcumin analogs. (A) Structural representation of curcumin and the FLLL32 and FLLL62 diketone analogs. (B) Computational representation of FLLL32 and FLLL62 binding to the STAT3 SH2 domain. The SH2 surface was shown with electrostatic potential, with blue representing positive charged surface and red representing negative charges. Both compounds bind to the pTyr705 site identically with a slight difference occurring at the Leu706 site.

    Article Snippet: For fluorescence polarization assays, STAT3 protein ( > 90% purity) was purchased from Abcam (Cambridge, MA).

    Techniques: Binding Assay

    LLL12 inhibited STAT3 DNA binding activity and expression of downstream targets associated with proliferation and survival of MM tumor cells (A) U266 and ARH-77 MM cells were treated in LLL12 (2.5μM) or DMSO for 24 hours and nuclear extracts were examined for DNA binding activity. LLL12 induced statistically significant (*) reductions in STAT3 DNA binding activity, results are representative of two independent experiments in each cell line. (B) U266 and ARH-77 human MM cell lines were cultured in LLL12 (5 μM) or DMSO for 24 hours. Reverse transcriptase PCR revealed decreased expression of STAT3 target genes in LLL12-treated cells as compared to DMSO control following treatment with LLL12. (C) Downstream STAT3 target proteins, Cyclin D1, Survivin, Bcl-2, and DNMT1 were downregulated by LLL12 as shown by western blot analysis. (D) MM.1S cells were stimulated with IL-6 (2.5–10 ng/ml) for 48 hours in the presence or absence of LLL12 (2.5μM) for the last 24 hours. Reverse transcriptase PCR showed IL-6 enhanced expression of STAT3 target genes, which was blocked by LLL12.

    Journal: International Journal of Cancer. Journal International du Cancer

    Article Title: A small molecule, LLL12 inhibits constitutive STAT3 and IL-6-induced STAT3 signaling and exhibits potent growth suppressive activity in human multiple myeloma cells

    doi: 10.1002/ijc.26152

    Figure Lengend Snippet: LLL12 inhibited STAT3 DNA binding activity and expression of downstream targets associated with proliferation and survival of MM tumor cells (A) U266 and ARH-77 MM cells were treated in LLL12 (2.5μM) or DMSO for 24 hours and nuclear extracts were examined for DNA binding activity. LLL12 induced statistically significant (*) reductions in STAT3 DNA binding activity, results are representative of two independent experiments in each cell line. (B) U266 and ARH-77 human MM cell lines were cultured in LLL12 (5 μM) or DMSO for 24 hours. Reverse transcriptase PCR revealed decreased expression of STAT3 target genes in LLL12-treated cells as compared to DMSO control following treatment with LLL12. (C) Downstream STAT3 target proteins, Cyclin D1, Survivin, Bcl-2, and DNMT1 were downregulated by LLL12 as shown by western blot analysis. (D) MM.1S cells were stimulated with IL-6 (2.5–10 ng/ml) for 48 hours in the presence or absence of LLL12 (2.5μM) for the last 24 hours. Reverse transcriptase PCR showed IL-6 enhanced expression of STAT3 target genes, which was blocked by LLL12.

    Article Snippet: The nuclear extracts were analyzed for STAT3 DNA binding activity using a STAT3 Transcription Factor Kit (Clontech Inc, Mountain View, CA).

    Techniques: Binding Assay, Activity Assay, Expressing, Cell Culture, Polymerase Chain Reaction, Western Blot

    LLL12 inhibited cell proliferation induced by IL-6 and blocked IL-6 mediated drug resistance in human MM cells (A) MM.1S cells were treated with IL-6 (25ng/ml) with or without LLL12. IL-6 can increase cell proliferation, which could be blocked by LLL12. (B) Treatments of MM1.S cells with lenalidomide caused a dose-dependent reduction of cell viability. IL-6 reversed lenalidomide induced inhibition of cell viability. Pre-treatment of MM1.S cells with LLL12, further reversed the rescue of lenalidomide-mediated inhibition of cell viability by IL-6. (C) U266 cells secreted higher levels of IL-6 than MM1.S cells. (D) U266 cells expressed higher levels of IL-6 than MM1.S cells. Both MM cell lines expressed similar levels of IL-6R and Gp130. (E) U266 cells expressed higher levels of STAT3 phosphoryltion than MM1.S cells. (F) U266 cells were more resistance to lenalidomide than MM1.S cells. (G) LLL12 enhanced lenalidomide-mediated inhibition of cell proliferation in U266 MM cells.

    Journal: International Journal of Cancer. Journal International du Cancer

    Article Title: A small molecule, LLL12 inhibits constitutive STAT3 and IL-6-induced STAT3 signaling and exhibits potent growth suppressive activity in human multiple myeloma cells

    doi: 10.1002/ijc.26152

    Figure Lengend Snippet: LLL12 inhibited cell proliferation induced by IL-6 and blocked IL-6 mediated drug resistance in human MM cells (A) MM.1S cells were treated with IL-6 (25ng/ml) with or without LLL12. IL-6 can increase cell proliferation, which could be blocked by LLL12. (B) Treatments of MM1.S cells with lenalidomide caused a dose-dependent reduction of cell viability. IL-6 reversed lenalidomide induced inhibition of cell viability. Pre-treatment of MM1.S cells with LLL12, further reversed the rescue of lenalidomide-mediated inhibition of cell viability by IL-6. (C) U266 cells secreted higher levels of IL-6 than MM1.S cells. (D) U266 cells expressed higher levels of IL-6 than MM1.S cells. Both MM cell lines expressed similar levels of IL-6R and Gp130. (E) U266 cells expressed higher levels of STAT3 phosphoryltion than MM1.S cells. (F) U266 cells were more resistance to lenalidomide than MM1.S cells. (G) LLL12 enhanced lenalidomide-mediated inhibition of cell proliferation in U266 MM cells.

    Article Snippet: The nuclear extracts were analyzed for STAT3 DNA binding activity using a STAT3 Transcription Factor Kit (Clontech Inc, Mountain View, CA).

    Techniques: Inhibition