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  • 78
    Thermo Fisher dmso
    Proteasome degradation of p53 protein upon <t>PEITC</t> treatment in SK-BR-3 and A549 cells. ( a ) SK-BR-3 cells were treated with the indicated concentrations of PEITC and inhibitor (10 μ M Nutlin-3 or 20 μ M MG132) for 4 h. ( b ) SK-BR-3 cells were treated with PEITC (4 or 8 μ M), 20 μ M MG132 or both for 4 h. ( c ) SK-BR-3 cells were treated with PEITC (4 or 8 μ M), 10 μ M Nutlin-3 or both for 4 h. ( d ) A549 cells were treated with PEITC (4 or 8 μ M), inhibitor (10 μ M Nutlin-3 or 20 μ M MG132) or both for 4 h. Cells were harvested and lysates were prepared. Lysate fractions were resolved by SDS-PAGE and probed with p53 DO-1 antibody. ( e ) SK-BR-3 cells were treated with the indicated concentrations of PEITC or <t>DMSO</t> for 4 h. Cells were harvested and soluble and insoluble fractions were prepared. Thirty μg of the soluble and insoluble lysate fractions were resolved by SDS-PAGE and probed with p53 DO-1 antibody
    Dmso, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dmso/product/Thermo Fisher
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dmso - by Bioz Stars, 2019-10
    78/100 stars
      Buy from Supplier

    82
    Millipore dimethyl sulfoxide dmso
    Effects of <t>BPA</t> on the mRNA expression level of caspase8 ( Casp8 ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with <t>DMSO</t> or BPA (0.1, 1, 5, and 10 μg/ml). Casp8 expression was measured using qPCR. Relative
    Dimethyl Sulfoxide Dmso, supplied by Millipore, used in various techniques. Bioz Stars score: 82/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dimethyl sulfoxide dmso/product/Millipore
    Average 82 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    dimethyl sulfoxide dmso - by Bioz Stars, 2019-10
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    78
    Selleck Chemicals dmso
    Effects of CDK4/6 inhibition on breast cancer cell proliferation and apoptosis in vitro a , Relative numbers of breast cancer cells cultured in 250 nM (MDA-MB-453) or 500 nM (MDA-MB-361, BT474) <t>abemaciclib</t> or <t>DMSO</t> for 11d, followed by drug withdrawal (arrow). b , Representative SA-β-galactosidase staining of MDA-MB-453 cells (left) and BT474 cells (right) treated with DMSO or abemaciclib (MDA-MB-453, 250 nM; BT474, 500 nM) for 0, 4, and 7 days. c , Western blot of SKBR3, BT474, MDA-MB-453, and MDA-MB-361 cells treated with DMSO, lapatinib, or abemaciclib for 48h. d , Western blot of MDA-MB-453 cells pretreated with DMSO or abemaciclib (500 nM) for 0, 1, or 7 days prior to exposure to staurosporine (500 nM) for 4h. For western blot source images, see Supplementary Figure 1 .
    Dmso, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dmso/product/Selleck Chemicals
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dmso - by Bioz Stars, 2019-10
    78/100 stars
      Buy from Supplier

    Image Search Results


    Proteasome degradation of p53 protein upon PEITC treatment in SK-BR-3 and A549 cells. ( a ) SK-BR-3 cells were treated with the indicated concentrations of PEITC and inhibitor (10 μ M Nutlin-3 or 20 μ M MG132) for 4 h. ( b ) SK-BR-3 cells were treated with PEITC (4 or 8 μ M), 20 μ M MG132 or both for 4 h. ( c ) SK-BR-3 cells were treated with PEITC (4 or 8 μ M), 10 μ M Nutlin-3 or both for 4 h. ( d ) A549 cells were treated with PEITC (4 or 8 μ M), inhibitor (10 μ M Nutlin-3 or 20 μ M MG132) or both for 4 h. Cells were harvested and lysates were prepared. Lysate fractions were resolved by SDS-PAGE and probed with p53 DO-1 antibody. ( e ) SK-BR-3 cells were treated with the indicated concentrations of PEITC or DMSO for 4 h. Cells were harvested and soluble and insoluble fractions were prepared. Thirty μg of the soluble and insoluble lysate fractions were resolved by SDS-PAGE and probed with p53 DO-1 antibody

    Journal: Cell Death and Differentiation

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth

    doi: 10.1038/cdd.2016.48

    Figure Lengend Snippet: Proteasome degradation of p53 protein upon PEITC treatment in SK-BR-3 and A549 cells. ( a ) SK-BR-3 cells were treated with the indicated concentrations of PEITC and inhibitor (10 μ M Nutlin-3 or 20 μ M MG132) for 4 h. ( b ) SK-BR-3 cells were treated with PEITC (4 or 8 μ M), 20 μ M MG132 or both for 4 h. ( c ) SK-BR-3 cells were treated with PEITC (4 or 8 μ M), 10 μ M Nutlin-3 or both for 4 h. ( d ) A549 cells were treated with PEITC (4 or 8 μ M), inhibitor (10 μ M Nutlin-3 or 20 μ M MG132) or both for 4 h. Cells were harvested and lysates were prepared. Lysate fractions were resolved by SDS-PAGE and probed with p53 DO-1 antibody. ( e ) SK-BR-3 cells were treated with the indicated concentrations of PEITC or DMSO for 4 h. Cells were harvested and soluble and insoluble fractions were prepared. Thirty μg of the soluble and insoluble lysate fractions were resolved by SDS-PAGE and probed with p53 DO-1 antibody

    Article Snippet: SK-BR-3, A549, H1299 and (10)3/175 cells were treated with PEITC (4 or 6 μ M) or 1% DMSO as a control for 6 h in slide chambers with four wells (ThermoFisher Scientific).

    Techniques: SDS Page

    Autophagy of p53 R175 protein upon PEITC treatment in SK-BR-3 cells. ( a ) SK-BR-3 cells were treated with PEITC (4 or 8 μ M), CHQ (50 μ M) or both for 4 h. Cell lysate fractions were resolved by SDS-PAGE and probed with p53 DO-1 antibody. ( b ) SK-BR-3 cells were transfected with ATG5 siRNA or NS siRNA. Thirty μg of the cell lysate was resolved by SDS-PAGE and probed with anti-ATG5 antibody. Blots were stripped and reprobed with anti-GAPDH antibody. ( c ) SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA were treated with DMSO or PEITC for 4 h. Protein levels were determined by western blotting using p53 DO-1 and GAPDH antibodies. ( d ) SK-BR-3 transfected with ATG5 siRNA or NS siRNA were treated with DMSO or PEITC for 3 days. Percentage of cell proliferation was determined by the WST-1 assay. ( e ) Effect of PEITC on apoptosis. ATG5 siRNA- or NS siRNA-transfected SK-BR-3 cells were treated with DMSO or PEITC for 3 days. Cells were assayed for histone-associated DNA fragments indicative of apoptosis

    Journal: Cell Death and Differentiation

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth

    doi: 10.1038/cdd.2016.48

    Figure Lengend Snippet: Autophagy of p53 R175 protein upon PEITC treatment in SK-BR-3 cells. ( a ) SK-BR-3 cells were treated with PEITC (4 or 8 μ M), CHQ (50 μ M) or both for 4 h. Cell lysate fractions were resolved by SDS-PAGE and probed with p53 DO-1 antibody. ( b ) SK-BR-3 cells were transfected with ATG5 siRNA or NS siRNA. Thirty μg of the cell lysate was resolved by SDS-PAGE and probed with anti-ATG5 antibody. Blots were stripped and reprobed with anti-GAPDH antibody. ( c ) SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA were treated with DMSO or PEITC for 4 h. Protein levels were determined by western blotting using p53 DO-1 and GAPDH antibodies. ( d ) SK-BR-3 transfected with ATG5 siRNA or NS siRNA were treated with DMSO or PEITC for 3 days. Percentage of cell proliferation was determined by the WST-1 assay. ( e ) Effect of PEITC on apoptosis. ATG5 siRNA- or NS siRNA-transfected SK-BR-3 cells were treated with DMSO or PEITC for 3 days. Cells were assayed for histone-associated DNA fragments indicative of apoptosis

    Article Snippet: SK-BR-3, A549, H1299 and (10)3/175 cells were treated with PEITC (4 or 6 μ M) or 1% DMSO as a control for 6 h in slide chambers with four wells (ThermoFisher Scientific).

    Techniques: SDS Page, Transfection, Western Blot, WST-1 Assay

    PEITC induces a ‘WT-like' conformational change in p53 R175 mutant protein. ( a ) ELISA to determine the effect of PEITC on conformation of recombinant-purified GST-p53 R175H by using conformation-specific antibodies PAB240 (mutant-specific) and PAB1620 (WT-specific). ( b ) SK-BR-3 cells were treated with DMSO or 4 μ M PEITC for 6 h. Immunofluorescence of the cells was performed using PAB240 and PAB1620 antibodies. The A549 cell line used as a control showed that p53 WT conformation was not changed by PEITC. The H1299 cell line was used as a control for anti-p53 antibodies. All scale bars represents a size of 20 μ m. ( c ) Quantification of PAB240 and PAB1620 staining shown in panel ( b ). *** P ≤0.0001 for PAB240 and PAB1620. ( d ) Immunoprecipitation of the p53 mutant protein from SK-BR-3 cell lysates using PAB240 antibody and detected by p53 (FL393) antibody

    Journal: Cell Death and Differentiation

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth

    doi: 10.1038/cdd.2016.48

    Figure Lengend Snippet: PEITC induces a ‘WT-like' conformational change in p53 R175 mutant protein. ( a ) ELISA to determine the effect of PEITC on conformation of recombinant-purified GST-p53 R175H by using conformation-specific antibodies PAB240 (mutant-specific) and PAB1620 (WT-specific). ( b ) SK-BR-3 cells were treated with DMSO or 4 μ M PEITC for 6 h. Immunofluorescence of the cells was performed using PAB240 and PAB1620 antibodies. The A549 cell line used as a control showed that p53 WT conformation was not changed by PEITC. The H1299 cell line was used as a control for anti-p53 antibodies. All scale bars represents a size of 20 μ m. ( c ) Quantification of PAB240 and PAB1620 staining shown in panel ( b ). *** P ≤0.0001 for PAB240 and PAB1620. ( d ) Immunoprecipitation of the p53 mutant protein from SK-BR-3 cell lysates using PAB240 antibody and detected by p53 (FL393) antibody

    Article Snippet: SK-BR-3, A549, H1299 and (10)3/175 cells were treated with PEITC (4 or 6 μ M) or 1% DMSO as a control for 6 h in slide chambers with four wells (ThermoFisher Scientific).

    Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Recombinant, Purification, Immunofluorescence, Staining, Immunoprecipitation

    PEITC induces γ -H2AX foci, activates ATM/CHK2, G2/M- and S-phase arrest and apoptosis. SK-BR-3 and A549 cells treated with PEITC or DMSO for 3 days were stained with anti- γ -H2AX antibody. ( a ) Merged images show cells stained with anti- γ -H2AX antibody (green) and DAPI (blue). All scale bars represents a size of 20 μ m. ( b ) Percentage of cells with γ -H2AX foci (≤10 or > 10, as indicated). ( c ) SK-BR-3 and A549 cells were treated with PEITC or DMSO for 4 h. Western blotting was performed using anti-pATM S1981 and anti-pCHK2 Thr68 antibodies. Blots were stripped and reprobed with anti-ATM and anti-CHK2 antibodies. ( d ) SK-BR-3 or ( e ) A549 cells were treated with PEITC, 10 μ M Nutlin-3 or both for 24 h and analyzed by flow cytometry. ( f ) SK-BR-3 and A549 cells were treated with 4μM PEITC, 10 μ M Nutlin-3 or both for 24 h. Apoptosis was measured by Annexin-V staining using a BD LSRFORTESSA instrument

    Journal: Cell Death and Differentiation

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth

    doi: 10.1038/cdd.2016.48

    Figure Lengend Snippet: PEITC induces γ -H2AX foci, activates ATM/CHK2, G2/M- and S-phase arrest and apoptosis. SK-BR-3 and A549 cells treated with PEITC or DMSO for 3 days were stained with anti- γ -H2AX antibody. ( a ) Merged images show cells stained with anti- γ -H2AX antibody (green) and DAPI (blue). All scale bars represents a size of 20 μ m. ( b ) Percentage of cells with γ -H2AX foci (≤10 or > 10, as indicated). ( c ) SK-BR-3 and A549 cells were treated with PEITC or DMSO for 4 h. Western blotting was performed using anti-pATM S1981 and anti-pCHK2 Thr68 antibodies. Blots were stripped and reprobed with anti-ATM and anti-CHK2 antibodies. ( d ) SK-BR-3 or ( e ) A549 cells were treated with PEITC, 10 μ M Nutlin-3 or both for 24 h and analyzed by flow cytometry. ( f ) SK-BR-3 and A549 cells were treated with 4μM PEITC, 10 μ M Nutlin-3 or both for 24 h. Apoptosis was measured by Annexin-V staining using a BD LSRFORTESSA instrument

    Article Snippet: SK-BR-3, A549, H1299 and (10)3/175 cells were treated with PEITC (4 or 6 μ M) or 1% DMSO as a control for 6 h in slide chambers with four wells (ThermoFisher Scientific).

    Techniques: Staining, Western Blot, Flow Cytometry, Cytometry

    PEITC restores the p53 R175 mutant protein transactivational functions. ( a ) PEITC induced p53 R175 mutant protein to bind chromatin. SK-BR-3 cells were treated with PEITC for 4 h and chromatin-bound and nuclear-soluble fractions were analyzed by immunoblotting. Histone H3 and Topoisomerase IIB served as markers for the chromatin and soluble nuclear fractions, respectively. ( b ) qRT-PCR of p53 regulated genes in SK-BR-3, H1299 and A549 cells treated with DMSO or 4 μ M PEITC for 4 h. RNA was extracted and gene expression level was measured using TaqMan gene expression assay. ( c ) qRT-PCR of p53-regulated genes in NS siRNA or p53 siRNA-transfected SK-BR-3 cells treated with DMSO or 4 μ M PEITC for 4 h. RNA was extracted and gene expression level was measured using TaqMan gene expression assay. ( d ) SK-BR-3, HOP92, AU565, H1299 and MEF ((10)3/175 and (10)3/273) cells were transfected with plasmid 16451 and were treated with PEITC (4 or 6 μ M) for 24 h, respectively, followed by a luciferase reporter assay. ( e ) Western blotting analysis of p21 expression in SK-BR-3 cells treated with 4 μ M PEITC or 20 μ M etoposide for 4 h. A549 cell line was treated with 4 μ M PEITC for 4 h as a control. Protein levels were determined by western blotting using p21, p53 DO-1 and GAPDH antibodies

    Journal: Cell Death and Differentiation

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth

    doi: 10.1038/cdd.2016.48

    Figure Lengend Snippet: PEITC restores the p53 R175 mutant protein transactivational functions. ( a ) PEITC induced p53 R175 mutant protein to bind chromatin. SK-BR-3 cells were treated with PEITC for 4 h and chromatin-bound and nuclear-soluble fractions were analyzed by immunoblotting. Histone H3 and Topoisomerase IIB served as markers for the chromatin and soluble nuclear fractions, respectively. ( b ) qRT-PCR of p53 regulated genes in SK-BR-3, H1299 and A549 cells treated with DMSO or 4 μ M PEITC for 4 h. RNA was extracted and gene expression level was measured using TaqMan gene expression assay. ( c ) qRT-PCR of p53-regulated genes in NS siRNA or p53 siRNA-transfected SK-BR-3 cells treated with DMSO or 4 μ M PEITC for 4 h. RNA was extracted and gene expression level was measured using TaqMan gene expression assay. ( d ) SK-BR-3, HOP92, AU565, H1299 and MEF ((10)3/175 and (10)3/273) cells were transfected with plasmid 16451 and were treated with PEITC (4 or 6 μ M) for 24 h, respectively, followed by a luciferase reporter assay. ( e ) Western blotting analysis of p21 expression in SK-BR-3 cells treated with 4 μ M PEITC or 20 μ M etoposide for 4 h. A549 cell line was treated with 4 μ M PEITC for 4 h as a control. Protein levels were determined by western blotting using p21, p53 DO-1 and GAPDH antibodies

    Article Snippet: SK-BR-3, A549, H1299 and (10)3/175 cells were treated with PEITC (4 or 6 μ M) or 1% DMSO as a control for 6 h in slide chambers with four wells (ThermoFisher Scientific).

    Techniques: Mutagenesis, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Western Blot

    PEITC inhibits cell proliferation and induces apoptosis in a p53 R175 mutant-dependent manner. ( a ) Human tumor cells lines with hotspot p53 mutations and p53 WT were treated with DMSO (control) or PEITC for 3 days. ( b ) SK-BR-3 and A549 cells transfected with siRNA were treated with DMSO or PEITC for 3 days. Percentage of cell proliferation was determined by the WST-1 assay. ( c ) Effect of PEITC on apoptosis. Untransfected (cells) or siRNA-transfected SK-BR-3 and A549 cells were treated with DMSO or 4 μ M PEITC for 3 days. Apoptosis was measured by Annexin-V staining using a BD LSRFORTESSA instrument. ( d ) The H1299 cells transfected with pcDNA3, pcDNA3-p53R175, pcDNA3-p53R273 or pcDNA3-wtp53 were treated with DMSO or PEITC for 3 days. Percentage of cell proliferation was determined by the WST-1assay. ( e ) Effect of PEITC on apoptosis. The H1299 cells transfected with pcDNA3, pcDNA3-p53R175, pcDNA3-p53R273 or pcDNA3-wtp53 were treated with DMSO or 8 μ M PEITC for 3 days. Apoptosis was measured by Annexin-V staining using a BD LSRFORTESSA instrument

    Journal: Cell Death and Differentiation

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth

    doi: 10.1038/cdd.2016.48

    Figure Lengend Snippet: PEITC inhibits cell proliferation and induces apoptosis in a p53 R175 mutant-dependent manner. ( a ) Human tumor cells lines with hotspot p53 mutations and p53 WT were treated with DMSO (control) or PEITC for 3 days. ( b ) SK-BR-3 and A549 cells transfected with siRNA were treated with DMSO or PEITC for 3 days. Percentage of cell proliferation was determined by the WST-1 assay. ( c ) Effect of PEITC on apoptosis. Untransfected (cells) or siRNA-transfected SK-BR-3 and A549 cells were treated with DMSO or 4 μ M PEITC for 3 days. Apoptosis was measured by Annexin-V staining using a BD LSRFORTESSA instrument. ( d ) The H1299 cells transfected with pcDNA3, pcDNA3-p53R175, pcDNA3-p53R273 or pcDNA3-wtp53 were treated with DMSO or PEITC for 3 days. Percentage of cell proliferation was determined by the WST-1assay. ( e ) Effect of PEITC on apoptosis. The H1299 cells transfected with pcDNA3, pcDNA3-p53R175, pcDNA3-p53R273 or pcDNA3-wtp53 were treated with DMSO or 8 μ M PEITC for 3 days. Apoptosis was measured by Annexin-V staining using a BD LSRFORTESSA instrument

    Article Snippet: SK-BR-3, A549, H1299 and (10)3/175 cells were treated with PEITC (4 or 6 μ M) or 1% DMSO as a control for 6 h in slide chambers with four wells (ThermoFisher Scientific).

    Techniques: Mutagenesis, Transfection, WST-1 Assay, Staining

    Effects of zinc and redox changes on PEITC-induced p53 R175 reactivation. ( a ) Effect of zinc on the activity of PEITC. SK-BR-3 cells were treated with PEITC, zinc or both. Percentage of cell proliferation was determined by the WST-1 assay. The PEITC activity is shown as 1/IC 50 for growth inhibition. ( b ) ELISA to determine the effect of zinc alone or zinc and PEITC on conformation of recombinant-purified GST-p53 R175H by using conformation-specific antibodies PAB240 (mutant-specific) and PAB1620 (WT-specific). ( c ) Effect of PEITC on the levels of reduced glutathione in SK-BR-3 cells. SK-BR-3 cells were treated with PEITC (4 or 8 μ M) or DMSO for 4 h. Ratio of reductant GSH and oxidative GSSG was then measured using the GSH/GSSG-Glo Glutathione Assay Kit. ( d ) Effect of NAC on PEITC activity. SK-BR-3 cells were treated with the indicated concentrations of PEITC or PEITC in combination with 3 mM NAC for 3 days. Percentage of cell proliferation was determined by the WST-1 assay. ( e ) SK-BR-3 cells were co-treated with the PEITC alone or in combination with 2 mM ATZ or 500 units PEG-Catalase for 3 days. Percentage of cell proliferation was determined by the WST-1 assay. ( f ) Effect on apoptosis. Untransfected (cells) or siRNA-transfected SK-BR-3 cells were treated with DMSO, ATZ, NAC or PEITC alone or PEITC in combination with ATZ or NAC for 3 days. Apoptosis was measured by Annexin-V staining using a BD LSRFORTESSA instrument

    Journal: Cell Death and Differentiation

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth

    doi: 10.1038/cdd.2016.48

    Figure Lengend Snippet: Effects of zinc and redox changes on PEITC-induced p53 R175 reactivation. ( a ) Effect of zinc on the activity of PEITC. SK-BR-3 cells were treated with PEITC, zinc or both. Percentage of cell proliferation was determined by the WST-1 assay. The PEITC activity is shown as 1/IC 50 for growth inhibition. ( b ) ELISA to determine the effect of zinc alone or zinc and PEITC on conformation of recombinant-purified GST-p53 R175H by using conformation-specific antibodies PAB240 (mutant-specific) and PAB1620 (WT-specific). ( c ) Effect of PEITC on the levels of reduced glutathione in SK-BR-3 cells. SK-BR-3 cells were treated with PEITC (4 or 8 μ M) or DMSO for 4 h. Ratio of reductant GSH and oxidative GSSG was then measured using the GSH/GSSG-Glo Glutathione Assay Kit. ( d ) Effect of NAC on PEITC activity. SK-BR-3 cells were treated with the indicated concentrations of PEITC or PEITC in combination with 3 mM NAC for 3 days. Percentage of cell proliferation was determined by the WST-1 assay. ( e ) SK-BR-3 cells were co-treated with the PEITC alone or in combination with 2 mM ATZ or 500 units PEG-Catalase for 3 days. Percentage of cell proliferation was determined by the WST-1 assay. ( f ) Effect on apoptosis. Untransfected (cells) or siRNA-transfected SK-BR-3 cells were treated with DMSO, ATZ, NAC or PEITC alone or PEITC in combination with ATZ or NAC for 3 days. Apoptosis was measured by Annexin-V staining using a BD LSRFORTESSA instrument

    Article Snippet: SK-BR-3, A549, H1299 and (10)3/175 cells were treated with PEITC (4 or 6 μ M) or 1% DMSO as a control for 6 h in slide chambers with four wells (ThermoFisher Scientific).

    Techniques: Activity Assay, WST-1 Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Recombinant, Purification, Mutagenesis, Glutathione Assay, Transfection, Staining

    Effects of BPA on the mRNA expression level of caspase8 ( Casp8 ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Casp8 expression was measured using qPCR. Relative

    Journal:

    Article Title: Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries

    doi: 10.1016/j.reprotox.2015.05.012

    Figure Lengend Snippet: Effects of BPA on the mRNA expression level of caspase8 ( Casp8 ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Casp8 expression was measured using qPCR. Relative

    Article Snippet: A stock solution of BPA was dissolved and diluted in dimethyl sulfoxide (DMSO) (Sigma–Aldrich) to achieve the selected BPA treatment concentrations (0.13, 1.3, 6.65, and 13.3 mg/ml) for final working concentrations of 0.1, 1.0, 5.0, and 10 μg of BPA per ml of culture media (0.44, 4.4, 22, and 44 μM BPA, respectively).

    Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    Effects of BPA on the ratios of Bcl2 family members. Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1 day with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Bcl2 , Bax , and Bad expression were measured using qPCR. Ratios of

    Journal:

    Article Title: Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries

    doi: 10.1016/j.reprotox.2015.05.012

    Figure Lengend Snippet: Effects of BPA on the ratios of Bcl2 family members. Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1 day with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Bcl2 , Bax , and Bad expression were measured using qPCR. Ratios of

    Article Snippet: A stock solution of BPA was dissolved and diluted in dimethyl sulfoxide (DMSO) (Sigma–Aldrich) to achieve the selected BPA treatment concentrations (0.13, 1.3, 6.65, and 13.3 mg/ml) for final working concentrations of 0.1, 1.0, 5.0, and 10 μg of BPA per ml of culture media (0.44, 4.4, 22, and 44 μM BPA, respectively).

    Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

    Effects of BPA on the mRNA expression level of Fas cell surface death receptor ( Fas ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Fas expression was measured

    Journal:

    Article Title: Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries

    doi: 10.1016/j.reprotox.2015.05.012

    Figure Lengend Snippet: Effects of BPA on the mRNA expression level of Fas cell surface death receptor ( Fas ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Fas expression was measured

    Article Snippet: A stock solution of BPA was dissolved and diluted in dimethyl sulfoxide (DMSO) (Sigma–Aldrich) to achieve the selected BPA treatment concentrations (0.13, 1.3, 6.65, and 13.3 mg/ml) for final working concentrations of 0.1, 1.0, 5.0, and 10 μg of BPA per ml of culture media (0.44, 4.4, 22, and 44 μM BPA, respectively).

    Techniques: Expressing, Cell Culture

    Effects of BPA on ROS levels in cultured neonatal ovaries. Neonatal ovaries were collected on postnatal day (PND) 0 and cultured with DMSO or BPA (0.1, 1, 5, and 10 μg/ml) for 2, 4, and 8 days and then subjected to in vitro ROS assays to measure

    Journal:

    Article Title: Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries

    doi: 10.1016/j.reprotox.2015.05.012

    Figure Lengend Snippet: Effects of BPA on ROS levels in cultured neonatal ovaries. Neonatal ovaries were collected on postnatal day (PND) 0 and cultured with DMSO or BPA (0.1, 1, 5, and 10 μg/ml) for 2, 4, and 8 days and then subjected to in vitro ROS assays to measure

    Article Snippet: A stock solution of BPA was dissolved and diluted in dimethyl sulfoxide (DMSO) (Sigma–Aldrich) to achieve the selected BPA treatment concentrations (0.13, 1.3, 6.65, and 13.3 mg/ml) for final working concentrations of 0.1, 1.0, 5.0, and 10 μg of BPA per ml of culture media (0.44, 4.4, 22, and 44 μM BPA, respectively).

    Techniques: Cell Culture, In Vitro

    Effects of BPA on percentage of germ cells and follicles in the ovary. Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 2, 4, and 8 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml), and subjected to histological evaluation

    Journal:

    Article Title: Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries

    doi: 10.1016/j.reprotox.2015.05.012

    Figure Lengend Snippet: Effects of BPA on percentage of germ cells and follicles in the ovary. Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 2, 4, and 8 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml), and subjected to histological evaluation

    Article Snippet: A stock solution of BPA was dissolved and diluted in dimethyl sulfoxide (DMSO) (Sigma–Aldrich) to achieve the selected BPA treatment concentrations (0.13, 1.3, 6.65, and 13.3 mg/ml) for final working concentrations of 0.1, 1.0, 5.0, and 10 μg of BPA per ml of culture media (0.44, 4.4, 22, and 44 μM BPA, respectively).

    Techniques: Cell Culture

    Effects of BPA on the mRNA expression level of catalase ( Cat ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Cat expression was measured in the cultured neonatal

    Journal:

    Article Title: Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries

    doi: 10.1016/j.reprotox.2015.05.012

    Figure Lengend Snippet: Effects of BPA on the mRNA expression level of catalase ( Cat ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Cat expression was measured in the cultured neonatal

    Article Snippet: A stock solution of BPA was dissolved and diluted in dimethyl sulfoxide (DMSO) (Sigma–Aldrich) to achieve the selected BPA treatment concentrations (0.13, 1.3, 6.65, and 13.3 mg/ml) for final working concentrations of 0.1, 1.0, 5.0, and 10 μg of BPA per ml of culture media (0.44, 4.4, 22, and 44 μM BPA, respectively).

    Techniques: Expressing, Cell Culture

    Effects of BPA on the mRNA expression level of glutathione peroxidase ( Gpx ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Gpx expression was measured in

    Journal:

    Article Title: Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries

    doi: 10.1016/j.reprotox.2015.05.012

    Figure Lengend Snippet: Effects of BPA on the mRNA expression level of glutathione peroxidase ( Gpx ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Gpx expression was measured in

    Article Snippet: A stock solution of BPA was dissolved and diluted in dimethyl sulfoxide (DMSO) (Sigma–Aldrich) to achieve the selected BPA treatment concentrations (0.13, 1.3, 6.65, and 13.3 mg/ml) for final working concentrations of 0.1, 1.0, 5.0, and 10 μg of BPA per ml of culture media (0.44, 4.4, 22, and 44 μM BPA, respectively).

    Techniques: Expressing, Cell Culture

    Effects of BPA on the mRNA expression level of B cell leukemia/lymphoma 2 ( Bcl2 ) and Bcl2-like 1 ( Bclxl ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Bcl2

    Journal:

    Article Title: Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries

    doi: 10.1016/j.reprotox.2015.05.012

    Figure Lengend Snippet: Effects of BPA on the mRNA expression level of B cell leukemia/lymphoma 2 ( Bcl2 ) and Bcl2-like 1 ( Bclxl ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Bcl2

    Article Snippet: A stock solution of BPA was dissolved and diluted in dimethyl sulfoxide (DMSO) (Sigma–Aldrich) to achieve the selected BPA treatment concentrations (0.13, 1.3, 6.65, and 13.3 mg/ml) for final working concentrations of 0.1, 1.0, 5.0, and 10 μg of BPA per ml of culture media (0.44, 4.4, 22, and 44 μM BPA, respectively).

    Techniques: Expressing, Cell Culture

    Effects of BPA on the mRNA expression level of glutathione reductase ( Gsr ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Gsr expression was measured in the

    Journal:

    Article Title: Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries

    doi: 10.1016/j.reprotox.2015.05.012

    Figure Lengend Snippet: Effects of BPA on the mRNA expression level of glutathione reductase ( Gsr ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Gsr expression was measured in the

    Article Snippet: A stock solution of BPA was dissolved and diluted in dimethyl sulfoxide (DMSO) (Sigma–Aldrich) to achieve the selected BPA treatment concentrations (0.13, 1.3, 6.65, and 13.3 mg/ml) for final working concentrations of 0.1, 1.0, 5.0, and 10 μg of BPA per ml of culture media (0.44, 4.4, 22, and 44 μM BPA, respectively).

    Techniques: Expressing, Cell Culture

    The inhibitor of BET proteins JQ1 inhibits STAT5 activity. Rested Ba/F3 cells were pre-treated 30 min with the indicated concentrations of JQ1, TSA or vehicle (0.02% DMSO in all conditions) and further stimulated 60 min with IL-3. Expression of STAT5 target ( Cis, c-Myc, Osm ) and control ( c-Fos, p21, 36b4 ) genes was evaluated by quantitative RT-PCR, as before.

    Journal: Nucleic Acids Research

    Article Title: Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function

    doi: 10.1093/nar/gkv188

    Figure Lengend Snippet: The inhibitor of BET proteins JQ1 inhibits STAT5 activity. Rested Ba/F3 cells were pre-treated 30 min with the indicated concentrations of JQ1, TSA or vehicle (0.02% DMSO in all conditions) and further stimulated 60 min with IL-3. Expression of STAT5 target ( Cis, c-Myc, Osm ) and control ( c-Fos, p21, 36b4 ) genes was evaluated by quantitative RT-PCR, as before.

    Article Snippet: Dimethyl sulfoxide (DMSO), TSA, MG132 and insulin were purchased from SIGMA (D-2650, T-8552, C-2211 and I-9278, respectively). (+)-JQ1 (BPS Bioscience #27401)—hereafter abbreviated to JQ1—was purchased from BIOMOL GmbH.

    Techniques: Activity Assay, Expressing, Quantitative RT-PCR

    STAT5 activity in Ba/F3 cells is differentially affected by class I-selective deacetylase inhibitors. Rested Ba/F3 cells were pre-treated with the indicated concentrations of the pan-inhibitor TSA and of the class I-selective deacetylase inhibitors VPA, apicidin, MGCD0103 and MS-275 for 15 min (TSA) or 30 min (all others), and further stimulated with IL-3 for 60 min, as described in Materials and Methods section. Final DMSO concentration was adjusted as detailed in legend to Supplementary Figure S2. Expression of STAT5 target ( Cis, c-Myc, Osm ) and control ( c-Fos, 36b4 ) genes was evaluated by quantitative RT-PCR as before. Data are expressed as mRNA levels in IL-3-stimulated cells relative to vehicle control. Non-normalized data are shown in Supplementary Figure S2.

    Journal: Nucleic Acids Research

    Article Title: Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function

    doi: 10.1093/nar/gkv188

    Figure Lengend Snippet: STAT5 activity in Ba/F3 cells is differentially affected by class I-selective deacetylase inhibitors. Rested Ba/F3 cells were pre-treated with the indicated concentrations of the pan-inhibitor TSA and of the class I-selective deacetylase inhibitors VPA, apicidin, MGCD0103 and MS-275 for 15 min (TSA) or 30 min (all others), and further stimulated with IL-3 for 60 min, as described in Materials and Methods section. Final DMSO concentration was adjusted as detailed in legend to Supplementary Figure S2. Expression of STAT5 target ( Cis, c-Myc, Osm ) and control ( c-Fos, 36b4 ) genes was evaluated by quantitative RT-PCR as before. Data are expressed as mRNA levels in IL-3-stimulated cells relative to vehicle control. Non-normalized data are shown in Supplementary Figure S2.

    Article Snippet: Dimethyl sulfoxide (DMSO), TSA, MG132 and insulin were purchased from SIGMA (D-2650, T-8552, C-2211 and I-9278, respectively). (+)-JQ1 (BPS Bioscience #27401)—hereafter abbreviated to JQ1—was purchased from BIOMOL GmbH.

    Techniques: Activity Assay, Histone Deacetylase Assay, Mass Spectrometry, Concentration Assay, Expressing, Quantitative RT-PCR

    Brd2 protein is present at the transcriptionally active Cis locus and is lost following both TSA and JQ1 treatment. ( A ) Rested Ba/F3 cells were either kept unstimulated (- IL-3; STAT5 activity off) or stimulated with IL-3 for 30 min (+ IL-3; STAT5 activity turned on). Brd2 ChIP was performed on sonified whole cell extracts from formaldehyde-cross-linked cells using 3 μg of either Brd2-specific antibodies or rabbit IgG (background control). Co-precipitated genomic DNA was analysed by quantitative PCR using the same Cis primers as in Figure 7 . ( B, C ) Ba/F3–1*6 cells were treated for 60 min with 200 nM TSA, 1 μM JQ1 or vehicle (0.02% DMSO). Brd2 ChIP was performed on sonified nuclear extracts from formaldehyde-cross-linked cells as described in Materials and Methods section using 3 μg of either Brd2-specific antibodies or rabbit IgG. Co-precipitated genomic DNA was analysed by quantitative PCR using the same primers as in panel A ( B ) or using primers specific for the transcription start site of STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes (Supplementary Table S1) ( C ).

    Journal: Nucleic Acids Research

    Article Title: Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function

    doi: 10.1093/nar/gkv188

    Figure Lengend Snippet: Brd2 protein is present at the transcriptionally active Cis locus and is lost following both TSA and JQ1 treatment. ( A ) Rested Ba/F3 cells were either kept unstimulated (- IL-3; STAT5 activity off) or stimulated with IL-3 for 30 min (+ IL-3; STAT5 activity turned on). Brd2 ChIP was performed on sonified whole cell extracts from formaldehyde-cross-linked cells using 3 μg of either Brd2-specific antibodies or rabbit IgG (background control). Co-precipitated genomic DNA was analysed by quantitative PCR using the same Cis primers as in Figure 7 . ( B, C ) Ba/F3–1*6 cells were treated for 60 min with 200 nM TSA, 1 μM JQ1 or vehicle (0.02% DMSO). Brd2 ChIP was performed on sonified nuclear extracts from formaldehyde-cross-linked cells as described in Materials and Methods section using 3 μg of either Brd2-specific antibodies or rabbit IgG. Co-precipitated genomic DNA was analysed by quantitative PCR using the same primers as in panel A ( B ) or using primers specific for the transcription start site of STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes (Supplementary Table S1) ( C ).

    Article Snippet: Dimethyl sulfoxide (DMSO), TSA, MG132 and insulin were purchased from SIGMA (D-2650, T-8552, C-2211 and I-9278, respectively). (+)-JQ1 (BPS Bioscience #27401)—hereafter abbreviated to JQ1—was purchased from BIOMOL GmbH.

    Techniques: Activity Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Lysine acetylation is not required for STAT5A-1*6 transcriptional activity in Ba/F3 cells. ( A–C ) Ba/F3 cells were transfected with empty pcDNA3 (-) or pcDNA3-based plasmids expressing wild-type mSTAT5A, constitutively active mSTAT5A-1*6 or mSTAT5A-1*6 mutants (K > Q, K > R and Y694F), as indicated. Transfected cells were maintained for 10 h in IL-3-free medium to prevent activation of endogenous STAT5. In (C), transfected cells were treated with 200 nM TSA or 0.02% DMSO (vehicle) for the last 90 min before harvest. Transgene expression and phosphorylation of STAT5 proteins were verified by western blot analysis of Brij whole-cell lysates using FLAG- and pSTAT5-specific antibodies, respectively. α-Tubulin was used as a loading control. Expression of the STAT5 target gene Cis was investigated by quantitative RT-PCR, as before. Of note, data not shown suggest that the weaker pSTAT5 signal consistently detected for the 3xQ mutant is likely due to epitope masking, probably as a result of multiple adjacent K > Q mutations surrounding the phospho-tyrosine residue. ( D ) Ba/F3-tet-on-1*6 cells were grown for 22 h in the absence or presence of 1 μg/ml doxycycline (Dox) to induce mSTAT5A-1*6 expression. Cells were kept in IL-3-containing medium for the first 12 h, washed twice in RPMI 1640 and further cultivated in IL-3-deprived medium for 10 h (±Dox, -IL-3) until harvest to inactivate endogenous STAT5 activity. As a positive control for endogenous STAT5 activity, non-induced cells were maintained in IL-3-containing medium (-Dox, +IL-3) for the duration of the experiment (22 h). Cytosolic and nuclear protein lysates were analysed by western blot to monitor endogenous nuclear STAT5B protein level (STAT5B) before and after induction of STAT5A-1*6 (FLAG). α-Tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to verify the quality of cell fractionation.

    Journal: Nucleic Acids Research

    Article Title: Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function

    doi: 10.1093/nar/gkv188

    Figure Lengend Snippet: Lysine acetylation is not required for STAT5A-1*6 transcriptional activity in Ba/F3 cells. ( A–C ) Ba/F3 cells were transfected with empty pcDNA3 (-) or pcDNA3-based plasmids expressing wild-type mSTAT5A, constitutively active mSTAT5A-1*6 or mSTAT5A-1*6 mutants (K > Q, K > R and Y694F), as indicated. Transfected cells were maintained for 10 h in IL-3-free medium to prevent activation of endogenous STAT5. In (C), transfected cells were treated with 200 nM TSA or 0.02% DMSO (vehicle) for the last 90 min before harvest. Transgene expression and phosphorylation of STAT5 proteins were verified by western blot analysis of Brij whole-cell lysates using FLAG- and pSTAT5-specific antibodies, respectively. α-Tubulin was used as a loading control. Expression of the STAT5 target gene Cis was investigated by quantitative RT-PCR, as before. Of note, data not shown suggest that the weaker pSTAT5 signal consistently detected for the 3xQ mutant is likely due to epitope masking, probably as a result of multiple adjacent K > Q mutations surrounding the phospho-tyrosine residue. ( D ) Ba/F3-tet-on-1*6 cells were grown for 22 h in the absence or presence of 1 μg/ml doxycycline (Dox) to induce mSTAT5A-1*6 expression. Cells were kept in IL-3-containing medium for the first 12 h, washed twice in RPMI 1640 and further cultivated in IL-3-deprived medium for 10 h (±Dox, -IL-3) until harvest to inactivate endogenous STAT5 activity. As a positive control for endogenous STAT5 activity, non-induced cells were maintained in IL-3-containing medium (-Dox, +IL-3) for the duration of the experiment (22 h). Cytosolic and nuclear protein lysates were analysed by western blot to monitor endogenous nuclear STAT5B protein level (STAT5B) before and after induction of STAT5A-1*6 (FLAG). α-Tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to verify the quality of cell fractionation.

    Article Snippet: Dimethyl sulfoxide (DMSO), TSA, MG132 and insulin were purchased from SIGMA (D-2650, T-8552, C-2211 and I-9278, respectively). (+)-JQ1 (BPS Bioscience #27401)—hereafter abbreviated to JQ1—was purchased from BIOMOL GmbH.

    Techniques: Activity Assay, Transfection, Expressing, Activation Assay, Western Blot, Quantitative RT-PCR, Mutagenesis, Positive Control, Cell Fractionation

    Histone occupancy is reduced and histone acetylation is altered all along the Cis gene upon TSA treatment. Rested Ba/F3 cells were pre-treated 30 min with 200 nM TSA or 0.02% DMSO (vehicle) and further stimulated 30 min with IL-3 before being processed for ChIP using antibodies directed against total histone H3, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4). Co-precipitated genomic DNA was analysed by quantitative PCR using primers specific for the -800 to +4000 Cis gene locus (Supplementary Table S1). Positions of amplicons investigated and of the four STAT5 binding sites arranged in two clusters are indicated along the x axis. Non-normalized Ac-H3 and Ac-H4 ChIP data are shown in Supplementary Figure S6.

    Journal: Nucleic Acids Research

    Article Title: Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function

    doi: 10.1093/nar/gkv188

    Figure Lengend Snippet: Histone occupancy is reduced and histone acetylation is altered all along the Cis gene upon TSA treatment. Rested Ba/F3 cells were pre-treated 30 min with 200 nM TSA or 0.02% DMSO (vehicle) and further stimulated 30 min with IL-3 before being processed for ChIP using antibodies directed against total histone H3, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4). Co-precipitated genomic DNA was analysed by quantitative PCR using primers specific for the -800 to +4000 Cis gene locus (Supplementary Table S1). Positions of amplicons investigated and of the four STAT5 binding sites arranged in two clusters are indicated along the x axis. Non-normalized Ac-H3 and Ac-H4 ChIP data are shown in Supplementary Figure S6.

    Article Snippet: Dimethyl sulfoxide (DMSO), TSA, MG132 and insulin were purchased from SIGMA (D-2650, T-8552, C-2211 and I-9278, respectively). (+)-JQ1 (BPS Bioscience #27401)—hereafter abbreviated to JQ1—was purchased from BIOMOL GmbH.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    Alterations in histone acetylation by TSA at different gene loci differentially affect gene activation. Rested Ba/F3 cells were pre-treated 30 min with 200 nM TSA or 0.02% DMSO (vehicle) and further stimulated 30 min with IL-3 before being processed for ChIP using antibodies directed against RNA polymerase II (RNA Pol II), histone H3, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4), as before. Co-precipitated genomic DNA was analysed by quantitative PCR at STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes, using the same primers as in Figure 6 . Non-normalized Ac-H3 and Ac-H4 ChIP data are shown in Supplementary Figure S7.

    Journal: Nucleic Acids Research

    Article Title: Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function

    doi: 10.1093/nar/gkv188

    Figure Lengend Snippet: Alterations in histone acetylation by TSA at different gene loci differentially affect gene activation. Rested Ba/F3 cells were pre-treated 30 min with 200 nM TSA or 0.02% DMSO (vehicle) and further stimulated 30 min with IL-3 before being processed for ChIP using antibodies directed against RNA polymerase II (RNA Pol II), histone H3, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4), as before. Co-precipitated genomic DNA was analysed by quantitative PCR at STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes, using the same primers as in Figure 6 . Non-normalized Ac-H3 and Ac-H4 ChIP data are shown in Supplementary Figure S7.

    Article Snippet: Dimethyl sulfoxide (DMSO), TSA, MG132 and insulin were purchased from SIGMA (D-2650, T-8552, C-2211 and I-9278, respectively). (+)-JQ1 (BPS Bioscience #27401)—hereafter abbreviated to JQ1—was purchased from BIOMOL GmbH.

    Techniques: Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Histone occupancy is reduced at promoters of both STAT5-dependent and -independent genes upon TSA treatment. Ba/F3 cells were withdrawn from IL-3 for 10 h and treated with 200 nM TSA or 0.02% DMSO (vehicle) for 5, 15, 30 and 60 min. As a control for IL-3 stimulation, Ba/F3 cells were stimulated with IL-3 for 30 min following a 30 min TSA pre-treatment (hence treated 60 min with TSA). Cells were harvested and processed for ChIP, as described in Materials and Methods section. ChIP was performed using antibodies directed against total histone H3 or RNA polymerase II (RNA Pol II). Co-precipitated genomic DNA was analysed by quantitative PCR using primers specific for proximal promoter regions (histone H3 ChIP) or transcription start sites (RNA Pol II ChIP) of STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes. Cis and Osm promoter amplicons overlap their respective STAT5-responsive elements (Supplementary Table S1).

    Journal: Nucleic Acids Research

    Article Title: Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function

    doi: 10.1093/nar/gkv188

    Figure Lengend Snippet: Histone occupancy is reduced at promoters of both STAT5-dependent and -independent genes upon TSA treatment. Ba/F3 cells were withdrawn from IL-3 for 10 h and treated with 200 nM TSA or 0.02% DMSO (vehicle) for 5, 15, 30 and 60 min. As a control for IL-3 stimulation, Ba/F3 cells were stimulated with IL-3 for 30 min following a 30 min TSA pre-treatment (hence treated 60 min with TSA). Cells were harvested and processed for ChIP, as described in Materials and Methods section. ChIP was performed using antibodies directed against total histone H3 or RNA polymerase II (RNA Pol II). Co-precipitated genomic DNA was analysed by quantitative PCR using primers specific for proximal promoter regions (histone H3 ChIP) or transcription start sites (RNA Pol II ChIP) of STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes. Cis and Osm promoter amplicons overlap their respective STAT5-responsive elements (Supplementary Table S1).

    Article Snippet: Dimethyl sulfoxide (DMSO), TSA, MG132 and insulin were purchased from SIGMA (D-2650, T-8552, C-2211 and I-9278, respectively). (+)-JQ1 (BPS Bioscience #27401)—hereafter abbreviated to JQ1—was purchased from BIOMOL GmbH.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    The nuclear BET protein Brd2 is rapidly relocalized to the chromatin fraction together with acetylated histones upon treatment with deacetylase inhibitors that inhibit STAT5 activity. ( A ) Ba/F3 cells were treated with 200 nM TSA, 10 μM MG132, both (T+MG) or vehicle (0.12% DMSO final in all conditions). Cells were treated for a total duration of ∼2 h, with a 45 min MG132 pre-treatment followed by a 90 min TSA treatment. Soluble and insoluble nuclear fractions were analysed by western blot using antibodies against Brd2 and the nuclear marker HDAC1 as loading control. Similar results were obtained upon 3 h pre-treatment with 10 μM MG132 followed by 90 min incubation with 200 nM TSA (4.5 h treatment in total; not shown). ( B ) Ba/F3 cells were rested for 6 h and stimulated with IL-3 for 60 min. Prior to IL-3 stimulation, cells were pre-treated 3.5 h with 10 μM MG132, 30 min with 200 nM TSA, both (3h MG132 followed by 30 min TSA) or vehicle (0.12% DMSO final in all conditions). Hence cells were incubated 4.5 h in total with 10 μM MG132 and 90 min with 200 nM TSA. Expression of the STAT5 target gene Cis , of the MG132-regulated gene hsp70 and of Brd2 and 36b4 genes as controls was monitored by quantitative RT-PCR. ( C ) Ba/F3 cells were treated for the indicated times with 200 nM TSA, 3 mM valproic acid (VPA), 500 nM apicidin (Api.), 1 μM MGCD0103 (MGC), 5 μM MS-275 (MS), or vehicle (Veh.; 0.02% DMSO final in all conditions). Cytosolic as well as soluble and insoluble nuclear fractions were analysed by western blot using the indicated antibodies. As before, α-tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to control cell fractionation.

    Journal: Nucleic Acids Research

    Article Title: Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function

    doi: 10.1093/nar/gkv188

    Figure Lengend Snippet: The nuclear BET protein Brd2 is rapidly relocalized to the chromatin fraction together with acetylated histones upon treatment with deacetylase inhibitors that inhibit STAT5 activity. ( A ) Ba/F3 cells were treated with 200 nM TSA, 10 μM MG132, both (T+MG) or vehicle (0.12% DMSO final in all conditions). Cells were treated for a total duration of ∼2 h, with a 45 min MG132 pre-treatment followed by a 90 min TSA treatment. Soluble and insoluble nuclear fractions were analysed by western blot using antibodies against Brd2 and the nuclear marker HDAC1 as loading control. Similar results were obtained upon 3 h pre-treatment with 10 μM MG132 followed by 90 min incubation with 200 nM TSA (4.5 h treatment in total; not shown). ( B ) Ba/F3 cells were rested for 6 h and stimulated with IL-3 for 60 min. Prior to IL-3 stimulation, cells were pre-treated 3.5 h with 10 μM MG132, 30 min with 200 nM TSA, both (3h MG132 followed by 30 min TSA) or vehicle (0.12% DMSO final in all conditions). Hence cells were incubated 4.5 h in total with 10 μM MG132 and 90 min with 200 nM TSA. Expression of the STAT5 target gene Cis , of the MG132-regulated gene hsp70 and of Brd2 and 36b4 genes as controls was monitored by quantitative RT-PCR. ( C ) Ba/F3 cells were treated for the indicated times with 200 nM TSA, 3 mM valproic acid (VPA), 500 nM apicidin (Api.), 1 μM MGCD0103 (MGC), 5 μM MS-275 (MS), or vehicle (Veh.; 0.02% DMSO final in all conditions). Cytosolic as well as soluble and insoluble nuclear fractions were analysed by western blot using the indicated antibodies. As before, α-tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to control cell fractionation.

    Article Snippet: Dimethyl sulfoxide (DMSO), TSA, MG132 and insulin were purchased from SIGMA (D-2650, T-8552, C-2211 and I-9278, respectively). (+)-JQ1 (BPS Bioscience #27401)—hereafter abbreviated to JQ1—was purchased from BIOMOL GmbH.

    Techniques: Histone Deacetylase Assay, Activity Assay, Western Blot, Marker, Incubation, Expressing, Quantitative RT-PCR, Mass Spectrometry, Cell Fractionation

    BPD and TFP suppress the growth of TNBC tumors in the mouse tumor model ( A – B ) MDA-MB-231 cells were injected into female nude mice ( n = 3/group). The tumor-bearing mice were given an i.v. injection of (A) BPD (10 mg/kg) or the vehicle control (DMSO), and (B) TFP (10 mg/kg) or DMSO twice per week. The tumor volumes [(L × W 2 )/2] were measured. The results are graphed as mean ± SEM numbers of tumor volumes. Significant P values are shown. * P

    Journal: Oncotarget

    Article Title: Pharmacological activation of FOXO3 suppresses triple-negative breast cancer in vitro and in vivo

    doi: 10.18632/oncotarget.9881

    Figure Lengend Snippet: BPD and TFP suppress the growth of TNBC tumors in the mouse tumor model ( A – B ) MDA-MB-231 cells were injected into female nude mice ( n = 3/group). The tumor-bearing mice were given an i.v. injection of (A) BPD (10 mg/kg) or the vehicle control (DMSO), and (B) TFP (10 mg/kg) or DMSO twice per week. The tumor volumes [(L × W 2 )/2] were measured. The results are graphed as mean ± SEM numbers of tumor volumes. Significant P values are shown. * P

    Article Snippet: TFP, dimethylsulfoxide (DMSO), 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 3,3′,5,5′-Tetramethylbenzidine (TMB) were purchased from Sigma (St. Louis, MO).

    Techniques: Multiple Displacement Amplification, Injection, Mouse Assay

    TFP and BPD significantly reduce the expression of oncogenic c-Myc, KLF5, and DRD2 in TNBC cells in a FOXO3-dependent manner ( A ) BT549 cells were transfected with control-siRNA or FOXO3-siRNA for 24 hours, transfected cells were treated with TFP, BPD or DMSO (control) for 48 hours, harvested, and fractionated for preparing cytoplasmic and nuclear extracts. Equal amount of each fraction was analyzed by immunoblotting (IB) analysis with the specific antibodies as indicated. Proteins β-Actin and PARP1 represent the fractionation and loading controls of the cytosolic and nuclear fractions, respectively. ( B ) A diagram represents the model for the FOXO3-dependent anticancer function of BPD and TFP. A schematic shows that BPD and TFP inhibit phosphor-Akt (pAkt) and lead to FOXO3 translocation from the cytoplasm into the nucleus, where FOXO3 can upregulate the expression of p27Kip1, Bax, and Bim proapoptotic proteins [ 17 , 19 , 41 ]. Moreover, BPD and TFP downregulate of the expression of c-Myc, KLF5 and DRD2 oncogenic survival proteins in a FOXO3-dependent manner. As a result of this FOXO3-mediated apoptotic pathway, BPD and TFP promote TNBC cell apoptosis.

    Journal: Oncotarget

    Article Title: Pharmacological activation of FOXO3 suppresses triple-negative breast cancer in vitro and in vivo

    doi: 10.18632/oncotarget.9881

    Figure Lengend Snippet: TFP and BPD significantly reduce the expression of oncogenic c-Myc, KLF5, and DRD2 in TNBC cells in a FOXO3-dependent manner ( A ) BT549 cells were transfected with control-siRNA or FOXO3-siRNA for 24 hours, transfected cells were treated with TFP, BPD or DMSO (control) for 48 hours, harvested, and fractionated for preparing cytoplasmic and nuclear extracts. Equal amount of each fraction was analyzed by immunoblotting (IB) analysis with the specific antibodies as indicated. Proteins β-Actin and PARP1 represent the fractionation and loading controls of the cytosolic and nuclear fractions, respectively. ( B ) A diagram represents the model for the FOXO3-dependent anticancer function of BPD and TFP. A schematic shows that BPD and TFP inhibit phosphor-Akt (pAkt) and lead to FOXO3 translocation from the cytoplasm into the nucleus, where FOXO3 can upregulate the expression of p27Kip1, Bax, and Bim proapoptotic proteins [ 17 , 19 , 41 ]. Moreover, BPD and TFP downregulate of the expression of c-Myc, KLF5 and DRD2 oncogenic survival proteins in a FOXO3-dependent manner. As a result of this FOXO3-mediated apoptotic pathway, BPD and TFP promote TNBC cell apoptosis.

    Article Snippet: TFP, dimethylsulfoxide (DMSO), 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 3,3′,5,5′-Tetramethylbenzidine (TMB) were purchased from Sigma (St. Louis, MO).

    Techniques: Expressing, Transfection, Fractionation, Translocation Assay

    The aberrant expression of oncogenic survival proteins in TNBC tumors and the effect of TFP or BPD on these proteins in TNBC cells ( A – B ) Oncoprint analysis data of Akt2, Akt3, c-Myc (MYC), Kruppel-like factor 5 (KLF5), and dopamine receptor D2 (DRD2) in TNBC patient samples are displayed. Data from the TCGA dataset containing molecular subtyping data of 123 TNBC subtype (A) and 104 non-TNBC subtype (B) were extracted and used to generate an oncoprint plot using the cBioPortal [ 39 , 40 ]. Gene amplification (red bars) and mRNA copy number gain (pink bars) are shown. ( C – D ) BT549 cells were treated with a dose response of TFP (C) or BPD (D) or DMSO control (denoted as 0 μM) for 24 hours, harvested, and fractionated for preparing cytoplasmic (Cyt.) and nuclear (Nuc.) extracts. Equal amount of each fraction was analyzed by immunoblotting (IB) analysis with specific antibodies as indicated. While α-Tubulin and GAPDH represent the loading controls of the Cyt. extract, Lamin A/C displays the loading control of the Nuc. Extract.

    Journal: Oncotarget

    Article Title: Pharmacological activation of FOXO3 suppresses triple-negative breast cancer in vitro and in vivo

    doi: 10.18632/oncotarget.9881

    Figure Lengend Snippet: The aberrant expression of oncogenic survival proteins in TNBC tumors and the effect of TFP or BPD on these proteins in TNBC cells ( A – B ) Oncoprint analysis data of Akt2, Akt3, c-Myc (MYC), Kruppel-like factor 5 (KLF5), and dopamine receptor D2 (DRD2) in TNBC patient samples are displayed. Data from the TCGA dataset containing molecular subtyping data of 123 TNBC subtype (A) and 104 non-TNBC subtype (B) were extracted and used to generate an oncoprint plot using the cBioPortal [ 39 , 40 ]. Gene amplification (red bars) and mRNA copy number gain (pink bars) are shown. ( C – D ) BT549 cells were treated with a dose response of TFP (C) or BPD (D) or DMSO control (denoted as 0 μM) for 24 hours, harvested, and fractionated for preparing cytoplasmic (Cyt.) and nuclear (Nuc.) extracts. Equal amount of each fraction was analyzed by immunoblotting (IB) analysis with specific antibodies as indicated. While α-Tubulin and GAPDH represent the loading controls of the Cyt. extract, Lamin A/C displays the loading control of the Nuc. Extract.

    Article Snippet: TFP, dimethylsulfoxide (DMSO), 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 3,3′,5,5′-Tetramethylbenzidine (TMB) were purchased from Sigma (St. Louis, MO).

    Techniques: Expressing, Amplification

    TFP or BPD treatment significantly promotes nuclear translocation and activation of endogenous FOXO3 in TNBC cells ( A – B ) MDA-MB-231 cells were treated with a dose response of TFP (A) or BPD (B) or dimethylsulfoxide (DMSO) control (denoted as 0 μM) for 24 hours, harvested, and fractionated for preparing cytoplasmic (Cyt.) and nuclear (Nuc.) extracts. Equal amount of each fraction was analyzed by immunoblotting (IB) analysis with specific antibodies as indicated. β-actin and PARP1 represent the fractionation and loading controls of the Cyt. and the Nuc. fractions, respectively. All these results shown above represented 3 independent experiments. ( C – D ) BT549 cells were treated with control (DMSO) or TFP (5 μM) or BPD (5 μM) for 24 hours. The treated cells were fixed and the subcellular localizations of endogenous FOXO3 and p53-pS15, a DNA damage marker, were detected using antibodies against FOXO3 and p53-pS15 and followed by an Alexa Fluor 594 (red)- or Alexa Fluor 488(green)-conjugated secondary antibody, and fluorescence microscopy. DAPI was used to show the nuclei, and co-localization of FOXO3 with p53-pS15 was shown as the merged images (orange or yellow color).

    Journal: Oncotarget

    Article Title: Pharmacological activation of FOXO3 suppresses triple-negative breast cancer in vitro and in vivo

    doi: 10.18632/oncotarget.9881

    Figure Lengend Snippet: TFP or BPD treatment significantly promotes nuclear translocation and activation of endogenous FOXO3 in TNBC cells ( A – B ) MDA-MB-231 cells were treated with a dose response of TFP (A) or BPD (B) or dimethylsulfoxide (DMSO) control (denoted as 0 μM) for 24 hours, harvested, and fractionated for preparing cytoplasmic (Cyt.) and nuclear (Nuc.) extracts. Equal amount of each fraction was analyzed by immunoblotting (IB) analysis with specific antibodies as indicated. β-actin and PARP1 represent the fractionation and loading controls of the Cyt. and the Nuc. fractions, respectively. All these results shown above represented 3 independent experiments. ( C – D ) BT549 cells were treated with control (DMSO) or TFP (5 μM) or BPD (5 μM) for 24 hours. The treated cells were fixed and the subcellular localizations of endogenous FOXO3 and p53-pS15, a DNA damage marker, were detected using antibodies against FOXO3 and p53-pS15 and followed by an Alexa Fluor 594 (red)- or Alexa Fluor 488(green)-conjugated secondary antibody, and fluorescence microscopy. DAPI was used to show the nuclei, and co-localization of FOXO3 with p53-pS15 was shown as the merged images (orange or yellow color).

    Article Snippet: TFP, dimethylsulfoxide (DMSO), 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 3,3′,5,5′-Tetramethylbenzidine (TMB) were purchased from Sigma (St. Louis, MO).

    Techniques: Translocation Assay, Activation Assay, Multiple Displacement Amplification, Fractionation, Marker, Fluorescence, Microscopy

    The cytotoxic effect of TFP and BPD on human TNBC cells The dose effects of TFP or BPD on cell viability of MDA-MB-231 ( A or C ) and BT549 ( B or D ) cells were determined. Cells were cultured in a 96-well plate (2,000 cells/well), treated with various doses of TFP or BPD as indicated, and incubated for 72 hours. The cell viability was determined by the MTT assay and the relative cell survival rate percentage was calculated by dividing the optical density of each drug treatment by that of the control (DMSO) treatment. Each data point represents the mean value from 3 independent experiments, with at least three replicates. The error bars represent standard deviation by paired t -test. The significant P -values between the treatment group versus the control group are indicated (* P

    Journal: Oncotarget

    Article Title: Pharmacological activation of FOXO3 suppresses triple-negative breast cancer in vitro and in vivo

    doi: 10.18632/oncotarget.9881

    Figure Lengend Snippet: The cytotoxic effect of TFP and BPD on human TNBC cells The dose effects of TFP or BPD on cell viability of MDA-MB-231 ( A or C ) and BT549 ( B or D ) cells were determined. Cells were cultured in a 96-well plate (2,000 cells/well), treated with various doses of TFP or BPD as indicated, and incubated for 72 hours. The cell viability was determined by the MTT assay and the relative cell survival rate percentage was calculated by dividing the optical density of each drug treatment by that of the control (DMSO) treatment. Each data point represents the mean value from 3 independent experiments, with at least three replicates. The error bars represent standard deviation by paired t -test. The significant P -values between the treatment group versus the control group are indicated (* P

    Article Snippet: TFP, dimethylsulfoxide (DMSO), 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 3,3′,5,5′-Tetramethylbenzidine (TMB) were purchased from Sigma (St. Louis, MO).

    Techniques: Multiple Displacement Amplification, Cell Culture, Incubation, MTT Assay, Standard Deviation

    BPD and TFP promote cellular apoptosis in TNBC cells ( A – B ) MDA-MB-231 cells were treated with TFP or BPD or DMSO (negative control) or camptothecin (CPT) (positive control) for 48 hours, harvested, and were subjected to Annexin V and propidium iodide staining for determining apoptosis. (B) An average (%) of Annexin V-positive cells (apoptotic) cells was determined and shown in the diagram. Each data point represents the mean value from 3 independent experiments. The error bars represent standard deviation (SD). P values between the test-group treated with drugs versus the control-group treated with DMSO are shown. ( C – E ) MDA-MB-231 cells were treated with TFP or BPD or DMSO for 48 hours, and the cells were fixed on the slides for determining apoptosis by TUNEL assay. Nuclei were stained with DAPI (color-inverted to red), and merged images (yellow) were considered as apoptotic cells. Scale bar: 20 μm. (D, E) An average (%) of apoptotic (TUNEL-positive) cells was determined and shown in the diagram. The samples include three biological replicates. The error bars represent SD, and P values are shown. ( F – G ) MDA-MB-231 (F) or BT549 (G) cells were treated with TFP or BPD or DMSO for 48 hours. Total lysates of cells were analyzed by Immunoblotting analysis with specific antibodies against full-length PARP1 protein or cleaved PARP1 fragment (89 kDa) as indicated. β-actin represents the loading controls.

    Journal: Oncotarget

    Article Title: Pharmacological activation of FOXO3 suppresses triple-negative breast cancer in vitro and in vivo

    doi: 10.18632/oncotarget.9881

    Figure Lengend Snippet: BPD and TFP promote cellular apoptosis in TNBC cells ( A – B ) MDA-MB-231 cells were treated with TFP or BPD or DMSO (negative control) or camptothecin (CPT) (positive control) for 48 hours, harvested, and were subjected to Annexin V and propidium iodide staining for determining apoptosis. (B) An average (%) of Annexin V-positive cells (apoptotic) cells was determined and shown in the diagram. Each data point represents the mean value from 3 independent experiments. The error bars represent standard deviation (SD). P values between the test-group treated with drugs versus the control-group treated with DMSO are shown. ( C – E ) MDA-MB-231 cells were treated with TFP or BPD or DMSO for 48 hours, and the cells were fixed on the slides for determining apoptosis by TUNEL assay. Nuclei were stained with DAPI (color-inverted to red), and merged images (yellow) were considered as apoptotic cells. Scale bar: 20 μm. (D, E) An average (%) of apoptotic (TUNEL-positive) cells was determined and shown in the diagram. The samples include three biological replicates. The error bars represent SD, and P values are shown. ( F – G ) MDA-MB-231 (F) or BT549 (G) cells were treated with TFP or BPD or DMSO for 48 hours. Total lysates of cells were analyzed by Immunoblotting analysis with specific antibodies against full-length PARP1 protein or cleaved PARP1 fragment (89 kDa) as indicated. β-actin represents the loading controls.

    Article Snippet: TFP, dimethylsulfoxide (DMSO), 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 3,3′,5,5′-Tetramethylbenzidine (TMB) were purchased from Sigma (St. Louis, MO).

    Techniques: Multiple Displacement Amplification, Negative Control, Cycling Probe Technology, Positive Control, Staining, Standard Deviation, TUNEL Assay

    TFP and BPD suppress the colony-forming ability of TNBC cells in a FOXO3-dependent manner ( A – B ) (A) BT549 cells were transfected with control-siRNA or FOXO3-siRNA for 48 h. The indicated proteins were detected by immunoblotting with specific antibodies (Abs) against FOXO3 and GAPDH (loading control). (B) The transfected cells were treated with TFP, BPD or DMSO (negative control) for 14 days and stained with crystal violet solution. Top (upper panels): the representative pictures of the assays are shown. Bottom (lower panels): the numbers of colonies in the drug (TFP or BPD)-treated plates were compared with those of the DMSO-treated plates. The results are graphed as mean ± SEM numbers of cell colonies. The number of biological replicates is three. * P

    Journal: Oncotarget

    Article Title: Pharmacological activation of FOXO3 suppresses triple-negative breast cancer in vitro and in vivo

    doi: 10.18632/oncotarget.9881

    Figure Lengend Snippet: TFP and BPD suppress the colony-forming ability of TNBC cells in a FOXO3-dependent manner ( A – B ) (A) BT549 cells were transfected with control-siRNA or FOXO3-siRNA for 48 h. The indicated proteins were detected by immunoblotting with specific antibodies (Abs) against FOXO3 and GAPDH (loading control). (B) The transfected cells were treated with TFP, BPD or DMSO (negative control) for 14 days and stained with crystal violet solution. Top (upper panels): the representative pictures of the assays are shown. Bottom (lower panels): the numbers of colonies in the drug (TFP or BPD)-treated plates were compared with those of the DMSO-treated plates. The results are graphed as mean ± SEM numbers of cell colonies. The number of biological replicates is three. * P

    Article Snippet: TFP, dimethylsulfoxide (DMSO), 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 3,3′,5,5′-Tetramethylbenzidine (TMB) were purchased from Sigma (St. Louis, MO).

    Techniques: Transfection, Negative Control, Staining

    P4-CM increased neuronal survival against oxidative stress. Differentiated SH-SY5Y cells were exposed to no-treatment (control), BDNF, DMSO-CM, or P4-CM in the presence or absence of Y1036 for 24 hours. All groups were then exposed to 100μM H

    Journal:

    Article Title: Pgrmc1/BDNF Signaling Plays a Critical Role in Mediating Glia-Neuron Cross Talk

    doi: 10.1210/en.2015-1610

    Figure Lengend Snippet: P4-CM increased neuronal survival against oxidative stress. Differentiated SH-SY5Y cells were exposed to no-treatment (control), BDNF, DMSO-CM, or P4-CM in the presence or absence of Y1036 for 24 hours. All groups were then exposed to 100μM H

    Article Snippet: C6 cells were propagated in DMEM (Invitrogen Life Technologies) supplemented with 15% fetal bovine serum (noncharcoal-stripped) (HyClone) and maintained at 37°C in a humidified environment containing 5% CO2 for 24 hours, then treated with vehicle control-dimethylsulfoxide (DMSO) (0.1%), P4 (Sigma-Aldrich) at 10nM for the indicated time.

    Techniques:

    P4-CM triggers synaptic marker expression in mouse hippocampal neurons. Mouse primary hippocampal neurons were exposed with nontreated control, BDNF, DMSO-CM, or P4-CM for 24 hours at DIV 7. Double-label immunofluorescence staining shows that expression

    Journal:

    Article Title: Pgrmc1/BDNF Signaling Plays a Critical Role in Mediating Glia-Neuron Cross Talk

    doi: 10.1210/en.2015-1610

    Figure Lengend Snippet: P4-CM triggers synaptic marker expression in mouse hippocampal neurons. Mouse primary hippocampal neurons were exposed with nontreated control, BDNF, DMSO-CM, or P4-CM for 24 hours at DIV 7. Double-label immunofluorescence staining shows that expression

    Article Snippet: C6 cells were propagated in DMEM (Invitrogen Life Technologies) supplemented with 15% fetal bovine serum (noncharcoal-stripped) (HyClone) and maintained at 37°C in a humidified environment containing 5% CO2 for 24 hours, then treated with vehicle control-dimethylsulfoxide (DMSO) (0.1%), P4 (Sigma-Aldrich) at 10nM for the indicated time.

    Techniques: Marker, Expressing, Immunofluorescence, Staining

    Effect of glial-CM on the expression of synaptic markers. SH-SY5Y human neuroblastoma cells were differentiated with RA for 7 days and were exposed to BDNF, DMSO-CM, or P4-CM in the presence or absence of Y1036 for 24 hours. Nontreated cultures were used

    Journal:

    Article Title: Pgrmc1/BDNF Signaling Plays a Critical Role in Mediating Glia-Neuron Cross Talk

    doi: 10.1210/en.2015-1610

    Figure Lengend Snippet: Effect of glial-CM on the expression of synaptic markers. SH-SY5Y human neuroblastoma cells were differentiated with RA for 7 days and were exposed to BDNF, DMSO-CM, or P4-CM in the presence or absence of Y1036 for 24 hours. Nontreated cultures were used

    Article Snippet: C6 cells were propagated in DMEM (Invitrogen Life Technologies) supplemented with 15% fetal bovine serum (noncharcoal-stripped) (HyClone) and maintained at 37°C in a humidified environment containing 5% CO2 for 24 hours, then treated with vehicle control-dimethylsulfoxide (DMSO) (0.1%), P4 (Sigma-Aldrich) at 10nM for the indicated time.

    Techniques: Expressing

    P4-CM induced morphological change in SH-SY5Y cells. SH-SY5Y human neuroblastoma cells were differentiated with RA for 7 days and formed a network of long and smooth neuritis (long arrows) at 24 hours exposed with nontreated control, BDNF, DMSO-CM, or

    Journal:

    Article Title: Pgrmc1/BDNF Signaling Plays a Critical Role in Mediating Glia-Neuron Cross Talk

    doi: 10.1210/en.2015-1610

    Figure Lengend Snippet: P4-CM induced morphological change in SH-SY5Y cells. SH-SY5Y human neuroblastoma cells were differentiated with RA for 7 days and formed a network of long and smooth neuritis (long arrows) at 24 hours exposed with nontreated control, BDNF, DMSO-CM, or

    Article Snippet: C6 cells were propagated in DMEM (Invitrogen Life Technologies) supplemented with 15% fetal bovine serum (noncharcoal-stripped) (HyClone) and maintained at 37°C in a humidified environment containing 5% CO2 for 24 hours, then treated with vehicle control-dimethylsulfoxide (DMSO) (0.1%), P4 (Sigma-Aldrich) at 10nM for the indicated time.

    Techniques:

    Blocking of BDNF signaling inhibited glial P4-CM-induced GAP43 density in both proximal and distal neuritic segments of SH-SY5Y cells. A, Immunocytochemistry of SH-SY5Y cultures. Cells were treated with DMSO-CM or P4-CM in the presence of human IgG control

    Journal:

    Article Title: Pgrmc1/BDNF Signaling Plays a Critical Role in Mediating Glia-Neuron Cross Talk

    doi: 10.1210/en.2015-1610

    Figure Lengend Snippet: Blocking of BDNF signaling inhibited glial P4-CM-induced GAP43 density in both proximal and distal neuritic segments of SH-SY5Y cells. A, Immunocytochemistry of SH-SY5Y cultures. Cells were treated with DMSO-CM or P4-CM in the presence of human IgG control

    Article Snippet: C6 cells were propagated in DMEM (Invitrogen Life Technologies) supplemented with 15% fetal bovine serum (noncharcoal-stripped) (HyClone) and maintained at 37°C in a humidified environment containing 5% CO2 for 24 hours, then treated with vehicle control-dimethylsulfoxide (DMSO) (0.1%), P4 (Sigma-Aldrich) at 10nM for the indicated time.

    Techniques: Blocking Assay, Immunocytochemistry

    P4, when directly added into differentiated SH-SY5Ycells, did not affect the mRNA levels of GAP43 and SYP. P4 (diluted to a final concentration of 0.1nM, 1nM, 10nM, or 100nM in DMSO) was added into differentiated SH-SY5Y cultures for 24 hours. The same

    Journal:

    Article Title: Pgrmc1/BDNF Signaling Plays a Critical Role in Mediating Glia-Neuron Cross Talk

    doi: 10.1210/en.2015-1610

    Figure Lengend Snippet: P4, when directly added into differentiated SH-SY5Ycells, did not affect the mRNA levels of GAP43 and SYP. P4 (diluted to a final concentration of 0.1nM, 1nM, 10nM, or 100nM in DMSO) was added into differentiated SH-SY5Y cultures for 24 hours. The same

    Article Snippet: C6 cells were propagated in DMEM (Invitrogen Life Technologies) supplemented with 15% fetal bovine serum (noncharcoal-stripped) (HyClone) and maintained at 37°C in a humidified environment containing 5% CO2 for 24 hours, then treated with vehicle control-dimethylsulfoxide (DMSO) (0.1%), P4 (Sigma-Aldrich) at 10nM for the indicated time.

    Techniques: Concentration Assay

    Blocking of BDNF signaling inhibited glial P4-CM-induced GAP43 density in both proximal and distal neuritic segments of primary hippocampus neurons. A, Immunocytochemistry of primary neuronal cultures. Cells were treated with DMSO-CM or P4-CM in the presence

    Journal:

    Article Title: Pgrmc1/BDNF Signaling Plays a Critical Role in Mediating Glia-Neuron Cross Talk

    doi: 10.1210/en.2015-1610

    Figure Lengend Snippet: Blocking of BDNF signaling inhibited glial P4-CM-induced GAP43 density in both proximal and distal neuritic segments of primary hippocampus neurons. A, Immunocytochemistry of primary neuronal cultures. Cells were treated with DMSO-CM or P4-CM in the presence

    Article Snippet: C6 cells were propagated in DMEM (Invitrogen Life Technologies) supplemented with 15% fetal bovine serum (noncharcoal-stripped) (HyClone) and maintained at 37°C in a humidified environment containing 5% CO2 for 24 hours, then treated with vehicle control-dimethylsulfoxide (DMSO) (0.1%), P4 (Sigma-Aldrich) at 10nM for the indicated time.

    Techniques: Blocking Assay, Immunocytochemistry

    Glial P4-CM increased GAP43 density in both proximal and distal neuritic segments of SH-SY5Y cells. A, Immunocytochemistry of SH-SY5Y cultures. Cells were treated with DMSO-CM or P4-CM in the presence or absence of Y1036 for 24 hours. 50-ng/mL BDNF was

    Journal:

    Article Title: Pgrmc1/BDNF Signaling Plays a Critical Role in Mediating Glia-Neuron Cross Talk

    doi: 10.1210/en.2015-1610

    Figure Lengend Snippet: Glial P4-CM increased GAP43 density in both proximal and distal neuritic segments of SH-SY5Y cells. A, Immunocytochemistry of SH-SY5Y cultures. Cells were treated with DMSO-CM or P4-CM in the presence or absence of Y1036 for 24 hours. 50-ng/mL BDNF was

    Article Snippet: C6 cells were propagated in DMEM (Invitrogen Life Technologies) supplemented with 15% fetal bovine serum (noncharcoal-stripped) (HyClone) and maintained at 37°C in a humidified environment containing 5% CO2 for 24 hours, then treated with vehicle control-dimethylsulfoxide (DMSO) (0.1%), P4 (Sigma-Aldrich) at 10nM for the indicated time.

    Techniques: Immunocytochemistry

    Effect of API on DNA damage in MM cells. The expression of γ-H2AX was determined by Western blotting analysis in MM cell lines treated with API at 50 μM or with DMSO (CTR) for 24 h. Actin was used as an internal control. The intensity of the bands was quantified using the ImageJ software after blot scanning of two independent experiments. The densitometric ratios between γ-H2AX and actin and statistical analysis are reported. Data are expressed as the mean ± SD of two independent experiments ( x p ≤ 0.05, ∗ p ≤ 0.01 compared with CTR).

    Journal: Frontiers in Pharmacology

    Article Title: In Vitro and In Vivo Anti-tumoral Effects of the Flavonoid Apigenin in Malignant Mesothelioma

    doi: 10.3389/fphar.2017.00373

    Figure Lengend Snippet: Effect of API on DNA damage in MM cells. The expression of γ-H2AX was determined by Western blotting analysis in MM cell lines treated with API at 50 μM or with DMSO (CTR) for 24 h. Actin was used as an internal control. The intensity of the bands was quantified using the ImageJ software after blot scanning of two independent experiments. The densitometric ratios between γ-H2AX and actin and statistical analysis are reported. Data are expressed as the mean ± SD of two independent experiments ( x p ≤ 0.05, ∗ p ≤ 0.01 compared with CTR).

    Article Snippet: DMSO, 4′,5,7,-trihydroxyflavone (Apigenin, API), Sulforhodamine B (SRB), Hoechst 33342 and DAPI were purchased from Sigma–Aldrich (Milano, Italy).

    Techniques: Expressing, Western Blot, Software

    Effect of API on the expression and activation of signaling pathway molecules. Western blotting analysis was performed on MM cells treated with API (50 μM) or DMSO (CTR) for 24 h. (A) The levels of pERK1 and pERK2 proteins, as well as p-p38 protein were compared with that of total ERK and p38 proteins, respectively. (B) The levels of pJNK p54, pJNK p46, p-c-Jun, and pAKT were compared with that of total JNK, c-Jun and AKT proteins, respectively. The ratios and statistical analysis are reported. Data are expressed as the mean ± SD of two independent experiments ( x p ≤ 0.05, ∗ p ≤ 0.01, # p ≤ 0.001 compared with CTR). Actin and tubulin were used as an internal control.

    Journal: Frontiers in Pharmacology

    Article Title: In Vitro and In Vivo Anti-tumoral Effects of the Flavonoid Apigenin in Malignant Mesothelioma

    doi: 10.3389/fphar.2017.00373

    Figure Lengend Snippet: Effect of API on the expression and activation of signaling pathway molecules. Western blotting analysis was performed on MM cells treated with API (50 μM) or DMSO (CTR) for 24 h. (A) The levels of pERK1 and pERK2 proteins, as well as p-p38 protein were compared with that of total ERK and p38 proteins, respectively. (B) The levels of pJNK p54, pJNK p46, p-c-Jun, and pAKT were compared with that of total JNK, c-Jun and AKT proteins, respectively. The ratios and statistical analysis are reported. Data are expressed as the mean ± SD of two independent experiments ( x p ≤ 0.05, ∗ p ≤ 0.01, # p ≤ 0.001 compared with CTR). Actin and tubulin were used as an internal control.

    Article Snippet: DMSO, 4′,5,7,-trihydroxyflavone (Apigenin, API), Sulforhodamine B (SRB), Hoechst 33342 and DAPI were purchased from Sigma–Aldrich (Milano, Italy).

    Techniques: Expressing, Activation Assay, Western Blot

    Effect of API on NF-κB activation. (A) Western blotting analysis was performed on MM cells treated with API at 50 μM or with the DMSO vehicle (CTR) for 24 h. The densitometric ratios between NF-κB and actin, IκBα and actin, p-IκBα and IκBα and statistical analysis are reported. Data are expressed as the mean ± SD of two independent experiments ( x p ≤ 0.05, # p ≤ 0.001 compared with CTR). (B) Inhibition of nuclear translocation of NF-κB after treatment with API in MM cells was assessed by immunofluorescence analysis. Cells were fixed after treatment, and incubated with the anti-NF-κB antibody. After two washes with PBS, the cells were incubated with the goat anti-mouse IgG Alexa fluor-488-conjugated secondary antibody. Nuclei were stained with DAPI. Original magnification x400.

    Journal: Frontiers in Pharmacology

    Article Title: In Vitro and In Vivo Anti-tumoral Effects of the Flavonoid Apigenin in Malignant Mesothelioma

    doi: 10.3389/fphar.2017.00373

    Figure Lengend Snippet: Effect of API on NF-κB activation. (A) Western blotting analysis was performed on MM cells treated with API at 50 μM or with the DMSO vehicle (CTR) for 24 h. The densitometric ratios between NF-κB and actin, IκBα and actin, p-IκBα and IκBα and statistical analysis are reported. Data are expressed as the mean ± SD of two independent experiments ( x p ≤ 0.05, # p ≤ 0.001 compared with CTR). (B) Inhibition of nuclear translocation of NF-κB after treatment with API in MM cells was assessed by immunofluorescence analysis. Cells were fixed after treatment, and incubated with the anti-NF-κB antibody. After two washes with PBS, the cells were incubated with the goat anti-mouse IgG Alexa fluor-488-conjugated secondary antibody. Nuclei were stained with DAPI. Original magnification x400.

    Article Snippet: DMSO, 4′,5,7,-trihydroxyflavone (Apigenin, API), Sulforhodamine B (SRB), Hoechst 33342 and DAPI were purchased from Sigma–Aldrich (Milano, Italy).

    Techniques: Activation Assay, Western Blot, Inhibition, Translocation Assay, Immunofluorescence, Incubation, Staining

    Effect of API on the intracellular ROS production in MM cell lines. Results are reported as the mean of the fluorescence intensity ± SD values from three experiments performed in triplicate. API was used in the range 6.25–100 μM. Statistical significance of the effects obtained with API was calculated vs. those obtained with DMSO ( x p ≤ 0.05, ∗ p ≤ 0.01, # p ≤ 0.001).

    Journal: Frontiers in Pharmacology

    Article Title: In Vitro and In Vivo Anti-tumoral Effects of the Flavonoid Apigenin in Malignant Mesothelioma

    doi: 10.3389/fphar.2017.00373

    Figure Lengend Snippet: Effect of API on the intracellular ROS production in MM cell lines. Results are reported as the mean of the fluorescence intensity ± SD values from three experiments performed in triplicate. API was used in the range 6.25–100 μM. Statistical significance of the effects obtained with API was calculated vs. those obtained with DMSO ( x p ≤ 0.05, ∗ p ≤ 0.01, # p ≤ 0.001).

    Article Snippet: DMSO, 4′,5,7,-trihydroxyflavone (Apigenin, API), Sulforhodamine B (SRB), Hoechst 33342 and DAPI were purchased from Sigma–Aldrich (Milano, Italy).

    Techniques: Fluorescence

    Effect of API on the expression of molecules involved in apoptosis in MM cells. (A) The expression of Bax, Bcl-2, p53 was assessed by Western blotting analysis in MM cells treated for 24 h with API at 50 μM or with DMSO (CTR) as vehicle. Actin was used as an internal control. (B) The expression of procaspase 9, procaspase 8, cleaved caspase 8 fragments, and the cleavage of PARP-1 were assessed by Western blotting analysis in MM cells treated for 24 h with API at 50 μM or with DMSO (CTR) as vehicle. Actin was used as an internal control. The intensity of the bands was quantified using ImageJ software after blot scanning, obtained from two independent experiments. (C) The densitometric ratios between Bax and Bcl-2, between p53 and actin, and between procaspase 9 and actin and statistical analysis are reported. Data are expressed as the mean ± SD of two independent experiments ( x p ≤ 0.05, ∗ p ≤ 0.01 compared with CTR).

    Journal: Frontiers in Pharmacology

    Article Title: In Vitro and In Vivo Anti-tumoral Effects of the Flavonoid Apigenin in Malignant Mesothelioma

    doi: 10.3389/fphar.2017.00373

    Figure Lengend Snippet: Effect of API on the expression of molecules involved in apoptosis in MM cells. (A) The expression of Bax, Bcl-2, p53 was assessed by Western blotting analysis in MM cells treated for 24 h with API at 50 μM or with DMSO (CTR) as vehicle. Actin was used as an internal control. (B) The expression of procaspase 9, procaspase 8, cleaved caspase 8 fragments, and the cleavage of PARP-1 were assessed by Western blotting analysis in MM cells treated for 24 h with API at 50 μM or with DMSO (CTR) as vehicle. Actin was used as an internal control. The intensity of the bands was quantified using ImageJ software after blot scanning, obtained from two independent experiments. (C) The densitometric ratios between Bax and Bcl-2, between p53 and actin, and between procaspase 9 and actin and statistical analysis are reported. Data are expressed as the mean ± SD of two independent experiments ( x p ≤ 0.05, ∗ p ≤ 0.01 compared with CTR).

    Article Snippet: DMSO, 4′,5,7,-trihydroxyflavone (Apigenin, API), Sulforhodamine B (SRB), Hoechst 33342 and DAPI were purchased from Sigma–Aldrich (Milano, Italy).

    Techniques: Expressing, Western Blot, Software

    Effect of API on the autophagic flux in MM cells. The expression of Beclin-1, p62/SQSTM (p62), and LC3-I and LC3-II was assessed by Western blotting in MM cell lines treated with API at 50 μM or DMSO (CTR) for 24 h. Actin was used as an internal control. The intensity of the bands obtained was quantified using the ImageJ software after blot scanning of two independent experiments. The densitometric ratios between Beclin-1 and actin, LC3-I and actin, LC3-II and actin, p62 and actin and statistical analysis are reported. Data are expressed as the mean ± SD of two independent experiments ( ∗ p ≤ 0.01 compared with CTR).

    Journal: Frontiers in Pharmacology

    Article Title: In Vitro and In Vivo Anti-tumoral Effects of the Flavonoid Apigenin in Malignant Mesothelioma

    doi: 10.3389/fphar.2017.00373

    Figure Lengend Snippet: Effect of API on the autophagic flux in MM cells. The expression of Beclin-1, p62/SQSTM (p62), and LC3-I and LC3-II was assessed by Western blotting in MM cell lines treated with API at 50 μM or DMSO (CTR) for 24 h. Actin was used as an internal control. The intensity of the bands obtained was quantified using the ImageJ software after blot scanning of two independent experiments. The densitometric ratios between Beclin-1 and actin, LC3-I and actin, LC3-II and actin, p62 and actin and statistical analysis are reported. Data are expressed as the mean ± SD of two independent experiments ( ∗ p ≤ 0.01 compared with CTR).

    Article Snippet: DMSO, 4′,5,7,-trihydroxyflavone (Apigenin, API), Sulforhodamine B (SRB), Hoechst 33342 and DAPI were purchased from Sigma–Aldrich (Milano, Italy).

    Techniques: Expressing, Western Blot, Software

    Effect of API on cell cycle distribution. FACS analysis of DNA content was performed on asynchronized log phase growing MM cells treated for 48 hours with DMSO or API at 6.25–100 μM. A representative experiment is shown in the figure.

    Journal: Frontiers in Pharmacology

    Article Title: In Vitro and In Vivo Anti-tumoral Effects of the Flavonoid Apigenin in Malignant Mesothelioma

    doi: 10.3389/fphar.2017.00373

    Figure Lengend Snippet: Effect of API on cell cycle distribution. FACS analysis of DNA content was performed on asynchronized log phase growing MM cells treated for 48 hours with DMSO or API at 6.25–100 μM. A representative experiment is shown in the figure.

    Article Snippet: DMSO, 4′,5,7,-trihydroxyflavone (Apigenin, API), Sulforhodamine B (SRB), Hoechst 33342 and DAPI were purchased from Sigma–Aldrich (Milano, Italy).

    Techniques: FACS

    Effect of Apigenin (API) on malignant mesothelioma (MM) cell lines growth and death. (A) The growth of human (MM-B1, H-Meso-1, MM-F1) and mouse (#40a) MM cell lines were assessed by the SRB assay after 24, 48, and 72 h of treatment with DMSO or API. The percentage of cells growth treated with API was calculated by normalizing the O.D. value to that of the control cultures (DMSO). The results are expressed as the mean ± SD of three independent experiments performed in triplicate ( x p ≤ 0.05, ∗ p ≤ 0.01, # p ≤ 0.001 compared with the cultures treated with DMSO). (B) Trypan blue exclusion test was performed to determine the percentage of cell death of MM cells treated with API or DMSO after 24, 48, and 72 h of treatment. The results are expressed as the mean ± SD of three independent experiments performed in triplicate ( x p ≤ 0.05, ∗ p ≤ 0.01, # p ≤ 0.001 compared with the cultures treated with DMSO).

    Journal: Frontiers in Pharmacology

    Article Title: In Vitro and In Vivo Anti-tumoral Effects of the Flavonoid Apigenin in Malignant Mesothelioma

    doi: 10.3389/fphar.2017.00373

    Figure Lengend Snippet: Effect of Apigenin (API) on malignant mesothelioma (MM) cell lines growth and death. (A) The growth of human (MM-B1, H-Meso-1, MM-F1) and mouse (#40a) MM cell lines were assessed by the SRB assay after 24, 48, and 72 h of treatment with DMSO or API. The percentage of cells growth treated with API was calculated by normalizing the O.D. value to that of the control cultures (DMSO). The results are expressed as the mean ± SD of three independent experiments performed in triplicate ( x p ≤ 0.05, ∗ p ≤ 0.01, # p ≤ 0.001 compared with the cultures treated with DMSO). (B) Trypan blue exclusion test was performed to determine the percentage of cell death of MM cells treated with API or DMSO after 24, 48, and 72 h of treatment. The results are expressed as the mean ± SD of three independent experiments performed in triplicate ( x p ≤ 0.05, ∗ p ≤ 0.01, # p ≤ 0.001 compared with the cultures treated with DMSO).

    Article Snippet: DMSO, 4′,5,7,-trihydroxyflavone (Apigenin, API), Sulforhodamine B (SRB), Hoechst 33342 and DAPI were purchased from Sigma–Aldrich (Milano, Italy).

    Techniques: Sulforhodamine B Assay

    Apigenin reduced tumor growth and increased the survival in C57BL/6 mice intraperitoneally transplanted with MM #40a cells. (A) Differences in mean abdominal circumferences between C57BL/6 mice treated with API or with DMSO-PBS (CTR). (B) Differences in the mean survival duration of C57BL/6 mice treated with API or with DMSO-PBS (CTR). The numbers of inoculated mice are reported in the Section “Materials and Methods”.

    Journal: Frontiers in Pharmacology

    Article Title: In Vitro and In Vivo Anti-tumoral Effects of the Flavonoid Apigenin in Malignant Mesothelioma

    doi: 10.3389/fphar.2017.00373

    Figure Lengend Snippet: Apigenin reduced tumor growth and increased the survival in C57BL/6 mice intraperitoneally transplanted with MM #40a cells. (A) Differences in mean abdominal circumferences between C57BL/6 mice treated with API or with DMSO-PBS (CTR). (B) Differences in the mean survival duration of C57BL/6 mice treated with API or with DMSO-PBS (CTR). The numbers of inoculated mice are reported in the Section “Materials and Methods”.

    Article Snippet: DMSO, 4′,5,7,-trihydroxyflavone (Apigenin, API), Sulforhodamine B (SRB), Hoechst 33342 and DAPI were purchased from Sigma–Aldrich (Milano, Italy).

    Techniques: Mouse Assay

    Stacked bar graphs showing the percentage of cells in different phases of the cell cycle. Percentage of cells in subG1, G0/G1, S, and G2/M phases was calculated with CellQuest Pro 5.2 software. Results represent mean values from three independent experiments. Statistical significance of the effects obtained with API treatment was calculated vs. those obtained in DMSO-treated cells with one-way ANOVA analysis of variance ( x p ≤ 0.05, ∗ p ≤ 0.01, # p ≤ 0.001).

    Journal: Frontiers in Pharmacology

    Article Title: In Vitro and In Vivo Anti-tumoral Effects of the Flavonoid Apigenin in Malignant Mesothelioma

    doi: 10.3389/fphar.2017.00373

    Figure Lengend Snippet: Stacked bar graphs showing the percentage of cells in different phases of the cell cycle. Percentage of cells in subG1, G0/G1, S, and G2/M phases was calculated with CellQuest Pro 5.2 software. Results represent mean values from three independent experiments. Statistical significance of the effects obtained with API treatment was calculated vs. those obtained in DMSO-treated cells with one-way ANOVA analysis of variance ( x p ≤ 0.05, ∗ p ≤ 0.01, # p ≤ 0.001).

    Article Snippet: DMSO, 4′,5,7,-trihydroxyflavone (Apigenin, API), Sulforhodamine B (SRB), Hoechst 33342 and DAPI were purchased from Sigma–Aldrich (Milano, Italy).

    Techniques: Software

    Keratin IF are less sensitive to WFA than VIF. Human lung cancer cells, A549, were treated for 3 hrs with DMSO [ctrl] (A), 4.0 μM WFA (B), and 6.0 μM WFA (C), followed by staining with vimentin (A′, B′, C′) and pan-cytokeratin antibodies (A′′, B′′, C′′). Scale bars =10 μm.

    Journal: PLoS ONE

    Article Title: Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

    doi: 10.1371/journal.pone.0039065

    Figure Lengend Snippet: Keratin IF are less sensitive to WFA than VIF. Human lung cancer cells, A549, were treated for 3 hrs with DMSO [ctrl] (A), 4.0 μM WFA (B), and 6.0 μM WFA (C), followed by staining with vimentin (A′, B′, C′) and pan-cytokeratin antibodies (A′′, B′′, C′′). Scale bars =10 μm.

    Article Snippet: For cell based experiments withaferin A (WFA) was purchased from ChromaDex (Irvine, CA) and dissolved in DMSO (Sigma, St. Louis, MO) at a stock concentration of 10 mM.

    Techniques: Staining

    WFA has no effect on the in vitro assembly of human recombinant vimentin. (A) Recombinant human vimentin (0.2 mg/ml) was assembled for 10 min at 37°C in (i) 50 mM NaCl; (ii) with 0.25% DMSO; (iii) with 50 μM WFA; and (iv) for 30 min with 50 μM WFA at a protein concentration of 0.5 mg/ml. The filaments were fixed with glutaraldehyde and visualized by negative stain electron microscopy. The arrows in (ii) indicate lateral annealing and apparent fusion of individual filaments. (scale bars, 0.2 μm). (B) Viscometric analysis of vimentin assembly in the absence (ctrl) and presence of 50 μM WFA at 37°C in 50 mM NaCl. (C) Centrifugation assay of vimentin assembled in the absence (c) and presence of WFA (w). VIF were assembled for the indicated times (5 to 15 min) in 160 mM NaCl then centrifuged for 5 min at 10 psi in an Airfuge. Samples were separated by SDS-PAGE and stained with Coomassie. The position of vimentin is indicated (55 kDa).

    Journal: PLoS ONE

    Article Title: Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

    doi: 10.1371/journal.pone.0039065

    Figure Lengend Snippet: WFA has no effect on the in vitro assembly of human recombinant vimentin. (A) Recombinant human vimentin (0.2 mg/ml) was assembled for 10 min at 37°C in (i) 50 mM NaCl; (ii) with 0.25% DMSO; (iii) with 50 μM WFA; and (iv) for 30 min with 50 μM WFA at a protein concentration of 0.5 mg/ml. The filaments were fixed with glutaraldehyde and visualized by negative stain electron microscopy. The arrows in (ii) indicate lateral annealing and apparent fusion of individual filaments. (scale bars, 0.2 μm). (B) Viscometric analysis of vimentin assembly in the absence (ctrl) and presence of 50 μM WFA at 37°C in 50 mM NaCl. (C) Centrifugation assay of vimentin assembled in the absence (c) and presence of WFA (w). VIF were assembled for the indicated times (5 to 15 min) in 160 mM NaCl then centrifuged for 5 min at 10 psi in an Airfuge. Samples were separated by SDS-PAGE and stained with Coomassie. The position of vimentin is indicated (55 kDa).

    Article Snippet: For cell based experiments withaferin A (WFA) was purchased from ChromaDex (Irvine, CA) and dissolved in DMSO (Sigma, St. Louis, MO) at a stock concentration of 10 mM.

    Techniques: In Vitro, Recombinant, Protein Concentration, Staining, Electron Microscopy, Centrifugation, SDS Page

    WFA treatment induces an increase in the phosphorylation of vimentin serine-38. BJ-5ta cells were treated for 3 hrs with DMSO (A) or 2 μM (B) WFA, then fixed and double labeled with vimentin (A′ and B′) and pSer38 vimentin (A′′ and B′′) antibodies. Scale bars =10 μm. Arrow: a region depicted at higher magnification in the inset showing vimentin particles stained with pSer38 vimentin antibody. (C) Whole cell lysates of cells treated with DMSO (ctrl) or 2 μM WFA for 60 min, 120 min, and 180 min, were separated by SDS-PAGE and stained with anti-vimentin and anti-vimentin pSer38 vimentin antibodies.

    Journal: PLoS ONE

    Article Title: Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

    doi: 10.1371/journal.pone.0039065

    Figure Lengend Snippet: WFA treatment induces an increase in the phosphorylation of vimentin serine-38. BJ-5ta cells were treated for 3 hrs with DMSO (A) or 2 μM (B) WFA, then fixed and double labeled with vimentin (A′ and B′) and pSer38 vimentin (A′′ and B′′) antibodies. Scale bars =10 μm. Arrow: a region depicted at higher magnification in the inset showing vimentin particles stained with pSer38 vimentin antibody. (C) Whole cell lysates of cells treated with DMSO (ctrl) or 2 μM WFA for 60 min, 120 min, and 180 min, were separated by SDS-PAGE and stained with anti-vimentin and anti-vimentin pSer38 vimentin antibodies.

    Article Snippet: For cell based experiments withaferin A (WFA) was purchased from ChromaDex (Irvine, CA) and dissolved in DMSO (Sigma, St. Louis, MO) at a stock concentration of 10 mM.

    Techniques: Labeling, Staining, SDS Page

    Cysteine-328 is not required for the effects of WFA on VIF. SW13-1HF5 cells which are null for cytoplasmic IF, were transfected with wild-type vimentin (A) and vimentin C328N (C). The cells were then treated for 3 hrs with DMSO (A and C) or 9 μM WFA (B and D). Scale bars =10 μm.

    Journal: PLoS ONE

    Article Title: Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

    doi: 10.1371/journal.pone.0039065

    Figure Lengend Snippet: Cysteine-328 is not required for the effects of WFA on VIF. SW13-1HF5 cells which are null for cytoplasmic IF, were transfected with wild-type vimentin (A) and vimentin C328N (C). The cells were then treated for 3 hrs with DMSO (A and C) or 9 μM WFA (B and D). Scale bars =10 μm.

    Article Snippet: For cell based experiments withaferin A (WFA) was purchased from ChromaDex (Irvine, CA) and dissolved in DMSO (Sigma, St. Louis, MO) at a stock concentration of 10 mM.

    Techniques: Transfection

    WFA affects the organization of keratin and neuron specific IF. MCF-7 cells, which express only keratin IF, were treated for 3 hrs with DMSO (A) and 4.0 μM WFA (B; the lowest effective concentration in these cells) and stained with pan-cytokeratin antibodies. Differentiated PC12 cells were treated for 3 hrs with DMSO (C) or 1.0 μM WFA (D, the lowest effective concentration for these cells), then stained with neurofilament-M (C′ and D′) and peripherin antibodies (C′′ and D′′). Scale bars =10 μm.

    Journal: PLoS ONE

    Article Title: Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

    doi: 10.1371/journal.pone.0039065

    Figure Lengend Snippet: WFA affects the organization of keratin and neuron specific IF. MCF-7 cells, which express only keratin IF, were treated for 3 hrs with DMSO (A) and 4.0 μM WFA (B; the lowest effective concentration in these cells) and stained with pan-cytokeratin antibodies. Differentiated PC12 cells were treated for 3 hrs with DMSO (C) or 1.0 μM WFA (D, the lowest effective concentration for these cells), then stained with neurofilament-M (C′ and D′) and peripherin antibodies (C′′ and D′′). Scale bars =10 μm.

    Article Snippet: For cell based experiments withaferin A (WFA) was purchased from ChromaDex (Irvine, CA) and dissolved in DMSO (Sigma, St. Louis, MO) at a stock concentration of 10 mM.

    Techniques: Concentration Assay, Staining

    Longer exposure to WFA induces apoptosis or senescence. (A) BJ-5ta cells were treated for 24 hrs with DMSO and 2 μM WFA followed by staining with annexin V and assayed by FACS. (B) Whole cell lysates were separated by SDS-PAGE and blotted with anti-vimentin. BJ-5ta cells treated for 24 hrs with0.5 μM WFA (C) and1 μM WFA (D) were stained with vimentin antibodies. BJ-5ta fibroblasts were incubated with DMSO alone (E) or 1 μM WFA (F) continuously for 5 days and stained for senescence-associated ß-galactosidase. (H) Cell proliferation of BJ-5ta cells was monitored by counting cells every 3 days during continuous incubation with DMSO alone (black line) or 1.0 μM WFA (red line). Scale bars =10 μm.

    Journal: PLoS ONE

    Article Title: Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

    doi: 10.1371/journal.pone.0039065

    Figure Lengend Snippet: Longer exposure to WFA induces apoptosis or senescence. (A) BJ-5ta cells were treated for 24 hrs with DMSO and 2 μM WFA followed by staining with annexin V and assayed by FACS. (B) Whole cell lysates were separated by SDS-PAGE and blotted with anti-vimentin. BJ-5ta cells treated for 24 hrs with0.5 μM WFA (C) and1 μM WFA (D) were stained with vimentin antibodies. BJ-5ta fibroblasts were incubated with DMSO alone (E) or 1 μM WFA (F) continuously for 5 days and stained for senescence-associated ß-galactosidase. (H) Cell proliferation of BJ-5ta cells was monitored by counting cells every 3 days during continuous incubation with DMSO alone (black line) or 1.0 μM WFA (red line). Scale bars =10 μm.

    Article Snippet: For cell based experiments withaferin A (WFA) was purchased from ChromaDex (Irvine, CA) and dissolved in DMSO (Sigma, St. Louis, MO) at a stock concentration of 10 mM.

    Techniques: Staining, FACS, SDS Page, Incubation

    WFA also alters the organization of microtubules and microfilaments. BJ-5ta cell were treated with DMSO (A and C) and 2 μM WFA (B and D) for 3 hrs and stained with vimentin antibodies (A′, B′, C′, D′), tubulin antibodies (A′′ and B′′), and phalloidin to visualize to actin (C′′ and D′′). Scale bars =10 μm.

    Journal: PLoS ONE

    Article Title: Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

    doi: 10.1371/journal.pone.0039065

    Figure Lengend Snippet: WFA also alters the organization of microtubules and microfilaments. BJ-5ta cell were treated with DMSO (A and C) and 2 μM WFA (B and D) for 3 hrs and stained with vimentin antibodies (A′, B′, C′, D′), tubulin antibodies (A′′ and B′′), and phalloidin to visualize to actin (C′′ and D′′). Scale bars =10 μm.

    Article Snippet: For cell based experiments withaferin A (WFA) was purchased from ChromaDex (Irvine, CA) and dissolved in DMSO (Sigma, St. Louis, MO) at a stock concentration of 10 mM.

    Techniques: Staining

    WFA treatment alters the subcellular organization of VIF. BJ-5ta cells were treated for 3 hrs (A–C and F) with DMSO alone (A), 0.5 μM WFA (B), 1 μM WFA (C), and 2 μM WFA (F). In addition, cells treated with 2 μM WFA are depicted after 1 hr (D) and 2 hrs (E) which show that the changes in VIF organization take place gradually. Cells were fixed and processed for immunofluorescence with vimentin antibodies. Arrow: a region depicted at higher magnification in the inset, showing non-filamentous vimentin particles and short IF or squiggles. Scale bars =10 μm.

    Journal: PLoS ONE

    Article Title: Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

    doi: 10.1371/journal.pone.0039065

    Figure Lengend Snippet: WFA treatment alters the subcellular organization of VIF. BJ-5ta cells were treated for 3 hrs (A–C and F) with DMSO alone (A), 0.5 μM WFA (B), 1 μM WFA (C), and 2 μM WFA (F). In addition, cells treated with 2 μM WFA are depicted after 1 hr (D) and 2 hrs (E) which show that the changes in VIF organization take place gradually. Cells were fixed and processed for immunofluorescence with vimentin antibodies. Arrow: a region depicted at higher magnification in the inset, showing non-filamentous vimentin particles and short IF or squiggles. Scale bars =10 μm.

    Article Snippet: For cell based experiments withaferin A (WFA) was purchased from ChromaDex (Irvine, CA) and dissolved in DMSO (Sigma, St. Louis, MO) at a stock concentration of 10 mM.

    Techniques: Immunofluorescence

    WFA does not affect the sedimentation velocity of vimentin. Sedimentation velocity profile of vimentin (0.15 mg/ml) reconstituted in 5 mM Tris-HCl, pH 8.4 (dashed lines) or 2 mM NaCl, pH 7.5 buffer (solid lines) alone (red lines), with DMSO (green lines), or with 25 μM WFA dissolved in DMSO (blue lines). Note that the curves of the two groups of runs are practically superimposable indicating that DMSO and DMSO plus WFA do not have any effect on the complex formation of vimentin oligomers.

    Journal: PLoS ONE

    Article Title: Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

    doi: 10.1371/journal.pone.0039065

    Figure Lengend Snippet: WFA does not affect the sedimentation velocity of vimentin. Sedimentation velocity profile of vimentin (0.15 mg/ml) reconstituted in 5 mM Tris-HCl, pH 8.4 (dashed lines) or 2 mM NaCl, pH 7.5 buffer (solid lines) alone (red lines), with DMSO (green lines), or with 25 μM WFA dissolved in DMSO (blue lines). Note that the curves of the two groups of runs are practically superimposable indicating that DMSO and DMSO plus WFA do not have any effect on the complex formation of vimentin oligomers.

    Article Snippet: For cell based experiments withaferin A (WFA) was purchased from ChromaDex (Irvine, CA) and dissolved in DMSO (Sigma, St. Louis, MO) at a stock concentration of 10 mM.

    Techniques: Sedimentation

    Overexpression of p21 rescued tigecycline-induced cell growth and proliferation inhibition in human melanoma cells A, C. RT-PCR assay was used to show the expression of cdkn1a (p21) mRNA after tigecycline treatment. B, D. Western blot assay was used to show the expression of p21 after tigecycline treatment. E. Western blot assay was used to show the expression of p21 in DMSO or tigecycline-treated p21/vector-overexpressed A375 and MV3 cells. F, G. The effect of DMSO or tigecycline on the viability of p21/vector-overexpressed A375 and MV3 cells. H. Image and quantification of p21-overexpressed A375 or MV3 cells as well as vector cells positive for Brdu staining after treating with DMSO or 10 μM tigcycline for 24 h, Scale bar, 100 μm. I. Colony formation was examined by soft agar assay (1000 cells/well) in p21-overexpressed A375 and MV3 cells as well as vector cells after treating with DMSO or 10 μM tigecycline for 14 to 21 days, Scale bar, 100 μm. Colony number was counted using counter. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Journal: Oncotarget

    Article Title: Antibiotic drug tigecycline inhibits melanoma progression and metastasis in a p21CIP1/Waf1-dependent manner

    doi: 10.18632/oncotarget.6419

    Figure Lengend Snippet: Overexpression of p21 rescued tigecycline-induced cell growth and proliferation inhibition in human melanoma cells A, C. RT-PCR assay was used to show the expression of cdkn1a (p21) mRNA after tigecycline treatment. B, D. Western blot assay was used to show the expression of p21 after tigecycline treatment. E. Western blot assay was used to show the expression of p21 in DMSO or tigecycline-treated p21/vector-overexpressed A375 and MV3 cells. F, G. The effect of DMSO or tigecycline on the viability of p21/vector-overexpressed A375 and MV3 cells. H. Image and quantification of p21-overexpressed A375 or MV3 cells as well as vector cells positive for Brdu staining after treating with DMSO or 10 μM tigcycline for 24 h, Scale bar, 100 μm. I. Colony formation was examined by soft agar assay (1000 cells/well) in p21-overexpressed A375 and MV3 cells as well as vector cells after treating with DMSO or 10 μM tigecycline for 14 to 21 days, Scale bar, 100 μm. Colony number was counted using counter. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Article Snippet: After incubated with tigecycline or DMSO for the indicated time, 20 μl MTT (5 μg/ml MTT in PBS; Sigma) was added to each well, then incubated at 37°C for 2 h and removed the formazan complex.

    Techniques: Over Expression, Inhibition, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Plasmid Preparation, BrdU Staining, Soft Agar Assay

    Overexpression of p21 recovered tigecycline induced cell cycle arrest in human melanoma cells A. Western blot analysis of cell cycle-related proteins in p21-overexpressed A375 and MV3 cells as well as vector cells after treated with DMSO or 10 μM tigcycline for 48 h. B, C. The cell cycle of A375 and MV3 cells was analyzed by flow cytometry in p21-overexpressed A375 and MV3 cells as well as vector cells after treated with DMSO or 10 μM tigcycline for 48 h. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Journal: Oncotarget

    Article Title: Antibiotic drug tigecycline inhibits melanoma progression and metastasis in a p21CIP1/Waf1-dependent manner

    doi: 10.18632/oncotarget.6419

    Figure Lengend Snippet: Overexpression of p21 recovered tigecycline induced cell cycle arrest in human melanoma cells A. Western blot analysis of cell cycle-related proteins in p21-overexpressed A375 and MV3 cells as well as vector cells after treated with DMSO or 10 μM tigcycline for 48 h. B, C. The cell cycle of A375 and MV3 cells was analyzed by flow cytometry in p21-overexpressed A375 and MV3 cells as well as vector cells after treated with DMSO or 10 μM tigcycline for 48 h. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Article Snippet: After incubated with tigecycline or DMSO for the indicated time, 20 μl MTT (5 μg/ml MTT in PBS; Sigma) was added to each well, then incubated at 37°C for 2 h and removed the formazan complex.

    Techniques: Over Expression, Western Blot, Plasmid Preparation, Flow Cytometry, Cytometry

    Tigecycline induced cell cycle arrest at G1 phase in human melanoma cells A, B. The cell cycle of A375 and MV3 cells was analyzed by flow cytometry after treating with DMSO or 10 μM tigecycline for 48 h. C, D. Western blot assay was performed to assess the cell cycle-related protein levels at 48 h in A375 and MV3 cells, respectively. Cells were treated with the indicated concentration or the indicated times of tigecycline; GAPDH was used as a control. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Journal: Oncotarget

    Article Title: Antibiotic drug tigecycline inhibits melanoma progression and metastasis in a p21CIP1/Waf1-dependent manner

    doi: 10.18632/oncotarget.6419

    Figure Lengend Snippet: Tigecycline induced cell cycle arrest at G1 phase in human melanoma cells A, B. The cell cycle of A375 and MV3 cells was analyzed by flow cytometry after treating with DMSO or 10 μM tigecycline for 48 h. C, D. Western blot assay was performed to assess the cell cycle-related protein levels at 48 h in A375 and MV3 cells, respectively. Cells were treated with the indicated concentration or the indicated times of tigecycline; GAPDH was used as a control. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Article Snippet: After incubated with tigecycline or DMSO for the indicated time, 20 μl MTT (5 μg/ml MTT in PBS; Sigma) was added to each well, then incubated at 37°C for 2 h and removed the formazan complex.

    Techniques: Flow Cytometry, Cytometry, Western Blot, Concentration Assay

    Tigecycline inhibited cell migration and invasion in human melanoma cells A. The migration by wound-healing assay of A375 and MV3 cells after treating with DMSO or 10 μM tigcycline for the indicated time, Scale bar, 100 μm. B. The effect of tigecycline on the wound closure in A375 and MV3 cells. C. The effect of transwell migration assays in A375 and MV3 cells after treating with DMSO or 10 μM tigcycline for 24 h, Scale bar, 100 μm. Migration rates were normalized by proliferation. D. The effect of transwell invasion assays in A375 and MV3 cells after treating with DMSO or 10 μM tigcycline for 72 h, Scale bar, 100 μm. Invasion rates were normalized by proliferation. E, F. Western blot analysis of the EMT-related protein levels at 48 h in A375 and MV3 cells respectively. Cells were treated with the indicated concentration or the indicated times of tigecycline; GAPDH was used as a control. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Journal: Oncotarget

    Article Title: Antibiotic drug tigecycline inhibits melanoma progression and metastasis in a p21CIP1/Waf1-dependent manner

    doi: 10.18632/oncotarget.6419

    Figure Lengend Snippet: Tigecycline inhibited cell migration and invasion in human melanoma cells A. The migration by wound-healing assay of A375 and MV3 cells after treating with DMSO or 10 μM tigcycline for the indicated time, Scale bar, 100 μm. B. The effect of tigecycline on the wound closure in A375 and MV3 cells. C. The effect of transwell migration assays in A375 and MV3 cells after treating with DMSO or 10 μM tigcycline for 24 h, Scale bar, 100 μm. Migration rates were normalized by proliferation. D. The effect of transwell invasion assays in A375 and MV3 cells after treating with DMSO or 10 μM tigcycline for 72 h, Scale bar, 100 μm. Invasion rates were normalized by proliferation. E, F. Western blot analysis of the EMT-related protein levels at 48 h in A375 and MV3 cells respectively. Cells were treated with the indicated concentration or the indicated times of tigecycline; GAPDH was used as a control. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Article Snippet: After incubated with tigecycline or DMSO for the indicated time, 20 μl MTT (5 μg/ml MTT in PBS; Sigma) was added to each well, then incubated at 37°C for 2 h and removed the formazan complex.

    Techniques: Migration, Wound Healing Assay, Western Blot, Concentration Assay

    Tigecycline suppressed tumor growth in xenograft model of human melanoma cells A, B. The colony formation was examined by soft agar assay (1000 cells/well) in A375 or MV3 cells after treating with DMSO or 10 μM tigecycline for 14 to 21 days, Scale bar, 100 μm. C, D. Human melanoma cell A375 and MV3 were injected into the flank of BALA/c nude mice. When tumors were palpable, mice were treated with tigecycline (100 mg/kg) or DMSO every two days for 5 times, and tumor volume was measured. E, F. The weight of xenograft tumors formed by the A375 and MV3 cells which were subsquently treating with tigecycline (100 mg/kg) or DMSO. G, H. The weight of mice was measured after tigecycline treatment. I. Western blot assay was performed to assess cell cycle and EMT-related protein levels in the xenograft tumors of A375 and MV3 melanoma cells; GAPDH was used as a control. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Journal: Oncotarget

    Article Title: Antibiotic drug tigecycline inhibits melanoma progression and metastasis in a p21CIP1/Waf1-dependent manner

    doi: 10.18632/oncotarget.6419

    Figure Lengend Snippet: Tigecycline suppressed tumor growth in xenograft model of human melanoma cells A, B. The colony formation was examined by soft agar assay (1000 cells/well) in A375 or MV3 cells after treating with DMSO or 10 μM tigecycline for 14 to 21 days, Scale bar, 100 μm. C, D. Human melanoma cell A375 and MV3 were injected into the flank of BALA/c nude mice. When tumors were palpable, mice were treated with tigecycline (100 mg/kg) or DMSO every two days for 5 times, and tumor volume was measured. E, F. The weight of xenograft tumors formed by the A375 and MV3 cells which were subsquently treating with tigecycline (100 mg/kg) or DMSO. G, H. The weight of mice was measured after tigecycline treatment. I. Western blot assay was performed to assess cell cycle and EMT-related protein levels in the xenograft tumors of A375 and MV3 melanoma cells; GAPDH was used as a control. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Article Snippet: After incubated with tigecycline or DMSO for the indicated time, 20 μl MTT (5 μg/ml MTT in PBS; Sigma) was added to each well, then incubated at 37°C for 2 h and removed the formazan complex.

    Techniques: Soft Agar Assay, Injection, Mouse Assay, Western Blot

    Overexpression of p21 retrieved tigecycline-induced cell migration and invasion suppression in human melanoma cells A, B, C, D. The effect of the transwell migration and invasion assays in p21-overexpressed A375 or MV3 cells as well as vector cells after treating with DMSO or 10 μM tigcycline for 24 h and 72 h respectively, Scale bar, 100 μm. Migration/invasion rates were normalized by proliferation. E. Western blot assay was used to show the expression of EMT-related proteins at 48 h in p21-overexpressed A375 and MV3 cells as well as vector cells after treated with DMSO or 10 μM tigcycline for 48 h. GAPDH was used as a control. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Journal: Oncotarget

    Article Title: Antibiotic drug tigecycline inhibits melanoma progression and metastasis in a p21CIP1/Waf1-dependent manner

    doi: 10.18632/oncotarget.6419

    Figure Lengend Snippet: Overexpression of p21 retrieved tigecycline-induced cell migration and invasion suppression in human melanoma cells A, B, C, D. The effect of the transwell migration and invasion assays in p21-overexpressed A375 or MV3 cells as well as vector cells after treating with DMSO or 10 μM tigcycline for 24 h and 72 h respectively, Scale bar, 100 μm. Migration/invasion rates were normalized by proliferation. E. Western blot assay was used to show the expression of EMT-related proteins at 48 h in p21-overexpressed A375 and MV3 cells as well as vector cells after treated with DMSO or 10 μM tigcycline for 48 h. GAPDH was used as a control. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Article Snippet: After incubated with tigecycline or DMSO for the indicated time, 20 μl MTT (5 μg/ml MTT in PBS; Sigma) was added to each well, then incubated at 37°C for 2 h and removed the formazan complex.

    Techniques: Over Expression, Migration, Plasmid Preparation, Western Blot, Expressing

    Tigcycline inhibited cell growth and proliferation in human melanoma cells A. Cell morphology of A375 and MV3 melanoma cells after treating with DMSO or the indicated concentration of tigecycline for 48 h, Scale bar, 100 μm. B, C. The effect of tigecycline on the proliferation rate of A375 and MV3 cells. D, E. The effect of tigecycline on the viability of A375 and MV3 cells. F. Image and quantification of A375 and MV3 cells positive for Brdu staining after treating with DMSO or 10 μM tigecycline for 24 h, Scale bar, 100 μm. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Journal: Oncotarget

    Article Title: Antibiotic drug tigecycline inhibits melanoma progression and metastasis in a p21CIP1/Waf1-dependent manner

    doi: 10.18632/oncotarget.6419

    Figure Lengend Snippet: Tigcycline inhibited cell growth and proliferation in human melanoma cells A. Cell morphology of A375 and MV3 melanoma cells after treating with DMSO or the indicated concentration of tigecycline for 48 h, Scale bar, 100 μm. B, C. The effect of tigecycline on the proliferation rate of A375 and MV3 cells. D, E. The effect of tigecycline on the viability of A375 and MV3 cells. F. Image and quantification of A375 and MV3 cells positive for Brdu staining after treating with DMSO or 10 μM tigecycline for 24 h, Scale bar, 100 μm. All data are shown as the mean ± SD. Student's t -test was carried out. * p

    Article Snippet: After incubated with tigecycline or DMSO for the indicated time, 20 μl MTT (5 μg/ml MTT in PBS; Sigma) was added to each well, then incubated at 37°C for 2 h and removed the formazan complex.

    Techniques: Concentration Assay, BrdU Staining

    SU6668 prevents Shh-induced osteogenic differentiation of C3H10T1/2 cells. (A) C3H10T1/2 cells were treated as indicated for 3 d. Alkaline phosphatase (AP) activity was measured and normalized with the amount of total protein. The normalized level of AP activity in the control sample treated with DMSO was set as 1. The normalized data are expressed as an average mean ± S.D. ***—p

    Journal: Biochimica et Biophysica Acta

    Article Title: Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells

    doi: 10.1016/j.bbamcr.2014.01.003

    Figure Lengend Snippet: SU6668 prevents Shh-induced osteogenic differentiation of C3H10T1/2 cells. (A) C3H10T1/2 cells were treated as indicated for 3 d. Alkaline phosphatase (AP) activity was measured and normalized with the amount of total protein. The normalized level of AP activity in the control sample treated with DMSO was set as 1. The normalized data are expressed as an average mean ± S.D. ***—p

    Article Snippet: SU6668 was dissolved in DMSO (both from Sigma-Aldrich, Steinheim, Germany) and stored at − 70 °C prior use.

    Techniques: Activity Assay

    (A) C3H10T1/2 cells were transfected with empty vector pSUPER or Ulk3 shRNAs and incubated during the indicated periods of time. Expression levels of Ulk3 (a) and Gli2 (b) were analyzed using qRT-PCR. The normalized with Hprt level of the respective gene expression at the indicated time point was set as 1 in the control cells transfected with pSUPER, and the data from the samples transfected with Ulk3 shRNAs were calculated relative to the control. The data are presented as an average mean ± S.D. (B) Expression of Gli2 and GAPDH proteins was analyzed by Western-blotting at the indicated time points in the post-transfectional C3H10T1/2 cells electroporated with empty vector pSUPER or Ulk3 shRNAs. Gli2 was detected using AF3635 antibody. (C) C3H10T1/2 cells were electroporated with pSUPER or Ulk3 shRNAs encoding constructs, incubated for 24 h and treated with DMSO or SU6668 in the presence or absence of Shh for additional 24 h or 48 h. Gli1 mRNA expression level was measured by qRT-PCR after 24 h and 48 h of treatment initiation (after 48 h and 72 h of transfection, respectively). For each data set obtained from the cells transfected with the same construct, the level of Gli1 mRNA expression was accepted as 1 in the control samples treated with DMSO. Data from other samples were calculated relative to their respectively transfected controls. The data are presented as an average mean of induction of Gli1 mRNA expression over control ± S.D. (D) C3H10T1/2 cells were transfected with pSUPER or Ulk3 shRNAs encoding constructs, incubated for 24 h and treated as indicated for additional 48 h. Levels of Gli1 and GAPDH proteins were examined by immuno-blotting (48 h post-treatment, 72 h post-transfection); *—p

    Journal: Biochimica et Biophysica Acta

    Article Title: Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells

    doi: 10.1016/j.bbamcr.2014.01.003

    Figure Lengend Snippet: (A) C3H10T1/2 cells were transfected with empty vector pSUPER or Ulk3 shRNAs and incubated during the indicated periods of time. Expression levels of Ulk3 (a) and Gli2 (b) were analyzed using qRT-PCR. The normalized with Hprt level of the respective gene expression at the indicated time point was set as 1 in the control cells transfected with pSUPER, and the data from the samples transfected with Ulk3 shRNAs were calculated relative to the control. The data are presented as an average mean ± S.D. (B) Expression of Gli2 and GAPDH proteins was analyzed by Western-blotting at the indicated time points in the post-transfectional C3H10T1/2 cells electroporated with empty vector pSUPER or Ulk3 shRNAs. Gli2 was detected using AF3635 antibody. (C) C3H10T1/2 cells were electroporated with pSUPER or Ulk3 shRNAs encoding constructs, incubated for 24 h and treated with DMSO or SU6668 in the presence or absence of Shh for additional 24 h or 48 h. Gli1 mRNA expression level was measured by qRT-PCR after 24 h and 48 h of treatment initiation (after 48 h and 72 h of transfection, respectively). For each data set obtained from the cells transfected with the same construct, the level of Gli1 mRNA expression was accepted as 1 in the control samples treated with DMSO. Data from other samples were calculated relative to their respectively transfected controls. The data are presented as an average mean of induction of Gli1 mRNA expression over control ± S.D. (D) C3H10T1/2 cells were transfected with pSUPER or Ulk3 shRNAs encoding constructs, incubated for 24 h and treated as indicated for additional 48 h. Levels of Gli1 and GAPDH proteins were examined by immuno-blotting (48 h post-treatment, 72 h post-transfection); *—p

    Article Snippet: SU6668 was dissolved in DMSO (both from Sigma-Aldrich, Steinheim, Germany) and stored at − 70 °C prior use.

    Techniques: Transfection, Plasmid Preparation, Incubation, Expressing, Quantitative RT-PCR, Western Blot, Construct

    SU6668 prevents de novo generation of GLI1 and GLI2 proteins in ASCs. (A) ASCs were treated with Shh, TGF-β1 and 20 μM SU6668 or DMSO. (a) Levels of GLI2 and GLI1 transcripts were measured using qPCR. Data are presented as an average mean ± S.D.; *—p

    Journal: Biochimica et Biophysica Acta

    Article Title: Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells

    doi: 10.1016/j.bbamcr.2014.01.003

    Figure Lengend Snippet: SU6668 prevents de novo generation of GLI1 and GLI2 proteins in ASCs. (A) ASCs were treated with Shh, TGF-β1 and 20 μM SU6668 or DMSO. (a) Levels of GLI2 and GLI1 transcripts were measured using qPCR. Data are presented as an average mean ± S.D.; *—p

    Article Snippet: SU6668 was dissolved in DMSO (both from Sigma-Aldrich, Steinheim, Germany) and stored at − 70 °C prior use.

    Techniques: Real-time Polymerase Chain Reaction

    (A, B) MDA-MB-231 cells were treated with 10 ng/ml of TGF-β3 in the presence of DMSO or 20 μM SU6668 during the indicated periods of time. Expression levels of ULK3 , SNAI1 , CSMAD7 and PAI were analyzed by qPCR, normalized with GAPDH mRNA levels and set as 1 in the control cells treated with DMSO at the respective time point (indicated as a baseline on panel B). The data from other samples were calculated relative to control. The data are presented as an average mean ± S.D.; *—p

    Journal: Biochimica et Biophysica Acta

    Article Title: Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells

    doi: 10.1016/j.bbamcr.2014.01.003

    Figure Lengend Snippet: (A, B) MDA-MB-231 cells were treated with 10 ng/ml of TGF-β3 in the presence of DMSO or 20 μM SU6668 during the indicated periods of time. Expression levels of ULK3 , SNAI1 , CSMAD7 and PAI were analyzed by qPCR, normalized with GAPDH mRNA levels and set as 1 in the control cells treated with DMSO at the respective time point (indicated as a baseline on panel B). The data from other samples were calculated relative to control. The data are presented as an average mean ± S.D.; *—p

    Article Snippet: SU6668 was dissolved in DMSO (both from Sigma-Aldrich, Steinheim, Germany) and stored at − 70 °C prior use.

    Techniques: Multiple Displacement Amplification, Expressing, Real-time Polymerase Chain Reaction

    SU6668 inhibits Shh dependent up-regulation of Gli1 via Ulk3. (A, B, C, D) 3 T3-L1 cells were transfected with Ulk3 -specific Silencer® Select siRNAs S89965 and S89966 using reverse followed by forward procedures resulted in single and double transfection, respectively. The cells were treated with DMSO, 20 μM SU6668 and Shh, if specified, and incubated during the indicated periods of time. (A) Levels of Ulk3 (a) and Gli2 (b) transcripts were measured by qPCR, normalized with Hprt mRNA expression level and set as 1 in the cells transfected with Negative siRNA. The data from samples transfected with Ulk3 -specific siRNAs were calculated relative to the negative control. (B) The cells were subjected to WB analysis after the indicated periods of time of the siRNA delivery using Gli2 G-20 antibody. (C) The cells subjected to double transfection by siRNAs were treated with Shh, DMSO or SU6668 for 24 and 48 h. Gli1 mRNA expression level was measured by qPCR, normalized with Hprt expression level and set as 1 in the respectively transfected control sample treated with DMSO. Gli1 mRNA expression level in the Shh-treated samples were calculated relative to the control. The data are presented as fold of induction of Gli1 mRNA level. Data of qPCR analyses are presented as an average mean ± S.D.; *—p

    Journal: Biochimica et Biophysica Acta

    Article Title: Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells

    doi: 10.1016/j.bbamcr.2014.01.003

    Figure Lengend Snippet: SU6668 inhibits Shh dependent up-regulation of Gli1 via Ulk3. (A, B, C, D) 3 T3-L1 cells were transfected with Ulk3 -specific Silencer® Select siRNAs S89965 and S89966 using reverse followed by forward procedures resulted in single and double transfection, respectively. The cells were treated with DMSO, 20 μM SU6668 and Shh, if specified, and incubated during the indicated periods of time. (A) Levels of Ulk3 (a) and Gli2 (b) transcripts were measured by qPCR, normalized with Hprt mRNA expression level and set as 1 in the cells transfected with Negative siRNA. The data from samples transfected with Ulk3 -specific siRNAs were calculated relative to the negative control. (B) The cells were subjected to WB analysis after the indicated periods of time of the siRNA delivery using Gli2 G-20 antibody. (C) The cells subjected to double transfection by siRNAs were treated with Shh, DMSO or SU6668 for 24 and 48 h. Gli1 mRNA expression level was measured by qPCR, normalized with Hprt expression level and set as 1 in the respectively transfected control sample treated with DMSO. Gli1 mRNA expression level in the Shh-treated samples were calculated relative to the control. The data are presented as fold of induction of Gli1 mRNA level. Data of qPCR analyses are presented as an average mean ± S.D.; *—p

    Article Snippet: SU6668 was dissolved in DMSO (both from Sigma-Aldrich, Steinheim, Germany) and stored at − 70 °C prior use.

    Techniques: Transfection, Incubation, Real-time Polymerase Chain Reaction, Expressing, Negative Control, Western Blot

    SU6668 hinders Shh signaling in C3H10T1/2 cells. (A, B) C3H10T1/2 cells were treated as indicated for 42 h. The levels of expression of Gli1 , Ptch1 , Gli2 and Ulk3 were analyzed by qPCR and normalized by the level of Hprt mRNA expression. The normalized level of the respective gene mRNA in the control sample treated with DMSO was set as 1. The data are presented as an average mean ± S.D.; *—p

    Journal: Biochimica et Biophysica Acta

    Article Title: Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells

    doi: 10.1016/j.bbamcr.2014.01.003

    Figure Lengend Snippet: SU6668 hinders Shh signaling in C3H10T1/2 cells. (A, B) C3H10T1/2 cells were treated as indicated for 42 h. The levels of expression of Gli1 , Ptch1 , Gli2 and Ulk3 were analyzed by qPCR and normalized by the level of Hprt mRNA expression. The normalized level of the respective gene mRNA in the control sample treated with DMSO was set as 1. The data are presented as an average mean ± S.D.; *—p

    Article Snippet: SU6668 was dissolved in DMSO (both from Sigma-Aldrich, Steinheim, Germany) and stored at − 70 °C prior use.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    TGF-β induced positive regulation of GLI2 protein is inhibited by SU6668 in MDA-MB-231 cells. (A, B) MDA-MB-231 cells were treated with DMSO or 20 μM SU6668 in the presence of TGF-β3 during the indicated periods of time. (A) GLI2 (a), ULK3 (b) and GLI1 (c) mRNA levels were analyzed by qPCR, normalized with GAPDH expression level and set as 1 in the control cells treated with DMSO (indicated by baseline on the panels a and b). The level of GLI1 mRNA expression in the cells treated with TGF-β was set as 100%. The data are presented as an average fold of induction of gene expression level over a control ± S.D. (B) Whole cell lysates were subjected to WB analysis. GLI2 protein was detected using AF3635 antibody. (C) MDA-MB-231 cells were transfected either with plasmids encoding mouse mGli2 or human hGLI2. The levels of the indicated gene expression were measured by qPCR after 18 h. The data are presented as an average fold of induction ± S.D. of the particular gene expression level over a control level measured in the cells transfected with an empty vector; (A, C) *—p

    Journal: Biochimica et Biophysica Acta

    Article Title: Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells

    doi: 10.1016/j.bbamcr.2014.01.003

    Figure Lengend Snippet: TGF-β induced positive regulation of GLI2 protein is inhibited by SU6668 in MDA-MB-231 cells. (A, B) MDA-MB-231 cells were treated with DMSO or 20 μM SU6668 in the presence of TGF-β3 during the indicated periods of time. (A) GLI2 (a), ULK3 (b) and GLI1 (c) mRNA levels were analyzed by qPCR, normalized with GAPDH expression level and set as 1 in the control cells treated with DMSO (indicated by baseline on the panels a and b). The level of GLI1 mRNA expression in the cells treated with TGF-β was set as 100%. The data are presented as an average fold of induction of gene expression level over a control ± S.D. (B) Whole cell lysates were subjected to WB analysis. GLI2 protein was detected using AF3635 antibody. (C) MDA-MB-231 cells were transfected either with plasmids encoding mouse mGli2 or human hGLI2. The levels of the indicated gene expression were measured by qPCR after 18 h. The data are presented as an average fold of induction ± S.D. of the particular gene expression level over a control level measured in the cells transfected with an empty vector; (A, C) *—p

    Article Snippet: SU6668 was dissolved in DMSO (both from Sigma-Aldrich, Steinheim, Germany) and stored at − 70 °C prior use.

    Techniques: Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Plasmid Preparation

    (A, B) 3 T3-L1 cells were treated as indicated for 30 h, trypsinized and split for total RNA and protein isolation. (A) Expression levels of GIH , Ptchl , Gli2 and Ulk3 were analyzed by qPCR and normalized with the level of Hprt expression. The level of the respective gene mRNA expression in the control sample was set as 1, and the data from other samples were calculated relative to 1. The level of Gli1 and Ptch1 expressions in the samples treated with Shh and DMSO was set as 100%. Gli1 and Ptch1 expression levels in the samples treated Shh and SU6668 were calculated relative to 100%. The data are presented as an average mean ± S.D.; *—p

    Journal: Biochimica et Biophysica Acta

    Article Title: Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells

    doi: 10.1016/j.bbamcr.2014.01.003

    Figure Lengend Snippet: (A, B) 3 T3-L1 cells were treated as indicated for 30 h, trypsinized and split for total RNA and protein isolation. (A) Expression levels of GIH , Ptchl , Gli2 and Ulk3 were analyzed by qPCR and normalized with the level of Hprt expression. The level of the respective gene mRNA expression in the control sample was set as 1, and the data from other samples were calculated relative to 1. The level of Gli1 and Ptch1 expressions in the samples treated with Shh and DMSO was set as 100%. Gli1 and Ptch1 expression levels in the samples treated Shh and SU6668 were calculated relative to 100%. The data are presented as an average mean ± S.D.; *—p

    Article Snippet: SU6668 was dissolved in DMSO (both from Sigma-Aldrich, Steinheim, Germany) and stored at − 70 °C prior use.

    Techniques: Isolation, Expressing, Real-time Polymerase Chain Reaction

    Quantitative real-time PCR analysis of select targets regulated by β-D-glucan in MCF-7 cells. Cells were grown in phenol red-free IMEM + 5% DCC for 48 h prior to addition of DMSO (vehicle control), 10 nM E 2 , 100 nM 4-OHT or the indicated concentrations of DMSO-dissolved β-D-glucan for 24 h. qPCR for each target gene was normalized to 18S rRNA and values were compared to fold expression in vehicle (DMSO)-treated MCF-7 cells. Values are the average of triplicate determinations ± SEM within one experiment.

    Journal: International Journal of Oncology

    Article Title: ?-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression

    doi: 10.3892/ijo.2014.2294

    Figure Lengend Snippet: Quantitative real-time PCR analysis of select targets regulated by β-D-glucan in MCF-7 cells. Cells were grown in phenol red-free IMEM + 5% DCC for 48 h prior to addition of DMSO (vehicle control), 10 nM E 2 , 100 nM 4-OHT or the indicated concentrations of DMSO-dissolved β-D-glucan for 24 h. qPCR for each target gene was normalized to 18S rRNA and values were compared to fold expression in vehicle (DMSO)-treated MCF-7 cells. Values are the average of triplicate determinations ± SEM within one experiment.

    Article Snippet: ICI 182,780 was from Tocris (Ellisville, MO, USA). β-D-glucan was purchased from Sigma (cat. no. G6513, purity ∼97%). β-D-glucan was dissolved either in water or in DMSO (Sigma) by heating in a waterbath at 90°C for 4–5 min. Once dissolved, the β-D-glucan stocks were stored at −20°C until use.

    Techniques: Real-time Polymerase Chain Reaction, Droplet Countercurrent Chromatography, Expressing

    β-D-glucan dissolved in DMSO but not water inhibits MCF-7 cell proliferation. MCF-7 cells were incubated in phenol red-free IMEM + 5% DCC and the indicated concentrations of β-D-glucan dissolved in water or DMSO for a total of 72 h with a medium/treatment change after 48 h. Values are the mean ± SEM for 4 separate values in one experiment for β-D-glucan in water and 6 separate experiments (biological replicates) for β-D-glucan in DMSO. Values of β-D-glucan in DMSO were corrected for the inhibitory effect of DMSO on cell proliferation. * p

    Journal: International Journal of Oncology

    Article Title: ?-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression

    doi: 10.3892/ijo.2014.2294

    Figure Lengend Snippet: β-D-glucan dissolved in DMSO but not water inhibits MCF-7 cell proliferation. MCF-7 cells were incubated in phenol red-free IMEM + 5% DCC and the indicated concentrations of β-D-glucan dissolved in water or DMSO for a total of 72 h with a medium/treatment change after 48 h. Values are the mean ± SEM for 4 separate values in one experiment for β-D-glucan in water and 6 separate experiments (biological replicates) for β-D-glucan in DMSO. Values of β-D-glucan in DMSO were corrected for the inhibitory effect of DMSO on cell proliferation. * p

    Article Snippet: ICI 182,780 was from Tocris (Ellisville, MO, USA). β-D-glucan was purchased from Sigma (cat. no. G6513, purity ∼97%). β-D-glucan was dissolved either in water or in DMSO (Sigma) by heating in a waterbath at 90°C for 4–5 min. Once dissolved, the β-D-glucan stocks were stored at −20°C until use.

    Techniques: Incubation, Droplet Countercurrent Chromatography

    β-D-glucan increases apoptosis and cell death in MCF-7 and LCC9 cells. (A) MCF-7 tamoxifen-sensitive and LCC9 tamoxifen-resistant breast cancer cells were incubated in phenol red-free IMEM + 5% DCC for 48 h prior to addition of the indicated concentrations of β-D-glucan dissolved in DMSO or DMSO as vehicle control for 24 h. BAX and BCL2 mRNA transcript expression was normalized by GAPDH (B) and the fold relative to DMSO (vehicle control) was set to one. (B) qPCR for GAPDH expression is given as CT values. For (A) and (B), the values are the average ± SEM of triplicate determinations within one experiment. (C) MCF-7 and LCC9 cells were incubated in phenol red-free IMEM + 5% DCC and the indicated concentrations of β-D-glucan dissolved in DMSO or DMSO as vehicle control for 72 h with a medium/treatment change after 48 h. Live/Dead Viability/Cytotoxicity assay was performed as described in Materials and methods. Values are the % of dead cells measured by uptake of ethidium homodimer-1 and fluorescence emission at 645 nm. Values are the average of 4 replicates within one experiment. * p

    Journal: International Journal of Oncology

    Article Title: ?-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression

    doi: 10.3892/ijo.2014.2294

    Figure Lengend Snippet: β-D-glucan increases apoptosis and cell death in MCF-7 and LCC9 cells. (A) MCF-7 tamoxifen-sensitive and LCC9 tamoxifen-resistant breast cancer cells were incubated in phenol red-free IMEM + 5% DCC for 48 h prior to addition of the indicated concentrations of β-D-glucan dissolved in DMSO or DMSO as vehicle control for 24 h. BAX and BCL2 mRNA transcript expression was normalized by GAPDH (B) and the fold relative to DMSO (vehicle control) was set to one. (B) qPCR for GAPDH expression is given as CT values. For (A) and (B), the values are the average ± SEM of triplicate determinations within one experiment. (C) MCF-7 and LCC9 cells were incubated in phenol red-free IMEM + 5% DCC and the indicated concentrations of β-D-glucan dissolved in DMSO or DMSO as vehicle control for 72 h with a medium/treatment change after 48 h. Live/Dead Viability/Cytotoxicity assay was performed as described in Materials and methods. Values are the % of dead cells measured by uptake of ethidium homodimer-1 and fluorescence emission at 645 nm. Values are the average of 4 replicates within one experiment. * p

    Article Snippet: ICI 182,780 was from Tocris (Ellisville, MO, USA). β-D-glucan was purchased from Sigma (cat. no. G6513, purity ∼97%). β-D-glucan was dissolved either in water or in DMSO (Sigma) by heating in a waterbath at 90°C for 4–5 min. Once dissolved, the β-D-glucan stocks were stored at −20°C until use.

    Techniques: Incubation, Droplet Countercurrent Chromatography, Expressing, Real-time Polymerase Chain Reaction, Cytotoxicity Assay, Fluorescence

    Quantitative real-time PCR analysis of select targets regulated by β-D-glucan in MCF-7 and LCC9 cells. Cells were grown in phenol red-free IMEM + 5% DCC for 48 h prior to addition of DMSO (vehicle), 10 nM E 2 , 100 nM 4-OHT or the indicated concentrations of DMSO-dissolved β-D-glucan for 24 h. qPCR for each target gene was normalized to 18S and values were compared to fold expression in vehicle (DMSO)-treated MCF-7 cells. Values are the average of triplicate determinations ± SEM within one experiment. (A) RASSF1, CTNNB1, IGFBP3, AR and NRF1 transcript expression in MCF-7 cells relative to DMSO control. (B) CTNNB1 , (C) IGFBP3 , (D) RASSF1 and (E) ESR2 (ERβ) transcript expression in MCF-7 and LCC9 cells relative to DMSO control.

    Journal: International Journal of Oncology

    Article Title: ?-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression

    doi: 10.3892/ijo.2014.2294

    Figure Lengend Snippet: Quantitative real-time PCR analysis of select targets regulated by β-D-glucan in MCF-7 and LCC9 cells. Cells were grown in phenol red-free IMEM + 5% DCC for 48 h prior to addition of DMSO (vehicle), 10 nM E 2 , 100 nM 4-OHT or the indicated concentrations of DMSO-dissolved β-D-glucan for 24 h. qPCR for each target gene was normalized to 18S and values were compared to fold expression in vehicle (DMSO)-treated MCF-7 cells. Values are the average of triplicate determinations ± SEM within one experiment. (A) RASSF1, CTNNB1, IGFBP3, AR and NRF1 transcript expression in MCF-7 cells relative to DMSO control. (B) CTNNB1 , (C) IGFBP3 , (D) RASSF1 and (E) ESR2 (ERβ) transcript expression in MCF-7 and LCC9 cells relative to DMSO control.

    Article Snippet: ICI 182,780 was from Tocris (Ellisville, MO, USA). β-D-glucan was purchased from Sigma (cat. no. G6513, purity ∼97%). β-D-glucan was dissolved either in water or in DMSO (Sigma) by heating in a waterbath at 90°C for 4–5 min. Once dissolved, the β-D-glucan stocks were stored at −20°C until use.

    Techniques: Real-time Polymerase Chain Reaction, Droplet Countercurrent Chromatography, Expressing

    β-D-glucan inhibits MCF-10A but not HEK-293 cell proliferation. MCF-10A and HEK-293 cells were incubated in phenol red-free IMEM + 5% DCC and the indicated concentrations of β-D-glucan dissolved in DMSO for a total of 72 h with a medium/treatment change after 48 h. Values are the BrdU incorporation absorbances normalized to DMSO (zero) and are the mean ± SEM for 4 separate values in one experiment. Values of β-D-glucan in DMSO were corrected for the inhibitory effect of DMSO on cell proliferation. * p

    Journal: International Journal of Oncology

    Article Title: ?-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression

    doi: 10.3892/ijo.2014.2294

    Figure Lengend Snippet: β-D-glucan inhibits MCF-10A but not HEK-293 cell proliferation. MCF-10A and HEK-293 cells were incubated in phenol red-free IMEM + 5% DCC and the indicated concentrations of β-D-glucan dissolved in DMSO for a total of 72 h with a medium/treatment change after 48 h. Values are the BrdU incorporation absorbances normalized to DMSO (zero) and are the mean ± SEM for 4 separate values in one experiment. Values of β-D-glucan in DMSO were corrected for the inhibitory effect of DMSO on cell proliferation. * p

    Article Snippet: ICI 182,780 was from Tocris (Ellisville, MO, USA). β-D-glucan was purchased from Sigma (cat. no. G6513, purity ∼97%). β-D-glucan was dissolved either in water or in DMSO (Sigma) by heating in a waterbath at 90°C for 4–5 min. Once dissolved, the β-D-glucan stocks were stored at −20°C until use.

    Techniques: Incubation, Droplet Countercurrent Chromatography, BrdU Incorporation Assay

    β-D-glucan inhibits the proliferation of endocrine-resistant breast cancer cells. LCC9 and LY2 endocrine-resistant breast cancer cells (A) and MDA-MB-231 triple negative breast cancer cells (B) were incubated in phenol red-free IMEM + 5% DCC and the indicated concentrations of β-D-glucan dissolved in DMSO for a total of 72 h with a medium/treatment change after 48 h. Values are the BrdU incorporation absorbances normalized to DMSO (zero) and are the mean ± SEM for 3 separate experiments. Values of β-D-glucan in DMSO were corrected for the inhibitory effect of DMSO on cell proliferation. * p

    Journal: International Journal of Oncology

    Article Title: ?-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression

    doi: 10.3892/ijo.2014.2294

    Figure Lengend Snippet: β-D-glucan inhibits the proliferation of endocrine-resistant breast cancer cells. LCC9 and LY2 endocrine-resistant breast cancer cells (A) and MDA-MB-231 triple negative breast cancer cells (B) were incubated in phenol red-free IMEM + 5% DCC and the indicated concentrations of β-D-glucan dissolved in DMSO for a total of 72 h with a medium/treatment change after 48 h. Values are the BrdU incorporation absorbances normalized to DMSO (zero) and are the mean ± SEM for 3 separate experiments. Values of β-D-glucan in DMSO were corrected for the inhibitory effect of DMSO on cell proliferation. * p

    Article Snippet: ICI 182,780 was from Tocris (Ellisville, MO, USA). β-D-glucan was purchased from Sigma (cat. no. G6513, purity ∼97%). β-D-glucan was dissolved either in water or in DMSO (Sigma) by heating in a waterbath at 90°C for 4–5 min. Once dissolved, the β-D-glucan stocks were stored at −20°C until use.

    Techniques: Multiple Displacement Amplification, Incubation, Droplet Countercurrent Chromatography, BrdU Incorporation Assay

    β-D-glucan rapidly inhibits NRF1 expression in MCF-7 cells. MCF-7 cells were grown in phenol red-free IMEM + 5% DCC for 48 h prior to addition of the indicated concentrations of DMSO-dissolved β-D-glucan for 45 min. (A) qPCR for NRF1 mRNA expression was normalized to 18S rRNA. * p

    Journal: International Journal of Oncology

    Article Title: ?-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression

    doi: 10.3892/ijo.2014.2294

    Figure Lengend Snippet: β-D-glucan rapidly inhibits NRF1 expression in MCF-7 cells. MCF-7 cells were grown in phenol red-free IMEM + 5% DCC for 48 h prior to addition of the indicated concentrations of DMSO-dissolved β-D-glucan for 45 min. (A) qPCR for NRF1 mRNA expression was normalized to 18S rRNA. * p

    Article Snippet: ICI 182,780 was from Tocris (Ellisville, MO, USA). β-D-glucan was purchased from Sigma (cat. no. G6513, purity ∼97%). β-D-glucan was dissolved either in water or in DMSO (Sigma) by heating in a waterbath at 90°C for 4–5 min. Once dissolved, the β-D-glucan stocks were stored at −20°C until use.

    Techniques: Expressing, Droplet Countercurrent Chromatography, Real-time Polymerase Chain Reaction

    β-D-glucan does not synergize with 4-hydroxytamoxifen to inhibit cell proliferation. MCF-7 tamoxifen-sensitive and LY2 tamoxifen-resistant breast cancer cells were incubated in phenol red-free IMEM + 5% DCC and the indicated concentrations of β-D-glucan dissolved in DMSO, 1 μ M 4-OHT, or the combination of 1 μ M 4-OHT + 10 or 50 μ g/ml β-D-glucan, as indicated, for a total of 72 h with a medium/treatment change after 48 h. Values are the BrdU incorporation absorbances normalized to DMSO (zero) and are the mean ± SEM for 3 separate experiments. Values of β-D-glucan in DMSO were corrected for the inhibitory effect of DMSO on cell proliferation. * p

    Journal: International Journal of Oncology

    Article Title: ?-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression

    doi: 10.3892/ijo.2014.2294

    Figure Lengend Snippet: β-D-glucan does not synergize with 4-hydroxytamoxifen to inhibit cell proliferation. MCF-7 tamoxifen-sensitive and LY2 tamoxifen-resistant breast cancer cells were incubated in phenol red-free IMEM + 5% DCC and the indicated concentrations of β-D-glucan dissolved in DMSO, 1 μ M 4-OHT, or the combination of 1 μ M 4-OHT + 10 or 50 μ g/ml β-D-glucan, as indicated, for a total of 72 h with a medium/treatment change after 48 h. Values are the BrdU incorporation absorbances normalized to DMSO (zero) and are the mean ± SEM for 3 separate experiments. Values of β-D-glucan in DMSO were corrected for the inhibitory effect of DMSO on cell proliferation. * p

    Article Snippet: ICI 182,780 was from Tocris (Ellisville, MO, USA). β-D-glucan was purchased from Sigma (cat. no. G6513, purity ∼97%). β-D-glucan was dissolved either in water or in DMSO (Sigma) by heating in a waterbath at 90°C for 4–5 min. Once dissolved, the β-D-glucan stocks were stored at −20°C until use.

    Techniques: Incubation, Droplet Countercurrent Chromatography, BrdU Incorporation Assay

    β-D-glucan affects ERα expression in MCF-7 and LCC9 cells. Cells were grown in phenol red-free IMEM + 5% DCC-stripped FBS for 48 h prior to addition of DMSO (vehicle control) or 10 or 50 μ g/ml β-D-glucan dissolved in DMSO for 24 h. (A) ESR1 transcript levels were measured by qPCR relative to 18S and are the average of triplicate determinations ± SEM within one experiment. Values are relative to MCF-7 cells treated with DMSO showing that ERα mRNA expression is lower in LCC9 relative to MCF-7 cells. (B) Whole cell extracts (30 μ g protein) were separated on 10% SDS-PAGE gels and the resulting western blot was probed with ERα antibody and the full length 66 kDa ERα band is shown. The PVDF membrane was stripped and re-probed for β-actin for normalization. Values are the ERα/β-actin ratio.

    Journal: International Journal of Oncology

    Article Title: ?-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression

    doi: 10.3892/ijo.2014.2294

    Figure Lengend Snippet: β-D-glucan affects ERα expression in MCF-7 and LCC9 cells. Cells were grown in phenol red-free IMEM + 5% DCC-stripped FBS for 48 h prior to addition of DMSO (vehicle control) or 10 or 50 μ g/ml β-D-glucan dissolved in DMSO for 24 h. (A) ESR1 transcript levels were measured by qPCR relative to 18S and are the average of triplicate determinations ± SEM within one experiment. Values are relative to MCF-7 cells treated with DMSO showing that ERα mRNA expression is lower in LCC9 relative to MCF-7 cells. (B) Whole cell extracts (30 μ g protein) were separated on 10% SDS-PAGE gels and the resulting western blot was probed with ERα antibody and the full length 66 kDa ERα band is shown. The PVDF membrane was stripped and re-probed for β-actin for normalization. Values are the ERα/β-actin ratio.

    Article Snippet: ICI 182,780 was from Tocris (Ellisville, MO, USA). β-D-glucan was purchased from Sigma (cat. no. G6513, purity ∼97%). β-D-glucan was dissolved either in water or in DMSO (Sigma) by heating in a waterbath at 90°C for 4–5 min. Once dissolved, the β-D-glucan stocks were stored at −20°C until use.

    Techniques: Expressing, Droplet Countercurrent Chromatography, Real-time Polymerase Chain Reaction, SDS Page, Western Blot

    Sequential addition of curcuminoids to 5% BSA or FCS . A) Solid or DMSO-dissolved curcuminoids were added individually or sequentially to 5% BSA (left bracket) or FCS (right bracket) as described in Methods. After each addition the concentration of total soluble curcumin in the protein solutions was determined. B) Elution profiles of curcuminoids after single (DMSO, SOLID) or sequential (DMSO-SOLID, SOLID-DMSO) addition of DMSO-dissolved (DMSO) or solid (SOLID) curcuminoids to 5% BSA. C) Elution profiles of curcuminoids after single or sequential addition of DMSO-dissolved or solid curcuminoids to FCS. Vertical bars represent mAUs and individual curcuminoids are designated as described in Fig. 1C.

    Journal: BMC Biotechnology

    Article Title: Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications

    doi: 10.1186/1472-6750-8-84

    Figure Lengend Snippet: Sequential addition of curcuminoids to 5% BSA or FCS . A) Solid or DMSO-dissolved curcuminoids were added individually or sequentially to 5% BSA (left bracket) or FCS (right bracket) as described in Methods. After each addition the concentration of total soluble curcumin in the protein solutions was determined. B) Elution profiles of curcuminoids after single (DMSO, SOLID) or sequential (DMSO-SOLID, SOLID-DMSO) addition of DMSO-dissolved (DMSO) or solid (SOLID) curcuminoids to 5% BSA. C) Elution profiles of curcuminoids after single or sequential addition of DMSO-dissolved or solid curcuminoids to FCS. Vertical bars represent mAUs and individual curcuminoids are designated as described in Fig. 1C.

    Article Snippet: BSA [Fraction V, 96–99% albumin], curcuminoids ('curcumin' > 95%) [Fluka], and DMSO [ACS reagent] were obtained from Sigma Chemical Company.

    Techniques: Concentration Assay

    Cell culture media preparation and curcuminoid solubilization in FCS produced by different mixing techniques . A) Solutions for cell culture media were prepared by adding either solid (white bars) or DMSO-dissolved (500 mM) curcuminoids (gray bars) to FCS. The FCS was subsequently added to DMEM at a final concentration of 5%. The total curcuminoid concentrations in the media were then determined by butanol extraction. The measured (meas.) values were compared to the calculated (calc.) values (brackets) for both media prepared with solid (white bars) and DMSO-dissolved (gray bars) curcuminoids. On the right side of the panel DMSO-dissolved (gray bars) or solid curcuminoids (white bars) were mixed with FCS either by stirring or by rotation. The total amount of total curcuminoids solubilized by either method was determined spectrophotometrically. B) Elution profiles after separation by reversed phase chromatography of DMSO-dissolved curcuminoids solubilized in FCS, mixed either by stirring (right) or rotation (left). C) Elution profiles of solid curcuminoids solubilized in FCS, mixed either by stirring (right) or rotation (left).

    Journal: BMC Biotechnology

    Article Title: Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications

    doi: 10.1186/1472-6750-8-84

    Figure Lengend Snippet: Cell culture media preparation and curcuminoid solubilization in FCS produced by different mixing techniques . A) Solutions for cell culture media were prepared by adding either solid (white bars) or DMSO-dissolved (500 mM) curcuminoids (gray bars) to FCS. The FCS was subsequently added to DMEM at a final concentration of 5%. The total curcuminoid concentrations in the media were then determined by butanol extraction. The measured (meas.) values were compared to the calculated (calc.) values (brackets) for both media prepared with solid (white bars) and DMSO-dissolved (gray bars) curcuminoids. On the right side of the panel DMSO-dissolved (gray bars) or solid curcuminoids (white bars) were mixed with FCS either by stirring or by rotation. The total amount of total curcuminoids solubilized by either method was determined spectrophotometrically. B) Elution profiles after separation by reversed phase chromatography of DMSO-dissolved curcuminoids solubilized in FCS, mixed either by stirring (right) or rotation (left). C) Elution profiles of solid curcuminoids solubilized in FCS, mixed either by stirring (right) or rotation (left).

    Article Snippet: BSA [Fraction V, 96–99% albumin], curcuminoids ('curcumin' > 95%) [Fluka], and DMSO [ACS reagent] were obtained from Sigma Chemical Company.

    Techniques: Cell Culture, Produced, Concentration Assay, Reversed-phase Chromatography

    Curcuminoid solubility in mammalian sera . Either 30 mg solid (white bars), or 10 μl of DMSO-dissolved curcuminoids at a 500 mM concentration (gray bars) were added to 1 ml of horse, rat, rabbit, human, fetal calf (FCS) serum or 5% BSA solution. Brackets on top of the bars show total protein/albumin concentrations as provided by the supplier's data sheets.

    Journal: BMC Biotechnology

    Article Title: Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications

    doi: 10.1186/1472-6750-8-84

    Figure Lengend Snippet: Curcuminoid solubility in mammalian sera . Either 30 mg solid (white bars), or 10 μl of DMSO-dissolved curcuminoids at a 500 mM concentration (gray bars) were added to 1 ml of horse, rat, rabbit, human, fetal calf (FCS) serum or 5% BSA solution. Brackets on top of the bars show total protein/albumin concentrations as provided by the supplier's data sheets.

    Article Snippet: BSA [Fraction V, 96–99% albumin], curcuminoids ('curcumin' > 95%) [Fluka], and DMSO [ACS reagent] were obtained from Sigma Chemical Company.

    Techniques: Solubility, Concentration Assay

    Stability of curcuminoids in cell culture media . A) Stability of curcuminoids in media prepared with solid curcuminoids. The media were either stored at 4°C (○) or in a cell culture incubator at 37°C, with (△) or without (▽) exposure to 5% CO 2 . B) Elution profiles after reversed phase chromatography of media prepared with solid curcuminoids at day 0, day 3, and day 7 of incubation at 37°C with 5% CO 2 . C) Same as in panel A, except that the media were prepared with DMSO-dissolved curcuminoids. The media were stored at 4°C (●) or in a cell culture incubator at 37°C with (▲) or without (▼) 5% CO 2 . D) Same as in panel B except that the media were prepared with DMSO-dissolved curcuminoids.

    Journal: BMC Biotechnology

    Article Title: Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications

    doi: 10.1186/1472-6750-8-84

    Figure Lengend Snippet: Stability of curcuminoids in cell culture media . A) Stability of curcuminoids in media prepared with solid curcuminoids. The media were either stored at 4°C (○) or in a cell culture incubator at 37°C, with (△) or without (▽) exposure to 5% CO 2 . B) Elution profiles after reversed phase chromatography of media prepared with solid curcuminoids at day 0, day 3, and day 7 of incubation at 37°C with 5% CO 2 . C) Same as in panel A, except that the media were prepared with DMSO-dissolved curcuminoids. The media were stored at 4°C (●) or in a cell culture incubator at 37°C with (▲) or without (▼) 5% CO 2 . D) Same as in panel B except that the media were prepared with DMSO-dissolved curcuminoids.

    Article Snippet: BSA [Fraction V, 96–99% albumin], curcuminoids ('curcumin' > 95%) [Fluka], and DMSO [ACS reagent] were obtained from Sigma Chemical Company.

    Techniques: Cell Culture, Reversed-phase Chromatography, Incubation

    Solubility of curcuminoids in FCS . A) Total curcuminoid solubility (μM) in 1 ml of FCS as a function of adding increasing amounts (1–70 mg) of solid curcuminoids. B) Curcuminoid solubility (μM) in 1 ml of FCS as a function of adding 10 μl aliquots of DMSO-dissolved curcuminoids at concentrations ranging from 10 to 500 mM.

    Journal: BMC Biotechnology

    Article Title: Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications

    doi: 10.1186/1472-6750-8-84

    Figure Lengend Snippet: Solubility of curcuminoids in FCS . A) Total curcuminoid solubility (μM) in 1 ml of FCS as a function of adding increasing amounts (1–70 mg) of solid curcuminoids. B) Curcuminoid solubility (μM) in 1 ml of FCS as a function of adding 10 μl aliquots of DMSO-dissolved curcuminoids at concentrations ranging from 10 to 500 mM.

    Article Snippet: BSA [Fraction V, 96–99% albumin], curcuminoids ('curcumin' > 95%) [Fluka], and DMSO [ACS reagent] were obtained from Sigma Chemical Company.

    Techniques: Solubility

    Solubility of curcuminoids in BSA solutions . A) Curcuminoid solubility (μM) in 1 ml of 5% BSA as a function of adding increasing amounts (1–70 mg) of solid curcumin. B) Curcuminoid solubility (μM) in 1 ml of 5% BSA as a function of adding 10 μl aliquots of DMSO-dissolved curcuminoids at concentrations ranging from 10 to 500 mM. C) Curcuminoid solubility (μM) as a function of adding 30 mg solid curcuminoids to 1 ml of BSA solutions ranging in concentrations from 0.2 to 10%. D) Curcuminoid solubility (μM) as a function of adding 10 μl aliquots 500 mM DMSO-solubilized curcuminoids to 1 ml of BSA solutions ranging in concentrations from 0.2 to 20%.

    Journal: BMC Biotechnology

    Article Title: Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications

    doi: 10.1186/1472-6750-8-84

    Figure Lengend Snippet: Solubility of curcuminoids in BSA solutions . A) Curcuminoid solubility (μM) in 1 ml of 5% BSA as a function of adding increasing amounts (1–70 mg) of solid curcumin. B) Curcuminoid solubility (μM) in 1 ml of 5% BSA as a function of adding 10 μl aliquots of DMSO-dissolved curcuminoids at concentrations ranging from 10 to 500 mM. C) Curcuminoid solubility (μM) as a function of adding 30 mg solid curcuminoids to 1 ml of BSA solutions ranging in concentrations from 0.2 to 10%. D) Curcuminoid solubility (μM) as a function of adding 10 μl aliquots 500 mM DMSO-solubilized curcuminoids to 1 ml of BSA solutions ranging in concentrations from 0.2 to 20%.

    Article Snippet: BSA [Fraction V, 96–99% albumin], curcuminoids ('curcumin' > 95%) [Fluka], and DMSO [ACS reagent] were obtained from Sigma Chemical Company.

    Techniques: Solubility

    Elution profiles of curcuminoids separated by reversed phase chromatography . A) 30 mg of solid (left profile) curcuminoids or 10 μl of 500 mM DMSO-dissolved (right profile) curcuminoids solubilized in 1 ml of FCS. B) 30 mg of solid (left profile) curcuminoids or 10 μl of 500 mM DMSO-dissolved (right profile) curcuminoids solubilized in 1 ml of 5% BSA. C) 30 mg of solid curcuminoids solubilized in 1 ml of 1% BSA (left profile) or 20% BSA (right profile). Vertical bars represent mAUs and individual curcuminoids are designated as described in Fig. 1C.

    Journal: BMC Biotechnology

    Article Title: Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications

    doi: 10.1186/1472-6750-8-84

    Figure Lengend Snippet: Elution profiles of curcuminoids separated by reversed phase chromatography . A) 30 mg of solid (left profile) curcuminoids or 10 μl of 500 mM DMSO-dissolved (right profile) curcuminoids solubilized in 1 ml of FCS. B) 30 mg of solid (left profile) curcuminoids or 10 μl of 500 mM DMSO-dissolved (right profile) curcuminoids solubilized in 1 ml of 5% BSA. C) 30 mg of solid curcuminoids solubilized in 1 ml of 1% BSA (left profile) or 20% BSA (right profile). Vertical bars represent mAUs and individual curcuminoids are designated as described in Fig. 1C.

    Article Snippet: BSA [Fraction V, 96–99% albumin], curcuminoids ('curcumin' > 95%) [Fluka], and DMSO [ACS reagent] were obtained from Sigma Chemical Company.

    Techniques: Reversed-phase Chromatography

    Sequential extraction of solid curcuminoids with 5% BSA . A) Solid curcuminoids in amounts of 10 mg (●), 20 mg (○), 30 mg (▼), 40 mg (▽), and 50 mg (■) were added to 1 ml of 5% BSA. After centrifugation, the supernatant containing BSA-soluble curcuminoids was removed and fresh 5% BSA was added to the remaining pellets containing insoluble curcuminoids (Methods). The procedure was repeated ten times and the concentration (μM) of total soluble curcuminoids determined. B) After the tenth extraction of solid curcumin with 5% BSA, the final pellets were washed and dissolved in 200 μl DMSO. A 10 μl aliquot of the DMSO-dissolved curcumin was added to 1 ml of 5% BSA and processed for concentration determination. The bars represent the concentration (μM) of total curcuminoids solubilized in 5% BSA. The numbers under the bars indicate the starting amount (mg) of the curcuminoids and the numbers in parentheses the final yield (mg) of DMSO-dissolved curcuminoids. C) Elution profiles of curcuminoids after first (ext.1), fifth (ext. 5), and tenth (ext. 10) extraction of 1 mg of solid curcuminoids. Also shown is the elution profile of the final pellet after ten extractions (residual) with 5% BSA. D) Same as in B, except that 50 mg of solid curcuminoids were sequentially extracted.

    Journal: BMC Biotechnology

    Article Title: Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications

    doi: 10.1186/1472-6750-8-84

    Figure Lengend Snippet: Sequential extraction of solid curcuminoids with 5% BSA . A) Solid curcuminoids in amounts of 10 mg (●), 20 mg (○), 30 mg (▼), 40 mg (▽), and 50 mg (■) were added to 1 ml of 5% BSA. After centrifugation, the supernatant containing BSA-soluble curcuminoids was removed and fresh 5% BSA was added to the remaining pellets containing insoluble curcuminoids (Methods). The procedure was repeated ten times and the concentration (μM) of total soluble curcuminoids determined. B) After the tenth extraction of solid curcumin with 5% BSA, the final pellets were washed and dissolved in 200 μl DMSO. A 10 μl aliquot of the DMSO-dissolved curcumin was added to 1 ml of 5% BSA and processed for concentration determination. The bars represent the concentration (μM) of total curcuminoids solubilized in 5% BSA. The numbers under the bars indicate the starting amount (mg) of the curcuminoids and the numbers in parentheses the final yield (mg) of DMSO-dissolved curcuminoids. C) Elution profiles of curcuminoids after first (ext.1), fifth (ext. 5), and tenth (ext. 10) extraction of 1 mg of solid curcuminoids. Also shown is the elution profile of the final pellet after ten extractions (residual) with 5% BSA. D) Same as in B, except that 50 mg of solid curcuminoids were sequentially extracted.

    Article Snippet: BSA [Fraction V, 96–99% albumin], curcuminoids ('curcumin' > 95%) [Fluka], and DMSO [ACS reagent] were obtained from Sigma Chemical Company.

    Techniques: Centrifugation, Concentration Assay

    Effect of curcuminoids on cell proliferation . A) Cell survival (%) after incubating HeLa cells in media without curcumin (○) or with media prepared with solid curcumin at concentrations of 10 (□), 20 (▽), 30 (△), 40 (◇), or 50 μM (+). The initial seeding density of the HeLa cells was about 20% confluence for all cells, which was assigned the relative value of 100%. Cells were counted daily for three days and the data points represent average cell numbers from four different viewing fields with standard deviations (error bars). B) Same as in panel A, except that cells were incubated in media prepared either without curcumin (●) or with medium prepared with DMSO-dissolved curcumin at concentrations of 10 (■), 20 (▼), 30 (▲), 40 (◆), or 50 μM (●).

    Journal: BMC Biotechnology

    Article Title: Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications

    doi: 10.1186/1472-6750-8-84

    Figure Lengend Snippet: Effect of curcuminoids on cell proliferation . A) Cell survival (%) after incubating HeLa cells in media without curcumin (○) or with media prepared with solid curcumin at concentrations of 10 (□), 20 (▽), 30 (△), 40 (◇), or 50 μM (+). The initial seeding density of the HeLa cells was about 20% confluence for all cells, which was assigned the relative value of 100%. Cells were counted daily for three days and the data points represent average cell numbers from four different viewing fields with standard deviations (error bars). B) Same as in panel A, except that cells were incubated in media prepared either without curcumin (●) or with medium prepared with DMSO-dissolved curcumin at concentrations of 10 (■), 20 (▼), 30 (▲), 40 (◆), or 50 μM (●).

    Article Snippet: BSA [Fraction V, 96–99% albumin], curcuminoids ('curcumin' > 95%) [Fluka], and DMSO [ACS reagent] were obtained from Sigma Chemical Company.

    Techniques: Incubation

    Immunoblot analysis of vimentin protein expression in PMA-induced U937 cells co-incubated with WIN55212-2 . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only (con) , 2.) Differentiation control = treatment with 25 nM PMA (0) , 3.) Experimental groups were treated additionally with 3 μM (3) or 5 μM (5) WIN55212-2, respectively. Vimentin protein expression was analysed by immunoblotting and quantified by Odyssey infrared imaging system (Li-Cor Biosciences) and shown as mean +/- SEM, n = 3. Relative intensities were normalized to a non-induced control (S0) and a PMA-induced control (S1) which served as internal control for quantification if different blot membranes have been used. *p

    Journal: Cell Communication and Signaling : CCS

    Article Title: The cannabinoid receptors agonist WIN55212-2 inhibits macrophageal differentiation and alters expression and phosphorylation of cell cycle control proteins

    doi: 10.1186/1478-811X-9-33

    Figure Lengend Snippet: Immunoblot analysis of vimentin protein expression in PMA-induced U937 cells co-incubated with WIN55212-2 . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only (con) , 2.) Differentiation control = treatment with 25 nM PMA (0) , 3.) Experimental groups were treated additionally with 3 μM (3) or 5 μM (5) WIN55212-2, respectively. Vimentin protein expression was analysed by immunoblotting and quantified by Odyssey infrared imaging system (Li-Cor Biosciences) and shown as mean +/- SEM, n = 3. Relative intensities were normalized to a non-induced control (S0) and a PMA-induced control (S1) which served as internal control for quantification if different blot membranes have been used. *p

    Article Snippet: PMA was diluted in DMSO (Sigma-Aldrich), the concentration of DMSO was 1:1000 at all times in every group.

    Techniques: Expressing, Incubation, Imaging

    Quantitative real time PCR analysis of cdc2 and p21 mRNA expression in PMA-induced U937 cells co-incubated with WIN55212-2 . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only (con) , 2.) Differentiation control = treatment with 25 nM PMA (0) , 3.) Experimental groups were treated additionally with 3 μM (3) or 5 μM (5) WIN55212-2, respectively. cdc2 (figure 7a) and p21 (figure 7b) mRNA expression were analysed after different time points by quantitative real-time PCR. Data are shown as mean +/- SEM, n = 3. Relative expression were normalized to GAPDH expression in non-induced cells. *p

    Journal: Cell Communication and Signaling : CCS

    Article Title: The cannabinoid receptors agonist WIN55212-2 inhibits macrophageal differentiation and alters expression and phosphorylation of cell cycle control proteins

    doi: 10.1186/1478-811X-9-33

    Figure Lengend Snippet: Quantitative real time PCR analysis of cdc2 and p21 mRNA expression in PMA-induced U937 cells co-incubated with WIN55212-2 . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only (con) , 2.) Differentiation control = treatment with 25 nM PMA (0) , 3.) Experimental groups were treated additionally with 3 μM (3) or 5 μM (5) WIN55212-2, respectively. cdc2 (figure 7a) and p21 (figure 7b) mRNA expression were analysed after different time points by quantitative real-time PCR. Data are shown as mean +/- SEM, n = 3. Relative expression were normalized to GAPDH expression in non-induced cells. *p

    Article Snippet: PMA was diluted in DMSO (Sigma-Aldrich), the concentration of DMSO was 1:1000 at all times in every group.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Incubation

    WIN55212-2 altered phosphorylation of cdc2 protein . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only (con) , 2.) Differentiation control = treatment with 25 nM PMA (PMA) , 3.) Experimental groups were treated additionally with 3 μM (PMA + 3 μM WIN) or 5 μM (PMA + 5 μM WIN) WIN55212-2, respectively. Tyr15-cdc2 (figure 6a) and Thr161-cdc2 (figure 6b) phosphorylation was analysed by immunoblotting and quantified by Odyssey infrared imaging system (Li-Cor Biosciences) and shown as mean +/- SEM, n = 3. Relative intensities were normalized to non-induced control cells (con) and an internal control (S0) for quantification if different blot membranes have been used. *p

    Journal: Cell Communication and Signaling : CCS

    Article Title: The cannabinoid receptors agonist WIN55212-2 inhibits macrophageal differentiation and alters expression and phosphorylation of cell cycle control proteins

    doi: 10.1186/1478-811X-9-33

    Figure Lengend Snippet: WIN55212-2 altered phosphorylation of cdc2 protein . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only (con) , 2.) Differentiation control = treatment with 25 nM PMA (PMA) , 3.) Experimental groups were treated additionally with 3 μM (PMA + 3 μM WIN) or 5 μM (PMA + 5 μM WIN) WIN55212-2, respectively. Tyr15-cdc2 (figure 6a) and Thr161-cdc2 (figure 6b) phosphorylation was analysed by immunoblotting and quantified by Odyssey infrared imaging system (Li-Cor Biosciences) and shown as mean +/- SEM, n = 3. Relative intensities were normalized to non-induced control cells (con) and an internal control (S0) for quantification if different blot membranes have been used. *p

    Article Snippet: PMA was diluted in DMSO (Sigma-Aldrich), the concentration of DMSO was 1:1000 at all times in every group.

    Techniques: Incubation, Imaging

    Quantitative real time PCR analysis of CB1 and CB2 receptor mRNA expression in PMA-induced U937 cells co-incubated with WIN55212-2 . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only (con) , 2.) Differentiation control = treatment with 25 nM PMA (0) , 3.) Experimental groups were treated additionally with 3 μM (3) or 5 μM (5) WIN55212-2, respectively. One group was treated with 5 μM (5) WIN55212-2 and 2 μM WIN AM630 (CB2 inhibition). CB1 (figure 9a) and CB2 (figure 9b) receptor mRNA expression were analysed after different time points by quantitative real-time PCR. Data are shown as mean +/- SD, n = 3. Relative expression were normalized to GAPDH expression in non-induced cells. *p

    Journal: Cell Communication and Signaling : CCS

    Article Title: The cannabinoid receptors agonist WIN55212-2 inhibits macrophageal differentiation and alters expression and phosphorylation of cell cycle control proteins

    doi: 10.1186/1478-811X-9-33

    Figure Lengend Snippet: Quantitative real time PCR analysis of CB1 and CB2 receptor mRNA expression in PMA-induced U937 cells co-incubated with WIN55212-2 . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only (con) , 2.) Differentiation control = treatment with 25 nM PMA (0) , 3.) Experimental groups were treated additionally with 3 μM (3) or 5 μM (5) WIN55212-2, respectively. One group was treated with 5 μM (5) WIN55212-2 and 2 μM WIN AM630 (CB2 inhibition). CB1 (figure 9a) and CB2 (figure 9b) receptor mRNA expression were analysed after different time points by quantitative real-time PCR. Data are shown as mean +/- SD, n = 3. Relative expression were normalized to GAPDH expression in non-induced cells. *p

    Article Snippet: PMA was diluted in DMSO (Sigma-Aldrich), the concentration of DMSO was 1:1000 at all times in every group.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Incubation, Inhibition

    WIN55212-2 did not enhance PMA-induced apoptosis . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only, 2.) Differentiation control = treatment with 25 nM PMA, 3.) Experimental groups were treated additionally with 3 μM or 5 μM WIN55212-2, respectively. Differentiated (a) and non-differentiated cells (b) were separated due to its adherence after 72 h incubation and apoptotic cells were detected by annexin V staining and FACS analysis (figure 3a) or immunocytochemical analysis after counterstaining with DAPI (figure 2b). Annexin V-positive cells in the non-differentiated cell fraction are quantified in figure 3a and displayed in figure 3b. Immunocytochemical analysis of adherent cells are shown in figure 3c. Data are shown as mean +/- SEM, n = 12; *p

    Journal: Cell Communication and Signaling : CCS

    Article Title: The cannabinoid receptors agonist WIN55212-2 inhibits macrophageal differentiation and alters expression and phosphorylation of cell cycle control proteins

    doi: 10.1186/1478-811X-9-33

    Figure Lengend Snippet: WIN55212-2 did not enhance PMA-induced apoptosis . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only, 2.) Differentiation control = treatment with 25 nM PMA, 3.) Experimental groups were treated additionally with 3 μM or 5 μM WIN55212-2, respectively. Differentiated (a) and non-differentiated cells (b) were separated due to its adherence after 72 h incubation and apoptotic cells were detected by annexin V staining and FACS analysis (figure 3a) or immunocytochemical analysis after counterstaining with DAPI (figure 2b). Annexin V-positive cells in the non-differentiated cell fraction are quantified in figure 3a and displayed in figure 3b. Immunocytochemical analysis of adherent cells are shown in figure 3c. Data are shown as mean +/- SEM, n = 12; *p

    Article Snippet: PMA was diluted in DMSO (Sigma-Aldrich), the concentration of DMSO was 1:1000 at all times in every group.

    Techniques: Incubation, Staining, FACS

    Immunocytochemical analysis of vimentin expression in PMA-induced U937 cells co-incubated with WIN55212-2 . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only (a, b, c, d), 2.) Differentiation control = treatment with 25 nM PMA (e, f, g, h), 3.) Experimental groups were treated additionally with 3 μM (I, j, k, l) or 5 μM (m, n, o, p) WIN55212-2, respectively. Cells were stain against vimentin (a, e, i, m), actin (b, f, j, n) and DAPI (c, g, k, o) and analysed by confocal laser scanning microscopy. Merged pictures are shown (d, h, l, p). Scale bar = 15 μM.

    Journal: Cell Communication and Signaling : CCS

    Article Title: The cannabinoid receptors agonist WIN55212-2 inhibits macrophageal differentiation and alters expression and phosphorylation of cell cycle control proteins

    doi: 10.1186/1478-811X-9-33

    Figure Lengend Snippet: Immunocytochemical analysis of vimentin expression in PMA-induced U937 cells co-incubated with WIN55212-2 . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only (a, b, c, d), 2.) Differentiation control = treatment with 25 nM PMA (e, f, g, h), 3.) Experimental groups were treated additionally with 3 μM (I, j, k, l) or 5 μM (m, n, o, p) WIN55212-2, respectively. Cells were stain against vimentin (a, e, i, m), actin (b, f, j, n) and DAPI (c, g, k, o) and analysed by confocal laser scanning microscopy. Merged pictures are shown (d, h, l, p). Scale bar = 15 μM.

    Article Snippet: PMA was diluted in DMSO (Sigma-Aldrich), the concentration of DMSO was 1:1000 at all times in every group.

    Techniques: Expressing, Incubation, Staining, Confocal Laser Scanning Microscopy

    Immunoblot analysis of c-jun and cdc2 protein expression in PMA-induced U937 cells co-incubated with WIN55212-2 . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only (con) , 2.) Differentiation control = treatment with 25 nM PMA (0) , 3.) Experimental groups were treated additionally with 3 μM (3) or 5 μM (5) WIN55212-2, respectively. C-jun (figure 5a) and cdc2 (figure 5b) protein expression was analysed by immunoblotting and quantified by Odyssey infrared imaging system (Li-Cor Biosciences) and shown as mean +/- SEM, n = 3. Relative intensities were normalized to non-induced control cells (con) and an internal control (S0) for quantification if different blot membranes have been used. *p

    Journal: Cell Communication and Signaling : CCS

    Article Title: The cannabinoid receptors agonist WIN55212-2 inhibits macrophageal differentiation and alters expression and phosphorylation of cell cycle control proteins

    doi: 10.1186/1478-811X-9-33

    Figure Lengend Snippet: Immunoblot analysis of c-jun and cdc2 protein expression in PMA-induced U937 cells co-incubated with WIN55212-2 . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only (con) , 2.) Differentiation control = treatment with 25 nM PMA (0) , 3.) Experimental groups were treated additionally with 3 μM (3) or 5 μM (5) WIN55212-2, respectively. C-jun (figure 5a) and cdc2 (figure 5b) protein expression was analysed by immunoblotting and quantified by Odyssey infrared imaging system (Li-Cor Biosciences) and shown as mean +/- SEM, n = 3. Relative intensities were normalized to non-induced control cells (con) and an internal control (S0) for quantification if different blot membranes have been used. *p

    Article Snippet: PMA was diluted in DMSO (Sigma-Aldrich), the concentration of DMSO was 1:1000 at all times in every group.

    Techniques: Expressing, Incubation, Imaging

    WIN55212-2 inhibited differentiation of human monocytic U937 cells into an adherent phenotype . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only, 2.) Differentiation control = treatment with 25 nM PMA, 3.) Experimental groups were treated additionally with 3 μM or 5 μM WIN55212-2, respectively. Adherent (figure 1a) and non-adherent cells (figure 1b) were separated and quantified after different time points up to 72 h by FACS analysis. Percentage of control group at time point zero is shown. Data are shown as mean +/- SEM, n = 12; *p

    Journal: Cell Communication and Signaling : CCS

    Article Title: The cannabinoid receptors agonist WIN55212-2 inhibits macrophageal differentiation and alters expression and phosphorylation of cell cycle control proteins

    doi: 10.1186/1478-811X-9-33

    Figure Lengend Snippet: WIN55212-2 inhibited differentiation of human monocytic U937 cells into an adherent phenotype . Differentiation of U937 cells was induced by 25 nM PMA with or without co-incubation with WIN55212-2. The experimental groups were: 1.) Vehicle control = treatment with 0.1% DMSO only, 2.) Differentiation control = treatment with 25 nM PMA, 3.) Experimental groups were treated additionally with 3 μM or 5 μM WIN55212-2, respectively. Adherent (figure 1a) and non-adherent cells (figure 1b) were separated and quantified after different time points up to 72 h by FACS analysis. Percentage of control group at time point zero is shown. Data are shown as mean +/- SEM, n = 12; *p

    Article Snippet: PMA was diluted in DMSO (Sigma-Aldrich), the concentration of DMSO was 1:1000 at all times in every group.

    Techniques: Incubation, FACS

    Quantitative analysis of gene expression in human retinal pigment epithelial cell line (ARPE-19) cells after fenretinide treatment. Real-time PCR was employed to examine quantitative differences in mRNA expression between dimethyl sulfoxide (DMSO)- and fenretinide (FR)-treated cells. Data shown are mean relative expression levels±standard error of the mean after normalization to the geometric mean of Gapdh , Tubb, and β2m (n=4).

    Journal: Molecular Vision

    Article Title: The expression of retinal cell markers in human retinal pigment epithelial cells and their augmentation by the synthetic retinoid fenretinide

    doi:

    Figure Lengend Snippet: Quantitative analysis of gene expression in human retinal pigment epithelial cell line (ARPE-19) cells after fenretinide treatment. Real-time PCR was employed to examine quantitative differences in mRNA expression between dimethyl sulfoxide (DMSO)- and fenretinide (FR)-treated cells. Data shown are mean relative expression levels±standard error of the mean after normalization to the geometric mean of Gapdh , Tubb, and β2m (n=4).

    Article Snippet: The following day cells were washed and treated with low-serum medium containing 3 μM fenretinide (Tocris Bioscience, Bristol, UK) dissolved in DMSO (Sigma-Aldrich Company Ltd, Dorset, UK).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Analysis of cell markers in ARPE-19 cells by reverse transciptase PCR. Dimethyl sulfoxide (DMSO)- and fenretinide (FR)-treated cells express mRNAs normally associated with neuronal and retinal cells in culture and lose retinal pigment epithelium (RPE) cell markers. mRNA expression was detected in DMSO- and FR-treated cells using reverse transcriptase PCR. Primer pairs based on human opsins, and retinal, neuronal, and RPE cell marker sequences were used to amplify products from equal amounts of cDNA. A no-template RNA control (NTC) and a reaction lacking reverse transcriptase (-RT) was included for each amplification. Tbp was amplified as a positive loading control.

    Journal: Molecular Vision

    Article Title: The expression of retinal cell markers in human retinal pigment epithelial cells and their augmentation by the synthetic retinoid fenretinide

    doi:

    Figure Lengend Snippet: Analysis of cell markers in ARPE-19 cells by reverse transciptase PCR. Dimethyl sulfoxide (DMSO)- and fenretinide (FR)-treated cells express mRNAs normally associated with neuronal and retinal cells in culture and lose retinal pigment epithelium (RPE) cell markers. mRNA expression was detected in DMSO- and FR-treated cells using reverse transcriptase PCR. Primer pairs based on human opsins, and retinal, neuronal, and RPE cell marker sequences were used to amplify products from equal amounts of cDNA. A no-template RNA control (NTC) and a reaction lacking reverse transcriptase (-RT) was included for each amplification. Tbp was amplified as a positive loading control.

    Article Snippet: The following day cells were washed and treated with low-serum medium containing 3 μM fenretinide (Tocris Bioscience, Bristol, UK) dissolved in DMSO (Sigma-Aldrich Company Ltd, Dorset, UK).

    Techniques: Polymerase Chain Reaction, Expressing, Marker, Amplification

    Neuronal markers are expressed in retinal pigment epithelium cells in culture but are lost after culture on a porcine lens capsule membrane (PLC). The expression of neurofilament heavy and medium polypeptides (NF-H and NF-M respectively) varied in dimethyl sulfoxide (DMSO)-treated cells. A : The first two panels show the variation of NF-H expression within the same dish of DMSO-treated ARPE-19 cells, which is increased across the whole dish after fenretinide (FR) treatment. Similarly, the expression of NF-M varies within cells of the same field and is increased by fenretinide. B : Passage 2 primary human RPE cells and an additional human RPE cell line (h1RPE7) express the neuronal markers NF-H and synaptophysin (SYP). C : Culturing ARPE-19 on a PLC decreases the expression of neuronal markers in control cells and prevents the fenretinide-induced increase of neuronal markers. Protein staining is indicated by the color of the text (red or green). All scale bars equal 50 μm.

    Journal: Molecular Vision

    Article Title: The expression of retinal cell markers in human retinal pigment epithelial cells and their augmentation by the synthetic retinoid fenretinide

    doi:

    Figure Lengend Snippet: Neuronal markers are expressed in retinal pigment epithelium cells in culture but are lost after culture on a porcine lens capsule membrane (PLC). The expression of neurofilament heavy and medium polypeptides (NF-H and NF-M respectively) varied in dimethyl sulfoxide (DMSO)-treated cells. A : The first two panels show the variation of NF-H expression within the same dish of DMSO-treated ARPE-19 cells, which is increased across the whole dish after fenretinide (FR) treatment. Similarly, the expression of NF-M varies within cells of the same field and is increased by fenretinide. B : Passage 2 primary human RPE cells and an additional human RPE cell line (h1RPE7) express the neuronal markers NF-H and synaptophysin (SYP). C : Culturing ARPE-19 on a PLC decreases the expression of neuronal markers in control cells and prevents the fenretinide-induced increase of neuronal markers. Protein staining is indicated by the color of the text (red or green). All scale bars equal 50 μm.

    Article Snippet: The following day cells were washed and treated with low-serum medium containing 3 μM fenretinide (Tocris Bioscience, Bristol, UK) dissolved in DMSO (Sigma-Aldrich Company Ltd, Dorset, UK).

    Techniques: Planar Chromatography, Expressing, Staining

    Immunocytochemical analysis of neuronal and photoreceptor cell markers in ARPE-19 cells after treatment with dimethyl sulfoxide or fenretinide. A-K : ARPE-19 cells were treated with 3 μM fenretinide or DMSO and processed for immunocytochemistry. The nuclei of cells are stained with 4',6-diamidino-2-phenylindole (blue) with the exception of cells where protein expression was detected within the nucleus (OPN4, CRX, and PAX6). Staining is indicated by the text color. L : Staining was not detected in control plates where only secondary antibodies were used. M : Final cell density did not affect cell morphology or changes in protein expression. ARPE-19 cells were seeded at various densities (i, 0.5×10 3 ; ii, 1×10 3 ; and iii, 2×10 3 cells/dish) and cultured for 7 days in DMSO-containing medium before staining for CALB2 (red). All scale bars are 50 μm.

    Journal: Molecular Vision

    Article Title: The expression of retinal cell markers in human retinal pigment epithelial cells and their augmentation by the synthetic retinoid fenretinide

    doi:

    Figure Lengend Snippet: Immunocytochemical analysis of neuronal and photoreceptor cell markers in ARPE-19 cells after treatment with dimethyl sulfoxide or fenretinide. A-K : ARPE-19 cells were treated with 3 μM fenretinide or DMSO and processed for immunocytochemistry. The nuclei of cells are stained with 4',6-diamidino-2-phenylindole (blue) with the exception of cells where protein expression was detected within the nucleus (OPN4, CRX, and PAX6). Staining is indicated by the text color. L : Staining was not detected in control plates where only secondary antibodies were used. M : Final cell density did not affect cell morphology or changes in protein expression. ARPE-19 cells were seeded at various densities (i, 0.5×10 3 ; ii, 1×10 3 ; and iii, 2×10 3 cells/dish) and cultured for 7 days in DMSO-containing medium before staining for CALB2 (red). All scale bars are 50 μm.

    Article Snippet: The following day cells were washed and treated with low-serum medium containing 3 μM fenretinide (Tocris Bioscience, Bristol, UK) dissolved in DMSO (Sigma-Aldrich Company Ltd, Dorset, UK).

    Techniques: Immunocytochemistry, Staining, Expressing, Cell Culture

    Detection of protein expression in ARPE-19 cells after dimethyl sulfoxide or fenretinide treatment by western blot analysis. Cells were treated with 3 μM fenretinide (FR) or dimethly sulfoxide (DMSO) for 7 days and extracts prepared for western blot. Equal amounts of protein were loaded onto a sodium dodecyl sulfate PAGE gel, transferred to membrane, and probed with antibodies generated against cone rod homeobox (CRX), opsin 3 (OPN3), melanopsin (OPN4), calbindin 2 (CALB2), cytokeratin 8 (KRT8), paired box 6 (PAX6), and sex determining region Y-box 2 (SOX2). Proteins were detected using chemiluminescence and visualized using autoradiographic film. Membranes were stripped and reprobed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. Representative blots from DMSO- and FR- treated cells are shown. Protein levels were quantified using densitometry and normalized relative to the GAPDH loading control. Data shown are mean±standard error of the mean normalized to GAPDH (n=4).

    Journal: Molecular Vision

    Article Title: The expression of retinal cell markers in human retinal pigment epithelial cells and their augmentation by the synthetic retinoid fenretinide

    doi:

    Figure Lengend Snippet: Detection of protein expression in ARPE-19 cells after dimethyl sulfoxide or fenretinide treatment by western blot analysis. Cells were treated with 3 μM fenretinide (FR) or dimethly sulfoxide (DMSO) for 7 days and extracts prepared for western blot. Equal amounts of protein were loaded onto a sodium dodecyl sulfate PAGE gel, transferred to membrane, and probed with antibodies generated against cone rod homeobox (CRX), opsin 3 (OPN3), melanopsin (OPN4), calbindin 2 (CALB2), cytokeratin 8 (KRT8), paired box 6 (PAX6), and sex determining region Y-box 2 (SOX2). Proteins were detected using chemiluminescence and visualized using autoradiographic film. Membranes were stripped and reprobed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. Representative blots from DMSO- and FR- treated cells are shown. Protein levels were quantified using densitometry and normalized relative to the GAPDH loading control. Data shown are mean±standard error of the mean normalized to GAPDH (n=4).

    Article Snippet: The following day cells were washed and treated with low-serum medium containing 3 μM fenretinide (Tocris Bioscience, Bristol, UK) dissolved in DMSO (Sigma-Aldrich Company Ltd, Dorset, UK).

    Techniques: Expressing, Western Blot, Polyacrylamide Gel Electrophoresis, Generated

    Fenretinide treatment induces a neuronal-like phenotype in ARPE-19 cells. Cells were treated on uncoated tissue culture plastic with media containing dimethyl sulfoxide ( A ) or 3 μM fenretinide (FR ; B ) once a day for 7 days, and the morphology was examined by phase microscopy. C : Scanning electron microscopy of FR-treated cells shows neural processes and the formation of varicosities and neurites (highlighted in the dashed boxes and shown at high magnification in D and E , respectively). F: Synaptic-like appositions were also present at the neurite terminal, shown at high magnification in G . Scale bars equal 200 μm in A and B , 100 μm in C , 4 μm in D , 20 μm in F , and 2 μm in E and G .

    Journal: Molecular Vision

    Article Title: The expression of retinal cell markers in human retinal pigment epithelial cells and their augmentation by the synthetic retinoid fenretinide

    doi:

    Figure Lengend Snippet: Fenretinide treatment induces a neuronal-like phenotype in ARPE-19 cells. Cells were treated on uncoated tissue culture plastic with media containing dimethyl sulfoxide ( A ) or 3 μM fenretinide (FR ; B ) once a day for 7 days, and the morphology was examined by phase microscopy. C : Scanning electron microscopy of FR-treated cells shows neural processes and the formation of varicosities and neurites (highlighted in the dashed boxes and shown at high magnification in D and E , respectively). F: Synaptic-like appositions were also present at the neurite terminal, shown at high magnification in G . Scale bars equal 200 μm in A and B , 100 μm in C , 4 μm in D , 20 μm in F , and 2 μm in E and G .

    Article Snippet: The following day cells were washed and treated with low-serum medium containing 3 μM fenretinide (Tocris Bioscience, Bristol, UK) dissolved in DMSO (Sigma-Aldrich Company Ltd, Dorset, UK).

    Techniques: Microscopy, Electron Microscopy

    A melanopsin (OPN4) peptide-blocking experiment was performed to test the specificity of the antibody for western blot. Due to the presence of multiple bands on the OPN4 western blot the OPN4 antibody was pre-incubated with a fivefold excess of 15 amino acid N-terminal blocking peptide in 10% milk/TBS-T for 2 h at room temperature before hybridization. Proteins extracted from fenretinide (FR) and dimethyl sulfoxide (DMSO) treated cells were incubated with OPN4 antibody pre-incubated with the blocking peptide (OPN4+BP) or OPN4 antibody alone. A single band of approximately 70 kDa specific to OPN4 was absent in the western blot containing the blocking peptide.

    Journal: Molecular Vision

    Article Title: The expression of retinal cell markers in human retinal pigment epithelial cells and their augmentation by the synthetic retinoid fenretinide

    doi:

    Figure Lengend Snippet: A melanopsin (OPN4) peptide-blocking experiment was performed to test the specificity of the antibody for western blot. Due to the presence of multiple bands on the OPN4 western blot the OPN4 antibody was pre-incubated with a fivefold excess of 15 amino acid N-terminal blocking peptide in 10% milk/TBS-T for 2 h at room temperature before hybridization. Proteins extracted from fenretinide (FR) and dimethyl sulfoxide (DMSO) treated cells were incubated with OPN4 antibody pre-incubated with the blocking peptide (OPN4+BP) or OPN4 antibody alone. A single band of approximately 70 kDa specific to OPN4 was absent in the western blot containing the blocking peptide.

    Article Snippet: The following day cells were washed and treated with low-serum medium containing 3 μM fenretinide (Tocris Bioscience, Bristol, UK) dissolved in DMSO (Sigma-Aldrich Company Ltd, Dorset, UK).

    Techniques: Blocking Assay, Western Blot, Incubation, Hybridization

    C10-treated mice maintain glucose tolerance despite obesity. A 3 h intraperitoneal glucose tolerance test was performed after 13 weeks of HF diet feeding. C10 prevented HF diet-induced glucose intolerance. Area under the curve was significantly lower in the LF sham group as well as the HF C10-treated group throughout the duration of the glucose challenge. Data points on the line graph indicate mean and error bars indicate +/− s.e.m. and bars on the bar graph indicate mean + s.e.m. Significance was determined using ANOVA followed by Tukey’s post hoc analysis for multiple comparison; *Different from HF sham and HF DMSO groups; P

    Journal: The Journal of Endocrinology

    Article Title: Phenylmethimazole abrogates diet-induced inflammation, glucose intolerance and NAFLD

    doi: 10.1530/JOE-18-0078

    Figure Lengend Snippet: C10-treated mice maintain glucose tolerance despite obesity. A 3 h intraperitoneal glucose tolerance test was performed after 13 weeks of HF diet feeding. C10 prevented HF diet-induced glucose intolerance. Area under the curve was significantly lower in the LF sham group as well as the HF C10-treated group throughout the duration of the glucose challenge. Data points on the line graph indicate mean and error bars indicate +/− s.e.m. and bars on the bar graph indicate mean + s.e.m. Significance was determined using ANOVA followed by Tukey’s post hoc analysis for multiple comparison; *Different from HF sham and HF DMSO groups; P

    Article Snippet: A working solution of C10 (100 µM) was prepared in 0.25% DMSO (Sigma-Aldrich).

    Techniques: Mouse Assay

    C10 does not prevent weight gain or an increase in fat mass due to HF-feeding. Seven-week old C57BL/6J male mice were fed either LF or HF diet and treated once daily with sham, DMSO, or C10 intraperitoneal injection for 18 weeks. Total body weights were measured weekly and body composition was measured every 2 weeks for the duration of the study. Adipose (mesenteric, subcutaneous, epididymal, and retroperitoneal) tissue weight was measured after tissue harvest at 18 weeks. (A) HF diet feeding promoted a marked increase in body weight when compared to LF-fed mice. (B) Additionally, % Fat mass was increased in HF-fed mice when compared to LF-fed controls. Percent Lean Mass was increased in LF-fed mice when compared to HF-fed groups. Percent Fluid Mass was no different between LF- and HF-fed mice. (C) HF-fed mice displayed increased adipose tissue weights after 18 weeks on HF diet when compared to LF-fed mice. Data points on line graphs (A and B) indicate mean and error bars indicate +/− s.e.m. and bars on bar graphs (C) indicate mean + s.e.m. Significance was determined using ANOVA followed by Tukey’s post hoc analysis for multiple comparison; *Different from HF-fed groups; P

    Journal: The Journal of Endocrinology

    Article Title: Phenylmethimazole abrogates diet-induced inflammation, glucose intolerance and NAFLD

    doi: 10.1530/JOE-18-0078

    Figure Lengend Snippet: C10 does not prevent weight gain or an increase in fat mass due to HF-feeding. Seven-week old C57BL/6J male mice were fed either LF or HF diet and treated once daily with sham, DMSO, or C10 intraperitoneal injection for 18 weeks. Total body weights were measured weekly and body composition was measured every 2 weeks for the duration of the study. Adipose (mesenteric, subcutaneous, epididymal, and retroperitoneal) tissue weight was measured after tissue harvest at 18 weeks. (A) HF diet feeding promoted a marked increase in body weight when compared to LF-fed mice. (B) Additionally, % Fat mass was increased in HF-fed mice when compared to LF-fed controls. Percent Lean Mass was increased in LF-fed mice when compared to HF-fed groups. Percent Fluid Mass was no different between LF- and HF-fed mice. (C) HF-fed mice displayed increased adipose tissue weights after 18 weeks on HF diet when compared to LF-fed mice. Data points on line graphs (A and B) indicate mean and error bars indicate +/− s.e.m. and bars on bar graphs (C) indicate mean + s.e.m. Significance was determined using ANOVA followed by Tukey’s post hoc analysis for multiple comparison; *Different from HF-fed groups; P

    Article Snippet: A working solution of C10 (100 µM) was prepared in 0.25% DMSO (Sigma-Aldrich).

    Techniques: Weight Gain, Mouse Assay, Injection

    C10 treatment reverses HF diet-induced hepatic steatosis and hepatic and adipose inflammation and reduces serum cholesterol. (A) Treatment with C10 decreased hepatic triglyceride content when compared to HF-fed sham and DMSO groups. (B) Total serum cholesterol levels were reduced in the HF-fed C10-treated mice when compared to HF-fed control mice, however, serum triglyceride levels were unchanged. Dotted lines represent the mean and error bars indicate + s.e.m. (C) Hepatic Ifnb1 , Tnfa and Il6 expression was reduced in C10-treated mice when compared to HF sham and DMSO groups. Bars indicate mean + s.e.m. Significance was determined using ANOVA followed by Tukey’s post hoc analysis for multiple comparison; # Different from HF sham and DMSO groups; P

    Journal: The Journal of Endocrinology

    Article Title: Phenylmethimazole abrogates diet-induced inflammation, glucose intolerance and NAFLD

    doi: 10.1530/JOE-18-0078

    Figure Lengend Snippet: C10 treatment reverses HF diet-induced hepatic steatosis and hepatic and adipose inflammation and reduces serum cholesterol. (A) Treatment with C10 decreased hepatic triglyceride content when compared to HF-fed sham and DMSO groups. (B) Total serum cholesterol levels were reduced in the HF-fed C10-treated mice when compared to HF-fed control mice, however, serum triglyceride levels were unchanged. Dotted lines represent the mean and error bars indicate + s.e.m. (C) Hepatic Ifnb1 , Tnfa and Il6 expression was reduced in C10-treated mice when compared to HF sham and DMSO groups. Bars indicate mean + s.e.m. Significance was determined using ANOVA followed by Tukey’s post hoc analysis for multiple comparison; # Different from HF sham and DMSO groups; P

    Article Snippet: A working solution of C10 (100 µM) was prepared in 0.25% DMSO (Sigma-Aldrich).

    Techniques: Mouse Assay, Expressing

    C10 treatment reverses HF diet-induced glucose intolerance. A 3 h intraperitoneal glucose tolerance test was performed just prior to the initiation of treatment (A) and after 12 weeks from the start date of the reversal study (B). (A) At the beginning of the study all HF-fed mice were glucose intolerant compared to LF-fed mice. (B) Following treatment, blood glucose levels remained elevated in the HF sham and HF DMSO groups when compared to the LF sham and HF C10-treated mice. Area under the curve was significantly lower in the C10-treated group when compared to the HF sham and DMSO groups and was nearly indistinguishable from the LF sham group. This indicates that C10 reverses glucose intolerance due to the HF diet feeding. Data points on line graphs indicate mean and error bars indicate +/− s.e.m. and bars on bar graphs indicate mean + s.e.m. Significance was determined using ANOVA followed by Tukey’s post hoc analysis for multiple comparison; (A) * Different from LF sham, (B) * Different from HF sham and DMSO groups; P

    Journal: The Journal of Endocrinology

    Article Title: Phenylmethimazole abrogates diet-induced inflammation, glucose intolerance and NAFLD

    doi: 10.1530/JOE-18-0078

    Figure Lengend Snippet: C10 treatment reverses HF diet-induced glucose intolerance. A 3 h intraperitoneal glucose tolerance test was performed just prior to the initiation of treatment (A) and after 12 weeks from the start date of the reversal study (B). (A) At the beginning of the study all HF-fed mice were glucose intolerant compared to LF-fed mice. (B) Following treatment, blood glucose levels remained elevated in the HF sham and HF DMSO groups when compared to the LF sham and HF C10-treated mice. Area under the curve was significantly lower in the C10-treated group when compared to the HF sham and DMSO groups and was nearly indistinguishable from the LF sham group. This indicates that C10 reverses glucose intolerance due to the HF diet feeding. Data points on line graphs indicate mean and error bars indicate +/− s.e.m. and bars on bar graphs indicate mean + s.e.m. Significance was determined using ANOVA followed by Tukey’s post hoc analysis for multiple comparison; (A) * Different from LF sham, (B) * Different from HF sham and DMSO groups; P

    Article Snippet: A working solution of C10 (100 µM) was prepared in 0.25% DMSO (Sigma-Aldrich).

    Techniques: Mouse Assay

    C10 prevents HF diet-induced hepatic steatosis. Hematoxylin and eosin staining was performed on liver tissue sections prepared after 18 weeks of HF diet feeding. Liver triglyceride content was determined by biochemical analysis (A) Histological examination revealed that C10 prevents hepatic lipid accumulation. All images in (A) were taken at 400× magnification. Scale bar, 40 µm. (B) Treatment with C10 decreased hepatic triglyceride content when compared to HF-fed sham and DMSO groups but had no effect on serum triglyceride levels. Dotted lines represent the mean and error bars indicate + s.e.m. Significance was determined using ANOVA followed by Tukey’s post hoc analysis for multiple comparison; *Different from HF-fed groups; P

    Journal: The Journal of Endocrinology

    Article Title: Phenylmethimazole abrogates diet-induced inflammation, glucose intolerance and NAFLD

    doi: 10.1530/JOE-18-0078

    Figure Lengend Snippet: C10 prevents HF diet-induced hepatic steatosis. Hematoxylin and eosin staining was performed on liver tissue sections prepared after 18 weeks of HF diet feeding. Liver triglyceride content was determined by biochemical analysis (A) Histological examination revealed that C10 prevents hepatic lipid accumulation. All images in (A) were taken at 400× magnification. Scale bar, 40 µm. (B) Treatment with C10 decreased hepatic triglyceride content when compared to HF-fed sham and DMSO groups but had no effect on serum triglyceride levels. Dotted lines represent the mean and error bars indicate + s.e.m. Significance was determined using ANOVA followed by Tukey’s post hoc analysis for multiple comparison; *Different from HF-fed groups; P

    Article Snippet: A working solution of C10 (100 µM) was prepared in 0.25% DMSO (Sigma-Aldrich).

    Techniques: Staining

    C10 inhibits hepatic inflammation in addition to triglyceride accumulation in cell culture. AML-12 and HepG2 cells were treated with 100 µM C10 or DMSO (control) to determine if C10 could prevent hepatic inflammation in the presence of 0.75 mM palmitate or 10 ng/mL LPS. Treatment with C10 prevented palmitate- (A) and LPS (B)-induced pro-inflammatory cytokine ( Ifnb1 and Tnfa ) expression. Inhibition of palmitate-induced pro-inflammatory cytokine expression was also observed in HepG2 cells (C). Treatment with C10 prevented palmitate-induced triglyceride accumulation in AML-12 cells (D). Bars indicate mean + s.e.m. Significance was determined using a one-way ANOVA followed by Tukey’s post hoc analysis for multiple comparison; * P

    Journal: The Journal of Endocrinology

    Article Title: Phenylmethimazole abrogates diet-induced inflammation, glucose intolerance and NAFLD

    doi: 10.1530/JOE-18-0078

    Figure Lengend Snippet: C10 inhibits hepatic inflammation in addition to triglyceride accumulation in cell culture. AML-12 and HepG2 cells were treated with 100 µM C10 or DMSO (control) to determine if C10 could prevent hepatic inflammation in the presence of 0.75 mM palmitate or 10 ng/mL LPS. Treatment with C10 prevented palmitate- (A) and LPS (B)-induced pro-inflammatory cytokine ( Ifnb1 and Tnfa ) expression. Inhibition of palmitate-induced pro-inflammatory cytokine expression was also observed in HepG2 cells (C). Treatment with C10 prevented palmitate-induced triglyceride accumulation in AML-12 cells (D). Bars indicate mean + s.e.m. Significance was determined using a one-way ANOVA followed by Tukey’s post hoc analysis for multiple comparison; * P

    Article Snippet: A working solution of C10 (100 µM) was prepared in 0.25% DMSO (Sigma-Aldrich).

    Techniques: Cell Culture, Expressing, Inhibition

    C10 prevents HF diet-induced inflammation in vivo . Inflammatory gene expression was measured in liver and mesenteric adipose tissue after 18 weeks of HF diet feeding. Hepatic Ifnb1 and Tnfa expression were reduced in C10-treated mice when compared to HF sham and DMSO groups (A). Additionally, C10 prevented an upregulation of Ifnb1 and Tnfa in mesenteric adipose tissue (B) as well as Emr1 ( F4/80 ), a macrophage marker (C). Bars indicate mean + s.e.m. Significance was determined using ANOVA followed by Tukey’s post hoc analysis for multiple comparison; *Different from HF sham and HF DMSO groups; P

    Journal: The Journal of Endocrinology

    Article Title: Phenylmethimazole abrogates diet-induced inflammation, glucose intolerance and NAFLD

    doi: 10.1530/JOE-18-0078

    Figure Lengend Snippet: C10 prevents HF diet-induced inflammation in vivo . Inflammatory gene expression was measured in liver and mesenteric adipose tissue after 18 weeks of HF diet feeding. Hepatic Ifnb1 and Tnfa expression were reduced in C10-treated mice when compared to HF sham and DMSO groups (A). Additionally, C10 prevented an upregulation of Ifnb1 and Tnfa in mesenteric adipose tissue (B) as well as Emr1 ( F4/80 ), a macrophage marker (C). Bars indicate mean + s.e.m. Significance was determined using ANOVA followed by Tukey’s post hoc analysis for multiple comparison; *Different from HF sham and HF DMSO groups; P

    Article Snippet: A working solution of C10 (100 µM) was prepared in 0.25% DMSO (Sigma-Aldrich).

    Techniques: In Vivo, Expressing, Mouse Assay, Marker

    Effects of CDK4/6 inhibition on breast cancer cell proliferation and apoptosis in vitro a , Relative numbers of breast cancer cells cultured in 250 nM (MDA-MB-453) or 500 nM (MDA-MB-361, BT474) abemaciclib or DMSO for 11d, followed by drug withdrawal (arrow). b , Representative SA-β-galactosidase staining of MDA-MB-453 cells (left) and BT474 cells (right) treated with DMSO or abemaciclib (MDA-MB-453, 250 nM; BT474, 500 nM) for 0, 4, and 7 days. c , Western blot of SKBR3, BT474, MDA-MB-453, and MDA-MB-361 cells treated with DMSO, lapatinib, or abemaciclib for 48h. d , Western blot of MDA-MB-453 cells pretreated with DMSO or abemaciclib (500 nM) for 0, 1, or 7 days prior to exposure to staurosporine (500 nM) for 4h. For western blot source images, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: Effects of CDK4/6 inhibition on breast cancer cell proliferation and apoptosis in vitro a , Relative numbers of breast cancer cells cultured in 250 nM (MDA-MB-453) or 500 nM (MDA-MB-361, BT474) abemaciclib or DMSO for 11d, followed by drug withdrawal (arrow). b , Representative SA-β-galactosidase staining of MDA-MB-453 cells (left) and BT474 cells (right) treated with DMSO or abemaciclib (MDA-MB-453, 250 nM; BT474, 500 nM) for 0, 4, and 7 days. c , Western blot of SKBR3, BT474, MDA-MB-453, and MDA-MB-361 cells treated with DMSO, lapatinib, or abemaciclib for 48h. d , Western blot of MDA-MB-453 cells pretreated with DMSO or abemaciclib (500 nM) for 0, 1, or 7 days prior to exposure to staurosporine (500 nM) for 4h. For western blot source images, see Supplementary Figure 1 .

    Article Snippet: For hormonal therapy studies, cells were treated with combinations of DMSO, abemaciclib (100 nM), and fulvestrant (100 nM, Sigma Aldrich).

    Techniques: Inhibition, In Vitro, Cell Culture, Multiple Displacement Amplification, Staining, Western Blot

    CDK4/6 inhibition enhances antigen presentation a , Antigen processing and presentation gene expression in MDA-MB-361 cells treated with 500 nM abemaciclib or DMSO (7d, n=3). b , Gene expression in cell lines treated with DMSO or palbociclib (7d, n=3). c , β2M/MHC-I flow cytometry in cell lines; grey, FMO control. (For MDA-MB-453, vehicle and abemaciclib, n=2; palbociclib, n=3. For MDA-MB-361, n=3) d , Gene expression in TCGA samples ( CCND1 shallow deletion, n=101; CCND1 diploid, n=503; CCND1 gain, n=203; CCND1 amplified, n=153). e , H-2K b SIINFEKL flow cytometry after 7d abemaciclib or DMSO (B16-OVA, n=9; MMTV-PyMT -S2WTP3-OVA, n=3). f , CD8 + T cell proliferation in response to abemaciclib-pretreated tumor cells (n=3). g , IFNγ and TNFα production in tumor cell/OT-I assay by ELISA (n=3). One-way ANOVA adjusted for multiple comparisons (c–d,f), unpaired two-tailed t-tests (a–b,e,g). Error bars, SD. *p

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: CDK4/6 inhibition enhances antigen presentation a , Antigen processing and presentation gene expression in MDA-MB-361 cells treated with 500 nM abemaciclib or DMSO (7d, n=3). b , Gene expression in cell lines treated with DMSO or palbociclib (7d, n=3). c , β2M/MHC-I flow cytometry in cell lines; grey, FMO control. (For MDA-MB-453, vehicle and abemaciclib, n=2; palbociclib, n=3. For MDA-MB-361, n=3) d , Gene expression in TCGA samples ( CCND1 shallow deletion, n=101; CCND1 diploid, n=503; CCND1 gain, n=203; CCND1 amplified, n=153). e , H-2K b SIINFEKL flow cytometry after 7d abemaciclib or DMSO (B16-OVA, n=9; MMTV-PyMT -S2WTP3-OVA, n=3). f , CD8 + T cell proliferation in response to abemaciclib-pretreated tumor cells (n=3). g , IFNγ and TNFα production in tumor cell/OT-I assay by ELISA (n=3). One-way ANOVA adjusted for multiple comparisons (c–d,f), unpaired two-tailed t-tests (a–b,e,g). Error bars, SD. *p

    Article Snippet: For hormonal therapy studies, cells were treated with combinations of DMSO, abemaciclib (100 nM), and fulvestrant (100 nM, Sigma Aldrich).

    Techniques: Inhibition, Expressing, Multiple Displacement Amplification, Flow Cytometry, Cytometry, Amplification, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Abemaciclib induces a ‘senescence-like’ phenotype without evidence of SASP a , Representative SA-β-galactosidase staining (left) of MMTV-rtTA/tetO-HER2 tumors treated with vehicle or abemaciclib for 12d (scale bar=500 μ m). Quantification of relative SA-β-galactosidase positive area (right, n=6 tumors/group). b , mRNA expression of SASP factors in MMTV-rtTA/tetO-HER2 tumors treated as in Fig. 1a . Il6 expression determined by qPCR (n=10 tumors/group); Il1a and Il1b by transcriptome analysis (vehicle, n=11; abemaciclib, n=12 tumors/group). c , MDA-MB-453 and BT474 cells treated with DMSO or abemaciclib (500 nM) for 7d, and expression determined by qPCR. d , mRNA expression of IL6 upon doxorubicin-induced senescence (n=3). MDA-MB-453 and BT474 cells were treated with doxorubicin (200 nM) for 24h, and mRNA extracted 3d later for qPCR. Unpaired two-tailed t tests (a,d). Error bars, SD. **p

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: Abemaciclib induces a ‘senescence-like’ phenotype without evidence of SASP a , Representative SA-β-galactosidase staining (left) of MMTV-rtTA/tetO-HER2 tumors treated with vehicle or abemaciclib for 12d (scale bar=500 μ m). Quantification of relative SA-β-galactosidase positive area (right, n=6 tumors/group). b , mRNA expression of SASP factors in MMTV-rtTA/tetO-HER2 tumors treated as in Fig. 1a . Il6 expression determined by qPCR (n=10 tumors/group); Il1a and Il1b by transcriptome analysis (vehicle, n=11; abemaciclib, n=12 tumors/group). c , MDA-MB-453 and BT474 cells treated with DMSO or abemaciclib (500 nM) for 7d, and expression determined by qPCR. d , mRNA expression of IL6 upon doxorubicin-induced senescence (n=3). MDA-MB-453 and BT474 cells were treated with doxorubicin (200 nM) for 24h, and mRNA extracted 3d later for qPCR. Unpaired two-tailed t tests (a,d). Error bars, SD. **p

    Article Snippet: For hormonal therapy studies, cells were treated with combinations of DMSO, abemaciclib (100 nM), and fulvestrant (100 nM, Sigma Aldrich).

    Techniques: Staining, Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Two Tailed Test

    Impact of CDK4/6 inhibition on immune cell populations and Treg biology a , Flow cytometric analysis of immune cell populations in MMTV-rtTA/tetO-HER2 tumors treated with vehicle or abemaciclib for 12d (vehicle, n=15; abemaciclib, n=17 tumors/group). b , Peripheral blood Tregs in MMTV-rtTA/tetO-HER2 mice (12d, n=4 mice/group). c–d , Tregs (CD4 + FoxP3 + ) quantified by flow cytometry of MMTV-PyMT tumors (c, vehicle, n=18; abemaciclib, n=16 tumors/group) and CT-26 tumors (d, n=12 tumors/group) treated as indicated for 12d. e , Tregs in lymph nodes of tumor-free mice (12d, one-way ANOVA corrected for multiple comparisons, vehicles and palbociclib, n=8; abemaciclib, n=7 mice/group). f , Treg:CD8 ratio in the spleens and lymph nodes (LN) of tumor-free FVB mice (12d, vehicles and palbociclib, n=8; abemaciclib, n=7 mice/group). g , Plasma autoantibodies in tumor-free and tumor-bearing mice treated with vehicle or abemaciclib for 12d (tumor-free vehicle, n=8; tumor-free abemaciclib, n=7; tumor-bearing vehicle, n=7; tumor-bearing abemaciclib, n=6 mice/group). h–l , Tumor-free FVB mice treated with abemaciclib or vehicle for 12d. Thymic mass (h). Thymic cell populations were quantified by flow cytometry. CD4 + CD8 + double positive (DP) thymocytes (i); CD4 + single positive (SP) thymocytes (j); CD8 + SP thymocytes (k); CD4 + FoxP3 + regulatory T cells (l). (vehicles and palbociclib, n=5; abemaciclib, n=4 mice/group). m , Effect of abemaciclib or DMSO on ex vivo differentiation of CD4 + CD25 − T cells into Tregs in the presence of TGF-β for 72hr (n=4). n , Effect of DMSO or abemaciclib treatment for 72hr on Treg apoptosis measured by Annexin V staining (n=2). o , Quantification of immunofluorescent staining of MMTV-rtTA/tetO-HER2 tumors (n=7 tumors/group). p – q , Dnmt1 (p) and Cdkn1a (q) in T cells from tumor-free mice (12d, two-way ANOVA corrected for multiple comparisons, n=7 mice/group [pooled]). Unpaired two-tailed t-tests (b–d), one-way ANOVA corrected for multiple comparisons (e–f,h–l). Error bars, SD. *p

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: Impact of CDK4/6 inhibition on immune cell populations and Treg biology a , Flow cytometric analysis of immune cell populations in MMTV-rtTA/tetO-HER2 tumors treated with vehicle or abemaciclib for 12d (vehicle, n=15; abemaciclib, n=17 tumors/group). b , Peripheral blood Tregs in MMTV-rtTA/tetO-HER2 mice (12d, n=4 mice/group). c–d , Tregs (CD4 + FoxP3 + ) quantified by flow cytometry of MMTV-PyMT tumors (c, vehicle, n=18; abemaciclib, n=16 tumors/group) and CT-26 tumors (d, n=12 tumors/group) treated as indicated for 12d. e , Tregs in lymph nodes of tumor-free mice (12d, one-way ANOVA corrected for multiple comparisons, vehicles and palbociclib, n=8; abemaciclib, n=7 mice/group). f , Treg:CD8 ratio in the spleens and lymph nodes (LN) of tumor-free FVB mice (12d, vehicles and palbociclib, n=8; abemaciclib, n=7 mice/group). g , Plasma autoantibodies in tumor-free and tumor-bearing mice treated with vehicle or abemaciclib for 12d (tumor-free vehicle, n=8; tumor-free abemaciclib, n=7; tumor-bearing vehicle, n=7; tumor-bearing abemaciclib, n=6 mice/group). h–l , Tumor-free FVB mice treated with abemaciclib or vehicle for 12d. Thymic mass (h). Thymic cell populations were quantified by flow cytometry. CD4 + CD8 + double positive (DP) thymocytes (i); CD4 + single positive (SP) thymocytes (j); CD8 + SP thymocytes (k); CD4 + FoxP3 + regulatory T cells (l). (vehicles and palbociclib, n=5; abemaciclib, n=4 mice/group). m , Effect of abemaciclib or DMSO on ex vivo differentiation of CD4 + CD25 − T cells into Tregs in the presence of TGF-β for 72hr (n=4). n , Effect of DMSO or abemaciclib treatment for 72hr on Treg apoptosis measured by Annexin V staining (n=2). o , Quantification of immunofluorescent staining of MMTV-rtTA/tetO-HER2 tumors (n=7 tumors/group). p – q , Dnmt1 (p) and Cdkn1a (q) in T cells from tumor-free mice (12d, two-way ANOVA corrected for multiple comparisons, n=7 mice/group [pooled]). Unpaired two-tailed t-tests (b–d), one-way ANOVA corrected for multiple comparisons (e–f,h–l). Error bars, SD. *p

    Article Snippet: For hormonal therapy studies, cells were treated with combinations of DMSO, abemaciclib (100 nM), and fulvestrant (100 nM, Sigma Aldrich).

    Techniques: Inhibition, Flow Cytometry, Mouse Assay, Cytometry, Ex Vivo, Staining, Two Tailed Test

    DNMT1 suppression mediates dsRNA response a , DNMT1 expression after treatment with abemaciclib (n=3). b , DNMT1 protein expression after treatment with abemaciclib. c , DNMT3A expression after treatment with abemaciclib (500 nM) for 7d (n=3, except abemaciclib 24 hrs, n=2). d , DNMT1 expression after abemaciclib or control in MMTV-rtTA/tetO-HER2 tumors, p=0.05, (12d, vehicle, n=11; abemaciclib, n=12 tumors/group). e , RB1 knockdown in MDA-MB-453 cells and its effect on indicated gene expression after 7d abemaciclib (500 nM) (two biological replicates each associated with three technical replicates). f , ERV3-1 methylation. g , ERV expression after abemaciclib or DMSO (7d, n=3). h , Relative dsRNA expression after 7d of abemaciclib compared to DMSO (n=3). i–j , Cytosolic pattern recognition receptors in cells (i) (7d, n=3) or PDX tumors (j) (21–28d, vehicle, n=4; abemaciclib, n=2 tumors/group). k , Western blot of overexpression of DNMT1 in MDA-MB-453 cells and quantification of mRNA expression (n=3). Unpaired two-tailed t-tests (d–e,g–k) adjusted for multiple comparisons (a). Error bars, SD. *p≤0.05, **p

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: DNMT1 suppression mediates dsRNA response a , DNMT1 expression after treatment with abemaciclib (n=3). b , DNMT1 protein expression after treatment with abemaciclib. c , DNMT3A expression after treatment with abemaciclib (500 nM) for 7d (n=3, except abemaciclib 24 hrs, n=2). d , DNMT1 expression after abemaciclib or control in MMTV-rtTA/tetO-HER2 tumors, p=0.05, (12d, vehicle, n=11; abemaciclib, n=12 tumors/group). e , RB1 knockdown in MDA-MB-453 cells and its effect on indicated gene expression after 7d abemaciclib (500 nM) (two biological replicates each associated with three technical replicates). f , ERV3-1 methylation. g , ERV expression after abemaciclib or DMSO (7d, n=3). h , Relative dsRNA expression after 7d of abemaciclib compared to DMSO (n=3). i–j , Cytosolic pattern recognition receptors in cells (i) (7d, n=3) or PDX tumors (j) (21–28d, vehicle, n=4; abemaciclib, n=2 tumors/group). k , Western blot of overexpression of DNMT1 in MDA-MB-453 cells and quantification of mRNA expression (n=3). Unpaired two-tailed t-tests (d–e,g–k) adjusted for multiple comparisons (a). Error bars, SD. *p≤0.05, **p

    Article Snippet: For hormonal therapy studies, cells were treated with combinations of DMSO, abemaciclib (100 nM), and fulvestrant (100 nM, Sigma Aldrich).

    Techniques: Expressing, Multiple Displacement Amplification, Methylation, Western Blot, Over Expression, Two Tailed Test

    CDK4/6 inhibition mediates Type III interferon production a , Phospho- and total STAT1 in MDA-MB-453 cells treated with abemaciclib +/− ruxolitinib for 7d. b , Effect of neutralization of IFN-α or IFN-γ on STAT1 mRNA expression (n=2–4). c – d , Impact of neutralization of IFN-α (c) and IFN-γ (d) on phospho-STAT1 and total STAT1 protein in indicated cell lines. e , Expression of type III interferon genes in indicated cell lines treated with abemaciclib for 7d compared to DMSO (n=3). f , Type III interferon production measured by ELISA (7d, n=2). Unpaired two-tailed t-tests (e–f) adjusted for multiple comparisons (b). Error bars, SD. *p

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: CDK4/6 inhibition mediates Type III interferon production a , Phospho- and total STAT1 in MDA-MB-453 cells treated with abemaciclib +/− ruxolitinib for 7d. b , Effect of neutralization of IFN-α or IFN-γ on STAT1 mRNA expression (n=2–4). c – d , Impact of neutralization of IFN-α (c) and IFN-γ (d) on phospho-STAT1 and total STAT1 protein in indicated cell lines. e , Expression of type III interferon genes in indicated cell lines treated with abemaciclib for 7d compared to DMSO (n=3). f , Type III interferon production measured by ELISA (7d, n=2). Unpaired two-tailed t-tests (e–f) adjusted for multiple comparisons (b). Error bars, SD. *p

    Article Snippet: For hormonal therapy studies, cells were treated with combinations of DMSO, abemaciclib (100 nM), and fulvestrant (100 nM, Sigma Aldrich).

    Techniques: Inhibition, Multiple Displacement Amplification, Neutralization, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    CDK4/6 inhibition increases interferon signaling a–b , Top ranked GO terms (a) and expression of interferon-responsive transcription factors (b) in MDA-MB-361 cells treated with 500 nM abemaciclib or DMSO (7d, n=3). c , Expression of interferon sensitive genes (ISGs) in MMTV-PyMT -S2WTP3 cells treated with DMSO or abemaciclib (500 nM, 7d) (n=3). d , Expression of ISGs in MDA-MB-361 and MCF7 cells treated with abemaciclib (100 nM), fulvestrant (100 nM), or the combination for 7d (n=3). e , Expression of ISGs in MDA-MB-453, MCF7, and MDA-MB-231 cells treated with abemaciclib or DMSO (7d, n=3). f , Expression of ISGs in PDX 14-07 tumors treated with abemaciclib or vehicle (21–28d, vehicle, n=4; abemaciclib, n=2 tumors/group). g , phospho- and total STAT1 in cells treated with 500 nM abemaciclib as indicated. h , Confirmation of p16-FLAG overexpression in MDA-MB-453 and BT474 cells (left) and gene expression in these cell lines by qPCR (right) (n=6). i–j , Gene expression changes in MMTV-rtTA/tetO-HER2 tumors from mice treated with vehicle or abemaciclib for 12d (vehicle, n=11; abemaciclib, n=12 tumors/group). Relative expression of interferon-responsive T cell chemoattractants (i); relative expression of ISGs (j). k , Correlation of expression of Stat1 and Nlrc5 with genes involved in antigen processing and presentation in MMTV-rtTA/tetO-HER2 tumors. Blue dots, vehicle-treated tumors; red dots, abemaciclib-treated tumors. (r is Pearson product-moment correlation coefficient). Unpaired two-tailed t-tests (b,d–f,h–j) adjusted for multiple comparisons (c). Error bars, SD. *p

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: CDK4/6 inhibition increases interferon signaling a–b , Top ranked GO terms (a) and expression of interferon-responsive transcription factors (b) in MDA-MB-361 cells treated with 500 nM abemaciclib or DMSO (7d, n=3). c , Expression of interferon sensitive genes (ISGs) in MMTV-PyMT -S2WTP3 cells treated with DMSO or abemaciclib (500 nM, 7d) (n=3). d , Expression of ISGs in MDA-MB-361 and MCF7 cells treated with abemaciclib (100 nM), fulvestrant (100 nM), or the combination for 7d (n=3). e , Expression of ISGs in MDA-MB-453, MCF7, and MDA-MB-231 cells treated with abemaciclib or DMSO (7d, n=3). f , Expression of ISGs in PDX 14-07 tumors treated with abemaciclib or vehicle (21–28d, vehicle, n=4; abemaciclib, n=2 tumors/group). g , phospho- and total STAT1 in cells treated with 500 nM abemaciclib as indicated. h , Confirmation of p16-FLAG overexpression in MDA-MB-453 and BT474 cells (left) and gene expression in these cell lines by qPCR (right) (n=6). i–j , Gene expression changes in MMTV-rtTA/tetO-HER2 tumors from mice treated with vehicle or abemaciclib for 12d (vehicle, n=11; abemaciclib, n=12 tumors/group). Relative expression of interferon-responsive T cell chemoattractants (i); relative expression of ISGs (j). k , Correlation of expression of Stat1 and Nlrc5 with genes involved in antigen processing and presentation in MMTV-rtTA/tetO-HER2 tumors. Blue dots, vehicle-treated tumors; red dots, abemaciclib-treated tumors. (r is Pearson product-moment correlation coefficient). Unpaired two-tailed t-tests (b,d–f,h–j) adjusted for multiple comparisons (c). Error bars, SD. *p

    Article Snippet: For hormonal therapy studies, cells were treated with combinations of DMSO, abemaciclib (100 nM), and fulvestrant (100 nM, Sigma Aldrich).

    Techniques: Inhibition, Expressing, Multiple Displacement Amplification, Over Expression, Real-time Polymerase Chain Reaction, Mouse Assay, Two Tailed Test

    Effect of 8 hr treatment of DMSO at different phases of infection. Cultured Vero cells (10 5 cells in 24-well plates) were infected with 1000 PFU of the dUTPase/LAT recombinant virus. Treatments were as follows: DMSO at the indicated concentrations were present for 8 h before adsorption (filled circles), 8 h immediately after adsorption (open circles) and 16 to 24 hr (triangles). Reporter gene activity was determined at 24 pi. Data are the averages ± S.D. of 3–6 duplicate determinations.

    Journal: BMC Infectious Diseases

    Article Title: Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis

    doi: 10.1186/1471-2334-2-9

    Figure Lengend Snippet: Effect of 8 hr treatment of DMSO at different phases of infection. Cultured Vero cells (10 5 cells in 24-well plates) were infected with 1000 PFU of the dUTPase/LAT recombinant virus. Treatments were as follows: DMSO at the indicated concentrations were present for 8 h before adsorption (filled circles), 8 h immediately after adsorption (open circles) and 16 to 24 hr (triangles). Reporter gene activity was determined at 24 pi. Data are the averages ± S.D. of 3–6 duplicate determinations.

    Article Snippet: The cytotoxic effect of DMSO (Sigma or Fisher) treatment on Vero cells was evaluated by the method of sulforhodamin B (SRB) [ , ].

    Techniques: Infection, Cell Culture, Recombinant, Adsorption, Activity Assay

    PCR analysis of HSV-1 DNA replication in the presence or absence of DMSO. Total DNA was isolated from cultures of Vero cells infected with 1000 PFU of the dUTPase/LAT recombinant and the indicated hrs post-infection and amplified in the conditions described in Material and Methods. Aliquots of the amplified products were fractionated in 6% polyacrylamide gels in Tris-borate-EDTA and evaluated by densitometry.

    Journal: BMC Infectious Diseases

    Article Title: Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis

    doi: 10.1186/1471-2334-2-9

    Figure Lengend Snippet: PCR analysis of HSV-1 DNA replication in the presence or absence of DMSO. Total DNA was isolated from cultures of Vero cells infected with 1000 PFU of the dUTPase/LAT recombinant and the indicated hrs post-infection and amplified in the conditions described in Material and Methods. Aliquots of the amplified products were fractionated in 6% polyacrylamide gels in Tris-borate-EDTA and evaluated by densitometry.

    Article Snippet: The cytotoxic effect of DMSO (Sigma or Fisher) treatment on Vero cells was evaluated by the method of sulforhodamin B (SRB) [ , ].

    Techniques: Polymerase Chain Reaction, Isolation, Infection, Recombinant, Amplification

    Cell toxicity of DMSO. Confluent cultures of Vero cells in 96-well plates were treated with the indicated concentrations of DMSO for either 24 h (filled circles) or for 8 hr (open circles) and cytotoxicity determined 28 hrs after the initiation of the treatment. Results are average ± SD of 4 duplicated determinations.

    Journal: BMC Infectious Diseases

    Article Title: Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis

    doi: 10.1186/1471-2334-2-9

    Figure Lengend Snippet: Cell toxicity of DMSO. Confluent cultures of Vero cells in 96-well plates were treated with the indicated concentrations of DMSO for either 24 h (filled circles) or for 8 hr (open circles) and cytotoxicity determined 28 hrs after the initiation of the treatment. Results are average ± SD of 4 duplicated determinations.

    Article Snippet: The cytotoxic effect of DMSO (Sigma or Fisher) treatment on Vero cells was evaluated by the method of sulforhodamin B (SRB) [ , ].

    Techniques:

    Effect of DMSO treatment on productive infection at different phases of infection. Cultured Vero cells (10 5 cells in 24-well plates) were infected with 1000 pfu of the dUTPase/LAT recombinant virus. Treatments were as follows: DMSO at the indicated concentrations was present for 24 h before adsorption (filled circles), during the adsorption phase (open circles) or for 23 hr after the adsorption period (filled triangles). Also included is the effect of DMSO present for 23 hr after adsorption on HFF cells infected under similar conditions (open triangles). Reporter gene activity was determined at 24 hr following infection. Data are the average ± SD of 3–6 duplicate determinations.

    Journal: BMC Infectious Diseases

    Article Title: Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis

    doi: 10.1186/1471-2334-2-9

    Figure Lengend Snippet: Effect of DMSO treatment on productive infection at different phases of infection. Cultured Vero cells (10 5 cells in 24-well plates) were infected with 1000 pfu of the dUTPase/LAT recombinant virus. Treatments were as follows: DMSO at the indicated concentrations was present for 24 h before adsorption (filled circles), during the adsorption phase (open circles) or for 23 hr after the adsorption period (filled triangles). Also included is the effect of DMSO present for 23 hr after adsorption on HFF cells infected under similar conditions (open triangles). Reporter gene activity was determined at 24 hr following infection. Data are the average ± SD of 3–6 duplicate determinations.

    Article Snippet: The cytotoxic effect of DMSO (Sigma or Fisher) treatment on Vero cells was evaluated by the method of sulforhodamin B (SRB) [ , ].

    Techniques: Infection, Cell Culture, Recombinant, Adsorption, Activity Assay

    Time course dependency of DMSO blockade of productive infection. Cultured Vero cells (10 5 cells in 24-well plates) were infected with 1000 PFU of the dUTPase/LAT recombinant virus. After the adsorption phase, 4% DMSO was added in the ovelay medium and kept for the indicated time periods. At those times, the media were replaced with fresh overlay medium without DMSO and the incubation continued 24 hr. Reporter gene activity was determined at 24 hr post-infection. Data are the average ± SD of 3–6 duplicate determinations.

    Journal: BMC Infectious Diseases

    Article Title: Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis

    doi: 10.1186/1471-2334-2-9

    Figure Lengend Snippet: Time course dependency of DMSO blockade of productive infection. Cultured Vero cells (10 5 cells in 24-well plates) were infected with 1000 PFU of the dUTPase/LAT recombinant virus. After the adsorption phase, 4% DMSO was added in the ovelay medium and kept for the indicated time periods. At those times, the media were replaced with fresh overlay medium without DMSO and the incubation continued 24 hr. Reporter gene activity was determined at 24 hr post-infection. Data are the average ± SD of 3–6 duplicate determinations.

    Article Snippet: The cytotoxic effect of DMSO (Sigma or Fisher) treatment on Vero cells was evaluated by the method of sulforhodamin B (SRB) [ , ].

    Techniques: Infection, Cell Culture, Recombinant, Adsorption, Incubation, Activity Assay

    Inactivation of HSV-1 virion infectivity by DMSO. The dUTPase/LAT recombinant virus was suspended in PBS-glucose medium at 2000 PFU/ml and incubated at 37°C for 7 hr. Aliquots of 0.5 ml of the suspension were then adsorbed to Vero cells (10 5 cells in 24-well plates) for 1 hr. The virus suspension was then replaced with overlay medium, and reporter gene activity was determined at 24 hr. Data are the averages ± S.D of 4 duplicate determinations.

    Journal: BMC Infectious Diseases

    Article Title: Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis

    doi: 10.1186/1471-2334-2-9

    Figure Lengend Snippet: Inactivation of HSV-1 virion infectivity by DMSO. The dUTPase/LAT recombinant virus was suspended in PBS-glucose medium at 2000 PFU/ml and incubated at 37°C for 7 hr. Aliquots of 0.5 ml of the suspension were then adsorbed to Vero cells (10 5 cells in 24-well plates) for 1 hr. The virus suspension was then replaced with overlay medium, and reporter gene activity was determined at 24 hr. Data are the averages ± S.D of 4 duplicate determinations.

    Article Snippet: The cytotoxic effect of DMSO (Sigma or Fisher) treatment on Vero cells was evaluated by the method of sulforhodamin B (SRB) [ , ].

    Techniques: Infection, Recombinant, Incubation, Activity Assay

    Effect of DMSO in the productive infection of HSV-1. Cultured Vero cells (10 5 cells in 24-well plates) were infected with 1 million PFU of the dUTPase/LAT recombinant virus. After 1 hr adsorption period, overlay medium was added containing the indicated concentrations of DMSO. At 24 hr post-infection, the cells were harvested and the virus yield was determined by plaque assay. Data are the average ± SD of 3 duplicate determinations.

    Journal: BMC Infectious Diseases

    Article Title: Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis

    doi: 10.1186/1471-2334-2-9

    Figure Lengend Snippet: Effect of DMSO in the productive infection of HSV-1. Cultured Vero cells (10 5 cells in 24-well plates) were infected with 1 million PFU of the dUTPase/LAT recombinant virus. After 1 hr adsorption period, overlay medium was added containing the indicated concentrations of DMSO. At 24 hr post-infection, the cells were harvested and the virus yield was determined by plaque assay. Data are the average ± SD of 3 duplicate determinations.

    Article Snippet: The cytotoxic effect of DMSO (Sigma or Fisher) treatment on Vero cells was evaluated by the method of sulforhodamin B (SRB) [ , ].

    Techniques: Infection, Cell Culture, Recombinant, Adsorption, Plaque Assay

    Induction of LC3 aggregations and autophagy-related genes by lapatinib in Huh7 (A), HA22T (B) and HepG2 (C) HCC cells. Cells were treated with DMSO or 5 or 10 μM lapatinib treatment for 4-48h as indicated. Cell lysates were then collected, subjected into 12% SDS-PAGE, and immunoblotted with antibodies against p62, Beclin, BNIP, ATG5, ATG7 LC3 and actin.

    Journal: Oncotarget

    Article Title: Lapatinib induces autophagic cell death and inhibits growth of human hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: Induction of LC3 aggregations and autophagy-related genes by lapatinib in Huh7 (A), HA22T (B) and HepG2 (C) HCC cells. Cells were treated with DMSO or 5 or 10 μM lapatinib treatment for 4-48h as indicated. Cell lysates were then collected, subjected into 12% SDS-PAGE, and immunoblotted with antibodies against p62, Beclin, BNIP, ATG5, ATG7 LC3 and actin.

    Article Snippet: One thousand-fold stock solution of lapatinib was prepared by dissolving a ground lapatinib tablet (GlaxoSmithKline) in DMSO (Sigma).

    Techniques: SDS Page

    Protection of HCC cells from lapatinib-induced cytotoxicity by the knockdown of autophagy-related proteins. After transduction with shRNA expression lentivirus as indicated, Huh7 or HepG2 cells were selected and were treated with DMSO or 2.5 - 10 μM lapatinib for 48h, and then the relative percentages of growth inhibition were detected using the MTS assay and calculated as described in Fig. 1

    Journal: Oncotarget

    Article Title: Lapatinib induces autophagic cell death and inhibits growth of human hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: Protection of HCC cells from lapatinib-induced cytotoxicity by the knockdown of autophagy-related proteins. After transduction with shRNA expression lentivirus as indicated, Huh7 or HepG2 cells were selected and were treated with DMSO or 2.5 - 10 μM lapatinib for 48h, and then the relative percentages of growth inhibition were detected using the MTS assay and calculated as described in Fig. 1

    Article Snippet: One thousand-fold stock solution of lapatinib was prepared by dissolving a ground lapatinib tablet (GlaxoSmithKline) in DMSO (Sigma).

    Techniques: Transduction, shRNA, Expressing, Inhibition, MTS Assay

    In vivo inhibition of HCC xenografts by lapatinib After inoculation of HepG2 cells (luciferase stably expressed) for 3 weeks, 12 mice were randomly divided into 3 groups and were orally administrated DMSO, 100 or 200 mg/kg of lapatinib for another 22 days (days 0-22). Tumor volumes were monitored every 2 days by detection of luciferase-activity-containing cells in vivo (A) or weighted after sacrificing on day 42 (B).

    Journal: Oncotarget

    Article Title: Lapatinib induces autophagic cell death and inhibits growth of human hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: In vivo inhibition of HCC xenografts by lapatinib After inoculation of HepG2 cells (luciferase stably expressed) for 3 weeks, 12 mice were randomly divided into 3 groups and were orally administrated DMSO, 100 or 200 mg/kg of lapatinib for another 22 days (days 0-22). Tumor volumes were monitored every 2 days by detection of luciferase-activity-containing cells in vivo (A) or weighted after sacrificing on day 42 (B).

    Article Snippet: One thousand-fold stock solution of lapatinib was prepared by dissolving a ground lapatinib tablet (GlaxoSmithKline) in DMSO (Sigma).

    Techniques: In Vivo, Inhibition, Luciferase, Stable Transfection, Mouse Assay, Activity Assay

    Induction of non-apoptotic cell death by lapatinib in HCC cells Cells were left untreated (unTx) treated, with 0.1% DMSO (vehicle, D) or 2.5–10 μM lapatinib for 3 days. After collection, cells were split into 2 tubes and resuspended in either PI containing PBS buffer (A or left panel of C, for detection of percent of total dead cells) or PI-containing hypotonic buffer (B or right panel of C, for detection of percent of hypodiploid or apoptotic cells) by flow cytometry.

    Journal: Oncotarget

    Article Title: Lapatinib induces autophagic cell death and inhibits growth of human hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: Induction of non-apoptotic cell death by lapatinib in HCC cells Cells were left untreated (unTx) treated, with 0.1% DMSO (vehicle, D) or 2.5–10 μM lapatinib for 3 days. After collection, cells were split into 2 tubes and resuspended in either PI containing PBS buffer (A or left panel of C, for detection of percent of total dead cells) or PI-containing hypotonic buffer (B or right panel of C, for detection of percent of hypodiploid or apoptotic cells) by flow cytometry.

    Article Snippet: One thousand-fold stock solution of lapatinib was prepared by dissolving a ground lapatinib tablet (GlaxoSmithKline) in DMSO (Sigma).

    Techniques: Flow Cytometry, Cytometry

    Induction of autophagy in lapatinib-treated HCC cells After DMSO, 5 or 10 μM lapatinib treatment for 3 days, Huh7 (A-B), HepG2 (C-D) and HA22T (E-G) HCC cells were stained with acridine orange, and the autophagic cells was analyzed by flow cytometry. Data are expressed as the mean fluorescence intensity (B or G) or percentage of acridine orange positive cells (C, D or F).

    Journal: Oncotarget

    Article Title: Lapatinib induces autophagic cell death and inhibits growth of human hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: Induction of autophagy in lapatinib-treated HCC cells After DMSO, 5 or 10 μM lapatinib treatment for 3 days, Huh7 (A-B), HepG2 (C-D) and HA22T (E-G) HCC cells were stained with acridine orange, and the autophagic cells was analyzed by flow cytometry. Data are expressed as the mean fluorescence intensity (B or G) or percentage of acridine orange positive cells (C, D or F).

    Article Snippet: One thousand-fold stock solution of lapatinib was prepared by dissolving a ground lapatinib tablet (GlaxoSmithKline) in DMSO (Sigma).

    Techniques: Staining, Flow Cytometry, Cytometry, Fluorescence

    Induction of LC3 aggregations by lapatinib in HCC cells The stable expression of GFP-LC3 in HCC cells was prepared by the transduction of a GFP-LC3-containing lentivirus. After DMSO, 5 or 10 μM lapatinib or 150 nM thapsigargin (TG, positive control) treatment for 3 days; Huh7 (A), HepG2 (B), or HA22T (C) cells were observed by fluorescence microscopy.

    Journal: Oncotarget

    Article Title: Lapatinib induces autophagic cell death and inhibits growth of human hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: Induction of LC3 aggregations by lapatinib in HCC cells The stable expression of GFP-LC3 in HCC cells was prepared by the transduction of a GFP-LC3-containing lentivirus. After DMSO, 5 or 10 μM lapatinib or 150 nM thapsigargin (TG, positive control) treatment for 3 days; Huh7 (A), HepG2 (B), or HA22T (C) cells were observed by fluorescence microscopy.

    Article Snippet: One thousand-fold stock solution of lapatinib was prepared by dissolving a ground lapatinib tablet (GlaxoSmithKline) in DMSO (Sigma).

    Techniques: Expressing, Transduction, Positive Control, Fluorescence, Microscopy

    Microphotographs of lapatinib-treated HCC cells (A) After DMSO or 5 or 10 μM lapatinib treatment for 2 days, Huh7 (A), HepG2 (B) or HA22T (C) HCC cells were fixed, stained and observed by transmission electron microscopy. Mitochondria with vacuolization were marked with red dots. The magnification information is as indicated in each figure.

    Journal: Oncotarget

    Article Title: Lapatinib induces autophagic cell death and inhibits growth of human hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: Microphotographs of lapatinib-treated HCC cells (A) After DMSO or 5 or 10 μM lapatinib treatment for 2 days, Huh7 (A), HepG2 (B) or HA22T (C) HCC cells were fixed, stained and observed by transmission electron microscopy. Mitochondria with vacuolization were marked with red dots. The magnification information is as indicated in each figure.

    Article Snippet: One thousand-fold stock solution of lapatinib was prepared by dissolving a ground lapatinib tablet (GlaxoSmithKline) in DMSO (Sigma).

    Techniques: Staining, Transmission Assay, Electron Microscopy

    Inhibition of hepatoma cell proliferation by lapatinib Huh7 (A), HepG2 (B), or HA22T (C) HCC cells were left untreated or treated with 0.1% DMSO (vehicle, D) or with DMSO containing various concentrations of lapatinib (1.25, 2.5, 5, or 10 μM) for 3 days. Relative amounts of viable cells were detected by MTS assay. O.D. values from DMSO-treated control cells were designated 100%.

    Journal: Oncotarget

    Article Title: Lapatinib induces autophagic cell death and inhibits growth of human hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: Inhibition of hepatoma cell proliferation by lapatinib Huh7 (A), HepG2 (B), or HA22T (C) HCC cells were left untreated or treated with 0.1% DMSO (vehicle, D) or with DMSO containing various concentrations of lapatinib (1.25, 2.5, 5, or 10 μM) for 3 days. Relative amounts of viable cells were detected by MTS assay. O.D. values from DMSO-treated control cells were designated 100%.

    Article Snippet: One thousand-fold stock solution of lapatinib was prepared by dissolving a ground lapatinib tablet (GlaxoSmithKline) in DMSO (Sigma).

    Techniques: Inhibition, MTS Assay

    Effects of CDK4/6 inhibition on breast cancer cell proliferation and apoptosis in vitro a , Relative numbers of breast cancer cells cultured in 250 nM (MDA-MB-453) or 500 nM (MDA-MB-361, BT474) abemaciclib or DMSO for 11d, followed by drug withdrawal (arrow). b , Representative SA-β-galactosidase staining of MDA-MB-453 cells (left) and BT474 cells (right) treated with DMSO or abemaciclib (MDA-MB-453, 250 nM; BT474, 500 nM) for 0, 4, and 7 days. c , Western blot of SKBR3, BT474, MDA-MB-453, and MDA-MB-361 cells treated with DMSO, lapatinib, or abemaciclib for 48h. d , Western blot of MDA-MB-453 cells pretreated with DMSO or abemaciclib (500 nM) for 0, 1, or 7 days prior to exposure to staurosporine (500 nM) for 4h. For western blot source images, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: Effects of CDK4/6 inhibition on breast cancer cell proliferation and apoptosis in vitro a , Relative numbers of breast cancer cells cultured in 250 nM (MDA-MB-453) or 500 nM (MDA-MB-361, BT474) abemaciclib or DMSO for 11d, followed by drug withdrawal (arrow). b , Representative SA-β-galactosidase staining of MDA-MB-453 cells (left) and BT474 cells (right) treated with DMSO or abemaciclib (MDA-MB-453, 250 nM; BT474, 500 nM) for 0, 4, and 7 days. c , Western blot of SKBR3, BT474, MDA-MB-453, and MDA-MB-361 cells treated with DMSO, lapatinib, or abemaciclib for 48h. d , Western blot of MDA-MB-453 cells pretreated with DMSO or abemaciclib (500 nM) for 0, 1, or 7 days prior to exposure to staurosporine (500 nM) for 4h. For western blot source images, see Supplementary Figure 1 .

    Article Snippet: To determine JAK dependency, cells were treated with combinations of DMSO, abemaciclib (500nM), and ruxolitinib (500nM, Selleckchem) for 7 days.

    Techniques: Inhibition, In Vitro, Cell Culture, Multiple Displacement Amplification, Staining, Western Blot

    CDK4/6 inhibition enhances antigen presentation a , Antigen processing and presentation gene expression in MDA-MB-361 cells treated with 500 nM abemaciclib or DMSO (7d, n=3). b , Gene expression in cell lines treated with DMSO or palbociclib (7d, n=3). c , β2M/MHC-I flow cytometry in cell lines; grey, FMO control. (For MDA-MB-453, vehicle and abemaciclib, n=2; palbociclib, n=3. For MDA-MB-361, n=3) d , Gene expression in TCGA samples ( CCND1 shallow deletion, n=101; CCND1 diploid, n=503; CCND1 gain, n=203; CCND1 amplified, n=153). e , H-2K b SIINFEKL flow cytometry after 7d abemaciclib or DMSO (B16-OVA, n=9; MMTV-PyMT -S2WTP3-OVA, n=3). f , CD8 + T cell proliferation in response to abemaciclib-pretreated tumor cells (n=3). g , IFNγ and TNFα production in tumor cell/OT-I assay by ELISA (n=3). One-way ANOVA adjusted for multiple comparisons (c–d,f), unpaired two-tailed t-tests (a–b,e,g). Error bars, SD. *p

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: CDK4/6 inhibition enhances antigen presentation a , Antigen processing and presentation gene expression in MDA-MB-361 cells treated with 500 nM abemaciclib or DMSO (7d, n=3). b , Gene expression in cell lines treated with DMSO or palbociclib (7d, n=3). c , β2M/MHC-I flow cytometry in cell lines; grey, FMO control. (For MDA-MB-453, vehicle and abemaciclib, n=2; palbociclib, n=3. For MDA-MB-361, n=3) d , Gene expression in TCGA samples ( CCND1 shallow deletion, n=101; CCND1 diploid, n=503; CCND1 gain, n=203; CCND1 amplified, n=153). e , H-2K b SIINFEKL flow cytometry after 7d abemaciclib or DMSO (B16-OVA, n=9; MMTV-PyMT -S2WTP3-OVA, n=3). f , CD8 + T cell proliferation in response to abemaciclib-pretreated tumor cells (n=3). g , IFNγ and TNFα production in tumor cell/OT-I assay by ELISA (n=3). One-way ANOVA adjusted for multiple comparisons (c–d,f), unpaired two-tailed t-tests (a–b,e,g). Error bars, SD. *p

    Article Snippet: To determine JAK dependency, cells were treated with combinations of DMSO, abemaciclib (500nM), and ruxolitinib (500nM, Selleckchem) for 7 days.

    Techniques: Inhibition, Expressing, Multiple Displacement Amplification, Flow Cytometry, Cytometry, Amplification, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Abemaciclib induces a ‘senescence-like’ phenotype without evidence of SASP a , Representative SA-β-galactosidase staining (left) of MMTV-rtTA/tetO-HER2 tumors treated with vehicle or abemaciclib for 12d (scale bar=500 μ m). Quantification of relative SA-β-galactosidase positive area (right, n=6 tumors/group). b , mRNA expression of SASP factors in MMTV-rtTA/tetO-HER2 tumors treated as in Fig. 1a . Il6 expression determined by qPCR (n=10 tumors/group); Il1a and Il1b by transcriptome analysis (vehicle, n=11; abemaciclib, n=12 tumors/group). c , MDA-MB-453 and BT474 cells treated with DMSO or abemaciclib (500 nM) for 7d, and expression determined by qPCR. d , mRNA expression of IL6 upon doxorubicin-induced senescence (n=3). MDA-MB-453 and BT474 cells were treated with doxorubicin (200 nM) for 24h, and mRNA extracted 3d later for qPCR. Unpaired two-tailed t tests (a,d). Error bars, SD. **p

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: Abemaciclib induces a ‘senescence-like’ phenotype without evidence of SASP a , Representative SA-β-galactosidase staining (left) of MMTV-rtTA/tetO-HER2 tumors treated with vehicle or abemaciclib for 12d (scale bar=500 μ m). Quantification of relative SA-β-galactosidase positive area (right, n=6 tumors/group). b , mRNA expression of SASP factors in MMTV-rtTA/tetO-HER2 tumors treated as in Fig. 1a . Il6 expression determined by qPCR (n=10 tumors/group); Il1a and Il1b by transcriptome analysis (vehicle, n=11; abemaciclib, n=12 tumors/group). c , MDA-MB-453 and BT474 cells treated with DMSO or abemaciclib (500 nM) for 7d, and expression determined by qPCR. d , mRNA expression of IL6 upon doxorubicin-induced senescence (n=3). MDA-MB-453 and BT474 cells were treated with doxorubicin (200 nM) for 24h, and mRNA extracted 3d later for qPCR. Unpaired two-tailed t tests (a,d). Error bars, SD. **p

    Article Snippet: To determine JAK dependency, cells were treated with combinations of DMSO, abemaciclib (500nM), and ruxolitinib (500nM, Selleckchem) for 7 days.

    Techniques: Staining, Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Two Tailed Test

    Impact of CDK4/6 inhibition on immune cell populations and Treg biology a , Flow cytometric analysis of immune cell populations in MMTV-rtTA/tetO-HER2 tumors treated with vehicle or abemaciclib for 12d (vehicle, n=15; abemaciclib, n=17 tumors/group). b , Peripheral blood Tregs in MMTV-rtTA/tetO-HER2 mice (12d, n=4 mice/group). c–d , Tregs (CD4 + FoxP3 + ) quantified by flow cytometry of MMTV-PyMT tumors (c, vehicle, n=18; abemaciclib, n=16 tumors/group) and CT-26 tumors (d, n=12 tumors/group) treated as indicated for 12d. e , Tregs in lymph nodes of tumor-free mice (12d, one-way ANOVA corrected for multiple comparisons, vehicles and palbociclib, n=8; abemaciclib, n=7 mice/group). f , Treg:CD8 ratio in the spleens and lymph nodes (LN) of tumor-free FVB mice (12d, vehicles and palbociclib, n=8; abemaciclib, n=7 mice/group). g , Plasma autoantibodies in tumor-free and tumor-bearing mice treated with vehicle or abemaciclib for 12d (tumor-free vehicle, n=8; tumor-free abemaciclib, n=7; tumor-bearing vehicle, n=7; tumor-bearing abemaciclib, n=6 mice/group). h–l , Tumor-free FVB mice treated with abemaciclib or vehicle for 12d. Thymic mass (h). Thymic cell populations were quantified by flow cytometry. CD4 + CD8 + double positive (DP) thymocytes (i); CD4 + single positive (SP) thymocytes (j); CD8 + SP thymocytes (k); CD4 + FoxP3 + regulatory T cells (l). (vehicles and palbociclib, n=5; abemaciclib, n=4 mice/group). m , Effect of abemaciclib or DMSO on ex vivo differentiation of CD4 + CD25 − T cells into Tregs in the presence of TGF-β for 72hr (n=4). n , Effect of DMSO or abemaciclib treatment for 72hr on Treg apoptosis measured by Annexin V staining (n=2). o , Quantification of immunofluorescent staining of MMTV-rtTA/tetO-HER2 tumors (n=7 tumors/group). p – q , Dnmt1 (p) and Cdkn1a (q) in T cells from tumor-free mice (12d, two-way ANOVA corrected for multiple comparisons, n=7 mice/group [pooled]). Unpaired two-tailed t-tests (b–d), one-way ANOVA corrected for multiple comparisons (e–f,h–l). Error bars, SD. *p

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: Impact of CDK4/6 inhibition on immune cell populations and Treg biology a , Flow cytometric analysis of immune cell populations in MMTV-rtTA/tetO-HER2 tumors treated with vehicle or abemaciclib for 12d (vehicle, n=15; abemaciclib, n=17 tumors/group). b , Peripheral blood Tregs in MMTV-rtTA/tetO-HER2 mice (12d, n=4 mice/group). c–d , Tregs (CD4 + FoxP3 + ) quantified by flow cytometry of MMTV-PyMT tumors (c, vehicle, n=18; abemaciclib, n=16 tumors/group) and CT-26 tumors (d, n=12 tumors/group) treated as indicated for 12d. e , Tregs in lymph nodes of tumor-free mice (12d, one-way ANOVA corrected for multiple comparisons, vehicles and palbociclib, n=8; abemaciclib, n=7 mice/group). f , Treg:CD8 ratio in the spleens and lymph nodes (LN) of tumor-free FVB mice (12d, vehicles and palbociclib, n=8; abemaciclib, n=7 mice/group). g , Plasma autoantibodies in tumor-free and tumor-bearing mice treated with vehicle or abemaciclib for 12d (tumor-free vehicle, n=8; tumor-free abemaciclib, n=7; tumor-bearing vehicle, n=7; tumor-bearing abemaciclib, n=6 mice/group). h–l , Tumor-free FVB mice treated with abemaciclib or vehicle for 12d. Thymic mass (h). Thymic cell populations were quantified by flow cytometry. CD4 + CD8 + double positive (DP) thymocytes (i); CD4 + single positive (SP) thymocytes (j); CD8 + SP thymocytes (k); CD4 + FoxP3 + regulatory T cells (l). (vehicles and palbociclib, n=5; abemaciclib, n=4 mice/group). m , Effect of abemaciclib or DMSO on ex vivo differentiation of CD4 + CD25 − T cells into Tregs in the presence of TGF-β for 72hr (n=4). n , Effect of DMSO or abemaciclib treatment for 72hr on Treg apoptosis measured by Annexin V staining (n=2). o , Quantification of immunofluorescent staining of MMTV-rtTA/tetO-HER2 tumors (n=7 tumors/group). p – q , Dnmt1 (p) and Cdkn1a (q) in T cells from tumor-free mice (12d, two-way ANOVA corrected for multiple comparisons, n=7 mice/group [pooled]). Unpaired two-tailed t-tests (b–d), one-way ANOVA corrected for multiple comparisons (e–f,h–l). Error bars, SD. *p

    Article Snippet: To determine JAK dependency, cells were treated with combinations of DMSO, abemaciclib (500nM), and ruxolitinib (500nM, Selleckchem) for 7 days.

    Techniques: Inhibition, Flow Cytometry, Mouse Assay, Cytometry, Ex Vivo, Staining, Two Tailed Test

    DNMT1 suppression mediates dsRNA response a , DNMT1 expression after treatment with abemaciclib (n=3). b , DNMT1 protein expression after treatment with abemaciclib. c , DNMT3A expression after treatment with abemaciclib (500 nM) for 7d (n=3, except abemaciclib 24 hrs, n=2). d , DNMT1 expression after abemaciclib or control in MMTV-rtTA/tetO-HER2 tumors, p=0.05, (12d, vehicle, n=11; abemaciclib, n=12 tumors/group). e , RB1 knockdown in MDA-MB-453 cells and its effect on indicated gene expression after 7d abemaciclib (500 nM) (two biological replicates each associated with three technical replicates). f , ERV3-1 methylation. g , ERV expression after abemaciclib or DMSO (7d, n=3). h , Relative dsRNA expression after 7d of abemaciclib compared to DMSO (n=3). i–j , Cytosolic pattern recognition receptors in cells (i) (7d, n=3) or PDX tumors (j) (21–28d, vehicle, n=4; abemaciclib, n=2 tumors/group). k , Western blot of overexpression of DNMT1 in MDA-MB-453 cells and quantification of mRNA expression (n=3). Unpaired two-tailed t-tests (d–e,g–k) adjusted for multiple comparisons (a). Error bars, SD. *p≤0.05, **p

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: DNMT1 suppression mediates dsRNA response a , DNMT1 expression after treatment with abemaciclib (n=3). b , DNMT1 protein expression after treatment with abemaciclib. c , DNMT3A expression after treatment with abemaciclib (500 nM) for 7d (n=3, except abemaciclib 24 hrs, n=2). d , DNMT1 expression after abemaciclib or control in MMTV-rtTA/tetO-HER2 tumors, p=0.05, (12d, vehicle, n=11; abemaciclib, n=12 tumors/group). e , RB1 knockdown in MDA-MB-453 cells and its effect on indicated gene expression after 7d abemaciclib (500 nM) (two biological replicates each associated with three technical replicates). f , ERV3-1 methylation. g , ERV expression after abemaciclib or DMSO (7d, n=3). h , Relative dsRNA expression after 7d of abemaciclib compared to DMSO (n=3). i–j , Cytosolic pattern recognition receptors in cells (i) (7d, n=3) or PDX tumors (j) (21–28d, vehicle, n=4; abemaciclib, n=2 tumors/group). k , Western blot of overexpression of DNMT1 in MDA-MB-453 cells and quantification of mRNA expression (n=3). Unpaired two-tailed t-tests (d–e,g–k) adjusted for multiple comparisons (a). Error bars, SD. *p≤0.05, **p

    Article Snippet: To determine JAK dependency, cells were treated with combinations of DMSO, abemaciclib (500nM), and ruxolitinib (500nM, Selleckchem) for 7 days.

    Techniques: Expressing, Multiple Displacement Amplification, Methylation, Western Blot, Over Expression, Two Tailed Test

    CDK4/6 inhibition mediates Type III interferon production a , Phospho- and total STAT1 in MDA-MB-453 cells treated with abemaciclib +/− ruxolitinib for 7d. b , Effect of neutralization of IFN-α or IFN-γ on STAT1 mRNA expression (n=2–4). c – d , Impact of neutralization of IFN-α (c) and IFN-γ (d) on phospho-STAT1 and total STAT1 protein in indicated cell lines. e , Expression of type III interferon genes in indicated cell lines treated with abemaciclib for 7d compared to DMSO (n=3). f , Type III interferon production measured by ELISA (7d, n=2). Unpaired two-tailed t-tests (e–f) adjusted for multiple comparisons (b). Error bars, SD. *p

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: CDK4/6 inhibition mediates Type III interferon production a , Phospho- and total STAT1 in MDA-MB-453 cells treated with abemaciclib +/− ruxolitinib for 7d. b , Effect of neutralization of IFN-α or IFN-γ on STAT1 mRNA expression (n=2–4). c – d , Impact of neutralization of IFN-α (c) and IFN-γ (d) on phospho-STAT1 and total STAT1 protein in indicated cell lines. e , Expression of type III interferon genes in indicated cell lines treated with abemaciclib for 7d compared to DMSO (n=3). f , Type III interferon production measured by ELISA (7d, n=2). Unpaired two-tailed t-tests (e–f) adjusted for multiple comparisons (b). Error bars, SD. *p

    Article Snippet: To determine JAK dependency, cells were treated with combinations of DMSO, abemaciclib (500nM), and ruxolitinib (500nM, Selleckchem) for 7 days.

    Techniques: Inhibition, Multiple Displacement Amplification, Neutralization, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    CDK4/6 inhibition increases interferon signaling a–b , Top ranked GO terms (a) and expression of interferon-responsive transcription factors (b) in MDA-MB-361 cells treated with 500 nM abemaciclib or DMSO (7d, n=3). c , Expression of interferon sensitive genes (ISGs) in MMTV-PyMT -S2WTP3 cells treated with DMSO or abemaciclib (500 nM, 7d) (n=3). d , Expression of ISGs in MDA-MB-361 and MCF7 cells treated with abemaciclib (100 nM), fulvestrant (100 nM), or the combination for 7d (n=3). e , Expression of ISGs in MDA-MB-453, MCF7, and MDA-MB-231 cells treated with abemaciclib or DMSO (7d, n=3). f , Expression of ISGs in PDX 14-07 tumors treated with abemaciclib or vehicle (21–28d, vehicle, n=4; abemaciclib, n=2 tumors/group). g , phospho- and total STAT1 in cells treated with 500 nM abemaciclib as indicated. h , Confirmation of p16-FLAG overexpression in MDA-MB-453 and BT474 cells (left) and gene expression in these cell lines by qPCR (right) (n=6). i–j , Gene expression changes in MMTV-rtTA/tetO-HER2 tumors from mice treated with vehicle or abemaciclib for 12d (vehicle, n=11; abemaciclib, n=12 tumors/group). Relative expression of interferon-responsive T cell chemoattractants (i); relative expression of ISGs (j). k , Correlation of expression of Stat1 and Nlrc5 with genes involved in antigen processing and presentation in MMTV-rtTA/tetO-HER2 tumors. Blue dots, vehicle-treated tumors; red dots, abemaciclib-treated tumors. (r is Pearson product-moment correlation coefficient). Unpaired two-tailed t-tests (b,d–f,h–j) adjusted for multiple comparisons (c). Error bars, SD. *p

    Journal: Nature

    Article Title: CDK4/6 inhibition triggers anti-tumor immunity

    doi: 10.1038/nature23465

    Figure Lengend Snippet: CDK4/6 inhibition increases interferon signaling a–b , Top ranked GO terms (a) and expression of interferon-responsive transcription factors (b) in MDA-MB-361 cells treated with 500 nM abemaciclib or DMSO (7d, n=3). c , Expression of interferon sensitive genes (ISGs) in MMTV-PyMT -S2WTP3 cells treated with DMSO or abemaciclib (500 nM, 7d) (n=3). d , Expression of ISGs in MDA-MB-361 and MCF7 cells treated with abemaciclib (100 nM), fulvestrant (100 nM), or the combination for 7d (n=3). e , Expression of ISGs in MDA-MB-453, MCF7, and MDA-MB-231 cells treated with abemaciclib or DMSO (7d, n=3). f , Expression of ISGs in PDX 14-07 tumors treated with abemaciclib or vehicle (21–28d, vehicle, n=4; abemaciclib, n=2 tumors/group). g , phospho- and total STAT1 in cells treated with 500 nM abemaciclib as indicated. h , Confirmation of p16-FLAG overexpression in MDA-MB-453 and BT474 cells (left) and gene expression in these cell lines by qPCR (right) (n=6). i–j , Gene expression changes in MMTV-rtTA/tetO-HER2 tumors from mice treated with vehicle or abemaciclib for 12d (vehicle, n=11; abemaciclib, n=12 tumors/group). Relative expression of interferon-responsive T cell chemoattractants (i); relative expression of ISGs (j). k , Correlation of expression of Stat1 and Nlrc5 with genes involved in antigen processing and presentation in MMTV-rtTA/tetO-HER2 tumors. Blue dots, vehicle-treated tumors; red dots, abemaciclib-treated tumors. (r is Pearson product-moment correlation coefficient). Unpaired two-tailed t-tests (b,d–f,h–j) adjusted for multiple comparisons (c). Error bars, SD. *p

    Article Snippet: To determine JAK dependency, cells were treated with combinations of DMSO, abemaciclib (500nM), and ruxolitinib (500nM, Selleckchem) for 7 days.

    Techniques: Inhibition, Expressing, Multiple Displacement Amplification, Over Expression, Real-time Polymerase Chain Reaction, Mouse Assay, Two Tailed Test