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  • 99
    Thermo Fisher transposon products
    Selection of a viable M-eGFP fusion protein. (A) cDNA of VSV M (blue) was subjected to <t>transposon</t> mutagenesis. The Tn (orange) was replaced with eGFP (green), and the resulting M-eGFP library was cloned into a VSV genomic analog consisting of 3′-Le-M-G-Tr-5′
    Transposon Products, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    transposon products - by Bioz Stars, 2020-04
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    99
    Millipore nitrite production the nitrite production
    Selection of a viable M-eGFP fusion protein. (A) cDNA of VSV M (blue) was subjected to <t>transposon</t> mutagenesis. The Tn (orange) was replaced with eGFP (green), and the resulting M-eGFP library was cloned into a VSV genomic analog consisting of 3′-Le-M-G-Tr-5′
    Nitrite Production The Nitrite Production, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Selleck Chemicals natural product library
    Selection of a viable M-eGFP fusion protein. (A) cDNA of VSV M (blue) was subjected to <t>transposon</t> mutagenesis. The Tn (orange) was replaced with eGFP (green), and the resulting M-eGFP library was cloned into a VSV genomic analog consisting of 3′-Le-M-G-Tr-5′
    Natural Product Library, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher oncomine ngs data product
    Selection of a viable M-eGFP fusion protein. (A) cDNA of VSV M (blue) was subjected to <t>transposon</t> mutagenesis. The Tn (orange) was replaced with eGFP (green), and the resulting M-eGFP library was cloned into a VSV genomic analog consisting of 3′-Le-M-G-Tr-5′
    Oncomine Ngs Data Product, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    OriGene product sc319092
    Selection of a viable M-eGFP fusion protein. (A) cDNA of VSV M (blue) was subjected to <t>transposon</t> mutagenesis. The Tn (orange) was replaced with eGFP (green), and the resulting M-eGFP library was cloned into a VSV genomic analog consisting of 3′-Le-M-G-Tr-5′
    Product Sc319092, supplied by OriGene, used in various techniques. Bioz Stars score: 86/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher in house viral production
    Selection of a viable M-eGFP fusion protein. (A) cDNA of VSV M (blue) was subjected to <t>transposon</t> mutagenesis. The Tn (orange) was replaced with eGFP (green), and the resulting M-eGFP library was cloned into a VSV genomic analog consisting of 3′-Le-M-G-Tr-5′
    In House Viral Production, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Syntax for Science productive syntax
    Selection of a viable M-eGFP fusion protein. (A) cDNA of VSV M (blue) was subjected to <t>transposon</t> mutagenesis. The Tn (orange) was replaced with eGFP (green), and the resulting M-eGFP library was cloned into a VSV genomic analog consisting of 3′-Le-M-G-Tr-5′
    Productive Syntax, supplied by Syntax for Science, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher exosap it product cleanup
    Selection of a viable M-eGFP fusion protein. (A) cDNA of VSV M (blue) was subjected to <t>transposon</t> mutagenesis. The Tn (orange) was replaced with eGFP (green), and the resulting M-eGFP library was cloned into a VSV genomic analog consisting of 3′-Le-M-G-Tr-5′
    Exosap It Product Cleanup, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher atp production production
    Selection of a viable M-eGFP fusion protein. (A) cDNA of VSV M (blue) was subjected to <t>transposon</t> mutagenesis. The Tn (orange) was replaced with eGFP (green), and the resulting M-eGFP library was cloned into a VSV genomic analog consisting of 3′-Le-M-G-Tr-5′
    Atp Production Production, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore lentiviral shrna production
    Cytoplasmic but not nuclear DcpS rescues miRNA degradation in vitro . ( A ) Localization of Flag-∆NLS-DcpS and Flag-∆NES-DcpS mutants detected by immunofluorescence using the anti-Flag M2 antibody (red). Dapi staining (Blue) represents nucleus. Cells transfected with the empty vector (pcDNA 3.1-Flag) are shown. ( B ) Cytoplasmic but not nuclear DcpS activates in vitro miRNA degradation. 5′- 32 P-labeled synthetic miRNA (32Psyn-miRNA) was incubated for 10 minutes with 10 μg of total protein extracts from cells infected with <t>lentivirus</t> encoding for either an <t>shRNA</t> targeting 3′UTR of endogenous DcpS or control shRNA (scramble) and transfected with vectors expressing either Flag tagged DcpS mutated for nuclear localization signal (Flag-DcpSΔNLS) or Flag tagged DcpS mutated for nuclear export signal (Flag-DcpSΔNES). Cells transfected with pcDNA 3.1-Flag empty vectors were used as control. Knockdown of endogenous DcpS (lower band) and overexpression of mutants (upper band) were confirmed by western blotting (bottom panel). The endogenous β-actin (marked with *) was probed and used as loading control (the lower band correspond to Flag-DcpS). The graph represents the quantification of three independent experiments. The error bars represent standard errors and significance was analyzed using a Student’s t-test.
    Lentiviral Shrna Production, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Virapur aav production
    Cytoplasmic but not nuclear DcpS rescues miRNA degradation in vitro . ( A ) Localization of Flag-∆NLS-DcpS and Flag-∆NES-DcpS mutants detected by immunofluorescence using the anti-Flag M2 antibody (red). Dapi staining (Blue) represents nucleus. Cells transfected with the empty vector (pcDNA 3.1-Flag) are shown. ( B ) Cytoplasmic but not nuclear DcpS activates in vitro miRNA degradation. 5′- 32 P-labeled synthetic miRNA (32Psyn-miRNA) was incubated for 10 minutes with 10 μg of total protein extracts from cells infected with <t>lentivirus</t> encoding for either an <t>shRNA</t> targeting 3′UTR of endogenous DcpS or control shRNA (scramble) and transfected with vectors expressing either Flag tagged DcpS mutated for nuclear localization signal (Flag-DcpSΔNLS) or Flag tagged DcpS mutated for nuclear export signal (Flag-DcpSΔNES). Cells transfected with pcDNA 3.1-Flag empty vectors were used as control. Knockdown of endogenous DcpS (lower band) and overexpression of mutants (upper band) were confirmed by western blotting (bottom panel). The endogenous β-actin (marked with *) was probed and used as loading control (the lower band correspond to Flag-DcpS). The graph represents the quantification of three independent experiments. The error bars represent standard errors and significance was analyzed using a Student’s t-test.
    Aav Production, supplied by Virapur, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore compozr product line
    Cytoplasmic but not nuclear DcpS rescues miRNA degradation in vitro . ( A ) Localization of Flag-∆NLS-DcpS and Flag-∆NES-DcpS mutants detected by immunofluorescence using the anti-Flag M2 antibody (red). Dapi staining (Blue) represents nucleus. Cells transfected with the empty vector (pcDNA 3.1-Flag) are shown. ( B ) Cytoplasmic but not nuclear DcpS activates in vitro miRNA degradation. 5′- 32 P-labeled synthetic miRNA (32Psyn-miRNA) was incubated for 10 minutes with 10 μg of total protein extracts from cells infected with <t>lentivirus</t> encoding for either an <t>shRNA</t> targeting 3′UTR of endogenous DcpS or control shRNA (scramble) and transfected with vectors expressing either Flag tagged DcpS mutated for nuclear localization signal (Flag-DcpSΔNLS) or Flag tagged DcpS mutated for nuclear export signal (Flag-DcpSΔNES). Cells transfected with pcDNA 3.1-Flag empty vectors were used as control. Knockdown of endogenous DcpS (lower band) and overexpression of mutants (upper band) were confirmed by western blotting (bottom panel). The endogenous β-actin (marked with *) was probed and used as loading control (the lower band correspond to Flag-DcpS). The graph represents the quantification of three independent experiments. The error bars represent standard errors and significance was analyzed using a Student’s t-test.
    Compozr Product Line, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Becton Dickinson cytokine production
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
    Cytokine Production, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 2610 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 2610 article reviews
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    93
    Cocalico antibody production
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
    Antibody Production, supplied by Cocalico, used in various techniques. Bioz Stars score: 93/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Covance antibody production
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
    Antibody Production, supplied by Covance, used in various techniques. Bioz Stars score: 87/100, based on 190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Enzymax antibody production
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
    Antibody Production, supplied by Enzymax, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibody production/product/Enzymax
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    80
    Eli Lilly dista products co
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
    Dista Products Co, supplied by Eli Lilly, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher dynabead product information
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
    Dynabead Product Information, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dynabead product information - by Bioz Stars, 2020-04
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    91
    Cell Signaling Technology Inc ptmscan product line
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
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    Reagents Direct wnt production
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
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    Covalab antibody production
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
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    Kaneka Corp antibody production
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
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    Millipore degradation product mca
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
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    Genetic Technologies Ltd ecm production
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
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    Clinical and Laboratory Standards Institute esbl production
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
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    Allergan juvéderm product line
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
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    Novartis pharmaceutica products lp
    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular <t>cytokine</t> staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.
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    Tocris wnt production
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    Reverdia biotechnological production
    A ) PEDF activates <t>Wnt</t> signaling in hMSCs. hMSCs were treated with Wnt3a (50 ng/ml) and PEDF (500 ng/ml) and immunoblotted for phosphorylated LRP6. B ) hMSCs were treated with <t>IWP-2</t> (2 μM) for 24 and 48 h and then challenged with PEDF (500 ng/ml
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    A ) PEDF activates <t>Wnt</t> signaling in hMSCs. hMSCs were treated with Wnt3a (50 ng/ml) and PEDF (500 ng/ml) and immunoblotted for phosphorylated LRP6. B ) hMSCs were treated with <t>IWP-2</t> (2 μM) for 24 and 48 h and then challenged with PEDF (500 ng/ml
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    Image Search Results


    Selection of a viable M-eGFP fusion protein. (A) cDNA of VSV M (blue) was subjected to transposon mutagenesis. The Tn (orange) was replaced with eGFP (green), and the resulting M-eGFP library was cloned into a VSV genomic analog consisting of 3′-Le-M-G-Tr-5′

    Journal: Journal of Virology

    Article Title: Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

    doi: 10.1128/JVI.01371-15

    Figure Lengend Snippet: Selection of a viable M-eGFP fusion protein. (A) cDNA of VSV M (blue) was subjected to transposon mutagenesis. The Tn (orange) was replaced with eGFP (green), and the resulting M-eGFP library was cloned into a VSV genomic analog consisting of 3′-Le-M-G-Tr-5′

    Article Snippet: The VSV M-coding sequence was mutagenized using a mutation generation system (F-701; Finnzymes, Vantaa, Finland) according to the instructions of the manufacturer.

    Techniques: Selection, Mutagenesis, Clone Assay

    Cytoplasmic but not nuclear DcpS rescues miRNA degradation in vitro . ( A ) Localization of Flag-∆NLS-DcpS and Flag-∆NES-DcpS mutants detected by immunofluorescence using the anti-Flag M2 antibody (red). Dapi staining (Blue) represents nucleus. Cells transfected with the empty vector (pcDNA 3.1-Flag) are shown. ( B ) Cytoplasmic but not nuclear DcpS activates in vitro miRNA degradation. 5′- 32 P-labeled synthetic miRNA (32Psyn-miRNA) was incubated for 10 minutes with 10 μg of total protein extracts from cells infected with lentivirus encoding for either an shRNA targeting 3′UTR of endogenous DcpS or control shRNA (scramble) and transfected with vectors expressing either Flag tagged DcpS mutated for nuclear localization signal (Flag-DcpSΔNLS) or Flag tagged DcpS mutated for nuclear export signal (Flag-DcpSΔNES). Cells transfected with pcDNA 3.1-Flag empty vectors were used as control. Knockdown of endogenous DcpS (lower band) and overexpression of mutants (upper band) were confirmed by western blotting (bottom panel). The endogenous β-actin (marked with *) was probed and used as loading control (the lower band correspond to Flag-DcpS). The graph represents the quantification of three independent experiments. The error bars represent standard errors and significance was analyzed using a Student’s t-test.

    Journal: Scientific Reports

    Article Title: The human decapping scavenger enzyme DcpS modulates microRNA turnover

    doi: 10.1038/srep16688

    Figure Lengend Snippet: Cytoplasmic but not nuclear DcpS rescues miRNA degradation in vitro . ( A ) Localization of Flag-∆NLS-DcpS and Flag-∆NES-DcpS mutants detected by immunofluorescence using the anti-Flag M2 antibody (red). Dapi staining (Blue) represents nucleus. Cells transfected with the empty vector (pcDNA 3.1-Flag) are shown. ( B ) Cytoplasmic but not nuclear DcpS activates in vitro miRNA degradation. 5′- 32 P-labeled synthetic miRNA (32Psyn-miRNA) was incubated for 10 minutes with 10 μg of total protein extracts from cells infected with lentivirus encoding for either an shRNA targeting 3′UTR of endogenous DcpS or control shRNA (scramble) and transfected with vectors expressing either Flag tagged DcpS mutated for nuclear localization signal (Flag-DcpSΔNLS) or Flag tagged DcpS mutated for nuclear export signal (Flag-DcpSΔNES). Cells transfected with pcDNA 3.1-Flag empty vectors were used as control. Knockdown of endogenous DcpS (lower band) and overexpression of mutants (upper band) were confirmed by western blotting (bottom panel). The endogenous β-actin (marked with *) was probed and used as loading control (the lower band correspond to Flag-DcpS). The graph represents the quantification of three independent experiments. The error bars represent standard errors and significance was analyzed using a Student’s t-test.

    Article Snippet: shRNA lentivirus production and cell transduction ShRNA was purchased from Sigma-Aldrich: TRCN0000005569 reference number for shRNA against 3′UTR of DcpS, TRCN0000296739 for Xrn1 shRNA, TRCN0000349677 for Xrn2 shRNA and SHC002 Non-Mammalian sh Control called ≪Sh Scramble≫.

    Techniques: In Vitro, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Labeling, Incubation, Infection, shRNA, Expressing, Over Expression, Western Blot

    DcpS activates miRNA degradation independent of its decapping scavenger activity. ( A ) Inhibition of DcpS alters in vitro miRNA degradation. 10 μg of total protein extracts from cells either infected with lentivirus encoding for an shRNA targeting DcpS (left panels) or treated with a DcpS inhibitor (DcpS-inh; right panels) were incubated for 15 minutes with 5′- 32 P-labeled small RNA oligonucleotide that corresponds to let-7 miRNA sequence (32Psyn-miRNA). Knockdown of DcpS was validated by Western blotting (bottom panels). The endogenous β-actin was probed and used as loading control. ( B ) Catalytically inactive DcpS rescues miRNA degradation in cell treated with DcpS-inhibitors. 5′- 32 P-labeled synthetic miRNA (32Psyn-miRNA) was incubated for 10 minutes with 10 μg of total protein extracts from cells treated. First, cells were treated with DcpS-inh or DMSO and 6 h after were transfected with Flag tagged catalytically inactive DcpS mutant (Flag DcpS-CatM) expressing vectors or pcDNA vectors. Cells were harvested and proteins were lysed 12 h after transfection using RIPA buffer. The graph represents the quantification of three independent experiments and the error bars represent standard errors. Significance was analyzed using Student’s t-test.

    Journal: Scientific Reports

    Article Title: The human decapping scavenger enzyme DcpS modulates microRNA turnover

    doi: 10.1038/srep16688

    Figure Lengend Snippet: DcpS activates miRNA degradation independent of its decapping scavenger activity. ( A ) Inhibition of DcpS alters in vitro miRNA degradation. 10 μg of total protein extracts from cells either infected with lentivirus encoding for an shRNA targeting DcpS (left panels) or treated with a DcpS inhibitor (DcpS-inh; right panels) were incubated for 15 minutes with 5′- 32 P-labeled small RNA oligonucleotide that corresponds to let-7 miRNA sequence (32Psyn-miRNA). Knockdown of DcpS was validated by Western blotting (bottom panels). The endogenous β-actin was probed and used as loading control. ( B ) Catalytically inactive DcpS rescues miRNA degradation in cell treated with DcpS-inhibitors. 5′- 32 P-labeled synthetic miRNA (32Psyn-miRNA) was incubated for 10 minutes with 10 μg of total protein extracts from cells treated. First, cells were treated with DcpS-inh or DMSO and 6 h after were transfected with Flag tagged catalytically inactive DcpS mutant (Flag DcpS-CatM) expressing vectors or pcDNA vectors. Cells were harvested and proteins were lysed 12 h after transfection using RIPA buffer. The graph represents the quantification of three independent experiments and the error bars represent standard errors. Significance was analyzed using Student’s t-test.

    Article Snippet: shRNA lentivirus production and cell transduction ShRNA was purchased from Sigma-Aldrich: TRCN0000005569 reference number for shRNA against 3′UTR of DcpS, TRCN0000296739 for Xrn1 shRNA, TRCN0000349677 for Xrn2 shRNA and SHC002 Non-Mammalian sh Control called ≪Sh Scramble≫.

    Techniques: Activity Assay, Inhibition, In Vitro, Infection, shRNA, Incubation, Labeling, Sequencing, Western Blot, Transfection, Mutagenesis, Expressing

    The 5′-3′ exonuclease Xrn2 contributes to DcpS-dependent miRNA degradation. ( A ) The knockdown of Xrn2 but not Xrn1 abrogates miRNA degradation in vitro . 5′- 32 P-labeled synthetic miRNA (32Psyn-miRNA) was incubated for 15 minutes with 10 μg of total protein extracts from cells infected with lentivirus encoding for shRNA targeting either Xrn1, Xrn2 or control shRNA (scramble). The efficacy of knockdowns was confirmed by western blotting (bottom panel). Dashed lines indicate that unrelated lanes have been removed between samples. ( B ) DcpS requires Xrn2 to support miRNA degradation. Cells were infected with shRNA targeting Xrn2 followed by transfection of plasmids expressing Flag-DcpS-∆NLS. Transfection with pcDNA 3.1-Flag empty vector served as control. Degradation assay was performed using 10 μg of total protein and incubated with a 5′- 32 P-labeled synthetic miRNA (32Psyn-miRNA) for 10 min. The efficacy of knockdown was validated by western blotting and the endogenous β-actin was probed and used as loading control (Bottom panel).

    Journal: Scientific Reports

    Article Title: The human decapping scavenger enzyme DcpS modulates microRNA turnover

    doi: 10.1038/srep16688

    Figure Lengend Snippet: The 5′-3′ exonuclease Xrn2 contributes to DcpS-dependent miRNA degradation. ( A ) The knockdown of Xrn2 but not Xrn1 abrogates miRNA degradation in vitro . 5′- 32 P-labeled synthetic miRNA (32Psyn-miRNA) was incubated for 15 minutes with 10 μg of total protein extracts from cells infected with lentivirus encoding for shRNA targeting either Xrn1, Xrn2 or control shRNA (scramble). The efficacy of knockdowns was confirmed by western blotting (bottom panel). Dashed lines indicate that unrelated lanes have been removed between samples. ( B ) DcpS requires Xrn2 to support miRNA degradation. Cells were infected with shRNA targeting Xrn2 followed by transfection of plasmids expressing Flag-DcpS-∆NLS. Transfection with pcDNA 3.1-Flag empty vector served as control. Degradation assay was performed using 10 μg of total protein and incubated with a 5′- 32 P-labeled synthetic miRNA (32Psyn-miRNA) for 10 min. The efficacy of knockdown was validated by western blotting and the endogenous β-actin was probed and used as loading control (Bottom panel).

    Article Snippet: shRNA lentivirus production and cell transduction ShRNA was purchased from Sigma-Aldrich: TRCN0000005569 reference number for shRNA against 3′UTR of DcpS, TRCN0000296739 for Xrn1 shRNA, TRCN0000349677 for Xrn2 shRNA and SHC002 Non-Mammalian sh Control called ≪Sh Scramble≫.

    Techniques: In Vitro, Labeling, Incubation, Infection, shRNA, Western Blot, Transfection, Expressing, Plasmid Preparation, Degradation Assay

    p53 deficiency increases secretory the function of the ER through the IRE1α/XBP1 pathway A. HCT116 p53 +/+ or HCT116 p53 −/− cells, MEF p53 +/+ or MEF p53 −/− cells, and U2OS shLuc or U2OS shp53 cells expressing secreted embryonic alkaline phosphatase (SEAP) were transduced with a pSEAP2 control vector and washed 24 h after transduction. The medium was then changed, and the cells were cultured for another 6 h. Culture media were analyzed for SEAP activity, and luminescence was normalized to cell number. The transfection efficiencies of HCT116 p53 +/+ and HCT116 p53 −/− cells were approximately 80% each (data not shown). B. Overexpression of wild-type p53 inhibited SEAP activity. SEAP activities of cells that constitutively expressed the indicated p53 molecules were analyzed using the same procedure described in (A). C. HCT116 p53 −/− cells that expressed SEAP were transfected with siControl, siATF6, siPERK, or siIRE1α, cultured for 24 h, and following a change of medium, the cells were cultured for another 6 h. Whole cell lysates were analyzed using western blotting with the indicated antibodies, and culture supernatants were analyzed for SEAP activity. Values shown are the mean ± s.d. of three different experiments simultaneously measured. The P value was calculated using two-way ANOVA.

    Journal: Oncotarget

    Article Title: Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway

    doi:

    Figure Lengend Snippet: p53 deficiency increases secretory the function of the ER through the IRE1α/XBP1 pathway A. HCT116 p53 +/+ or HCT116 p53 −/− cells, MEF p53 +/+ or MEF p53 −/− cells, and U2OS shLuc or U2OS shp53 cells expressing secreted embryonic alkaline phosphatase (SEAP) were transduced with a pSEAP2 control vector and washed 24 h after transduction. The medium was then changed, and the cells were cultured for another 6 h. Culture media were analyzed for SEAP activity, and luminescence was normalized to cell number. The transfection efficiencies of HCT116 p53 +/+ and HCT116 p53 −/− cells were approximately 80% each (data not shown). B. Overexpression of wild-type p53 inhibited SEAP activity. SEAP activities of cells that constitutively expressed the indicated p53 molecules were analyzed using the same procedure described in (A). C. HCT116 p53 −/− cells that expressed SEAP were transfected with siControl, siATF6, siPERK, or siIRE1α, cultured for 24 h, and following a change of medium, the cells were cultured for another 6 h. Whole cell lysates were analyzed using western blotting with the indicated antibodies, and culture supernatants were analyzed for SEAP activity. Values shown are the mean ± s.d. of three different experiments simultaneously measured. The P value was calculated using two-way ANOVA.

    Article Snippet: Wild-type p53, p53-G245S, p53-R248W, p53-R249S, p53-R273H, shp53 (pLKO.1 p53 shRNA-753 and -814 from Sigma-Aldrich (TRCN0000003753)), and shLuc (pLKO.1 Luciferase shRNA Control, Sigma-Aldrich) constructs were introduced into HCT116 p53 +/+ , U2OS, or H1299 cells using lipofection.

    Techniques: Expressing, Transduction, Plasmid Preparation, Cell Culture, Activity Assay, Transfection, Over Expression, Western Blot

    IRE1α expression is regulated by p53 A. Western blot analysis of the expression of endogenous IRE1α in 23 human cancer cell lines. Cell lines were grouped according to expression of wild-type or mutant p53 as indicated. (A well between wt-p53 and mutant-p53 cell lines was cut, from the gel as indicated by a black line, due to the controversial p53 status of the cell line). Right panel: The intensities of the IRE1α bands (left panel) are expressed relative to those of β-actin. Values shown are the mean ± standard deviation (s.d.). The P value was calculated using two-way ANOVA. B. Downregulation of p53 expression induces increased expression of IRE1α. HCT116 p53 +/+ and U2OS cells were transfected with shLuc, shp53 (753), or shp53 (814), and selected using puromycin. Whole cell lysates of a pool of transfectants were analyzed using western blotting with the indicated antibodies. C. Overexpression of wild-type p53 inhibits IRE1α expression in mutant-p53 cell lines. Cell lysates, prepared 48 h after transfection with wild-type p53, were analyzed for the expression of indicated proteins. D. Mutant p53 proteins do not inhibit IRE1α expression. Cell lysates were prepared from cells transfected with p53-G245S, p53-R248W, p53-249S, and p53-R273H expression vectors or from cells that constitutively expressed wild-type p53 and were analyzed for the expression of the indicated proteins.

    Journal: Oncotarget

    Article Title: Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway

    doi:

    Figure Lengend Snippet: IRE1α expression is regulated by p53 A. Western blot analysis of the expression of endogenous IRE1α in 23 human cancer cell lines. Cell lines were grouped according to expression of wild-type or mutant p53 as indicated. (A well between wt-p53 and mutant-p53 cell lines was cut, from the gel as indicated by a black line, due to the controversial p53 status of the cell line). Right panel: The intensities of the IRE1α bands (left panel) are expressed relative to those of β-actin. Values shown are the mean ± standard deviation (s.d.). The P value was calculated using two-way ANOVA. B. Downregulation of p53 expression induces increased expression of IRE1α. HCT116 p53 +/+ and U2OS cells were transfected with shLuc, shp53 (753), or shp53 (814), and selected using puromycin. Whole cell lysates of a pool of transfectants were analyzed using western blotting with the indicated antibodies. C. Overexpression of wild-type p53 inhibits IRE1α expression in mutant-p53 cell lines. Cell lysates, prepared 48 h after transfection with wild-type p53, were analyzed for the expression of indicated proteins. D. Mutant p53 proteins do not inhibit IRE1α expression. Cell lysates were prepared from cells transfected with p53-G245S, p53-R248W, p53-249S, and p53-R273H expression vectors or from cells that constitutively expressed wild-type p53 and were analyzed for the expression of the indicated proteins.

    Article Snippet: Wild-type p53, p53-G245S, p53-R248W, p53-R249S, p53-R273H, shp53 (pLKO.1 p53 shRNA-753 and -814 from Sigma-Aldrich (TRCN0000003753)), and shLuc (pLKO.1 Luciferase shRNA Control, Sigma-Aldrich) constructs were introduced into HCT116 p53 +/+ , U2OS, or H1299 cells using lipofection.

    Techniques: Expressing, Western Blot, Mutagenesis, Standard Deviation, Transfection, Over Expression

    The IRE1α inhibitor (STF-083010) suppresses the growth in vitro and in vivo of p53-deficient human cancer cells A. Effects of an IRE1α inhibitor on cell viability and on Tm-induced cell death in p53-deficient cells. HCT116 p53 +/+ or HCT116 p53 −/− cells, MEF p53 +/+ or MEF p53 −/− cells, and U2OS shLuc or U2OS shp53 cells were treated with Tm (0.5 mg/mL), STF-083010 (50 μM), or both for 24 h. Cell viability was determined using an MTT assay. Values shown are the mean ± s.d. of three different experiments measured simultaneously. B. An IRE1α inhibitor selectively suppresses the growth of p53-deficient tumors in nude mice. HCT116 p53 +/+ and HCT116 p53 −/− cells were used to engraft nude mice, and 4 days after injecting the cells, DMSO or STF-083010 (40 mg/kg) was intraperitoneally administered once every 3 days. Tumor volume was measured on the indicated days. After 15 days, the weights of the tumors (left panel) were measured. Values shown are the mean ± standard error of the mean of eight mice from each group. The P value was calculated using two-way ANOVA.

    Journal: Oncotarget

    Article Title: Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway

    doi:

    Figure Lengend Snippet: The IRE1α inhibitor (STF-083010) suppresses the growth in vitro and in vivo of p53-deficient human cancer cells A. Effects of an IRE1α inhibitor on cell viability and on Tm-induced cell death in p53-deficient cells. HCT116 p53 +/+ or HCT116 p53 −/− cells, MEF p53 +/+ or MEF p53 −/− cells, and U2OS shLuc or U2OS shp53 cells were treated with Tm (0.5 mg/mL), STF-083010 (50 μM), or both for 24 h. Cell viability was determined using an MTT assay. Values shown are the mean ± s.d. of three different experiments measured simultaneously. B. An IRE1α inhibitor selectively suppresses the growth of p53-deficient tumors in nude mice. HCT116 p53 +/+ and HCT116 p53 −/− cells were used to engraft nude mice, and 4 days after injecting the cells, DMSO or STF-083010 (40 mg/kg) was intraperitoneally administered once every 3 days. Tumor volume was measured on the indicated days. After 15 days, the weights of the tumors (left panel) were measured. Values shown are the mean ± standard error of the mean of eight mice from each group. The P value was calculated using two-way ANOVA.

    Article Snippet: Wild-type p53, p53-G245S, p53-R248W, p53-R249S, p53-R273H, shp53 (pLKO.1 p53 shRNA-753 and -814 from Sigma-Aldrich (TRCN0000003753)), and shLuc (pLKO.1 Luciferase shRNA Control, Sigma-Aldrich) constructs were introduced into HCT116 p53 +/+ , U2OS, or H1299 cells using lipofection.

    Techniques: In Vitro, In Vivo, MTT Assay, Mouse Assay

    ER stress response in p53-deficient or knockdown cells A. HCT116 p53 +/+ or HCT116 p53 −/− cells, B. MEF p53 +/+ or MEF p53 −/− cells, and C . U2OS shLuc or U2OS shp53 cells were incubated with Tm (0.5 μg/mL) or BFA (1 μg/mL) for the times indicated. Cell lysates were analyzed using western blotting with the indicated antibodies. The blot was cut based on the size of proteins or stripped. Total RNAs were extracted and subjected to RT-PCR analysis using specific primer sets for XBP1(U) and XBP1(S). Cell lysates were analyzed using western blotting with indicated antibodies.

    Journal: Oncotarget

    Article Title: Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway

    doi:

    Figure Lengend Snippet: ER stress response in p53-deficient or knockdown cells A. HCT116 p53 +/+ or HCT116 p53 −/− cells, B. MEF p53 +/+ or MEF p53 −/− cells, and C . U2OS shLuc or U2OS shp53 cells were incubated with Tm (0.5 μg/mL) or BFA (1 μg/mL) for the times indicated. Cell lysates were analyzed using western blotting with the indicated antibodies. The blot was cut based on the size of proteins or stripped. Total RNAs were extracted and subjected to RT-PCR analysis using specific primer sets for XBP1(U) and XBP1(S). Cell lysates were analyzed using western blotting with indicated antibodies.

    Article Snippet: Wild-type p53, p53-G245S, p53-R248W, p53-R249S, p53-R273H, shp53 (pLKO.1 p53 shRNA-753 and -814 from Sigma-Aldrich (TRCN0000003753)), and shLuc (pLKO.1 Luciferase shRNA Control, Sigma-Aldrich) constructs were introduced into HCT116 p53 +/+ , U2OS, or H1299 cells using lipofection.

    Techniques: Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Inhibition of cell proliferation or cell cycle synchronization promotes iCM reprogramming Related to Fig. 1i-p . ( a ) Comparison of the ratio of CCA: CCI cells in the three treatment groups: uninfected, DsRed-infected, and M+G+T-infected. Chi-square test suggests that proliferation states were not significantly different among the treatment groups at day 3. ( b, c ) Knockdown (KD) efficiency of shRNAs ( b ) or overexpression (OE) levels ( c ) of cell cycle-related genes were determined by qRT-PCR on day 4 lentiviral transduced cells. shNT, non-targeting control shRNA. Average ± SD was shown. n = 3 samples. ( d-i ) Cell cycle staging of CF cells simultaneously transduced with reprogramming factors and shRNA ( e, g ) or OE ( f, h ) constructs by PI staining. ( e-f ) Flow cytometry histogram of PI staining intensity. ( g-i) Percentages of cells in G0/G1, S, or G2/M phases were calculated based on (e) and (f). ( i ) Summary of (g) and (h). ( j-m ) Measurement of DNA synthesis in CF cells simultaneously transduced with reprogramming factors and shRNA ( k ) or OE ( l ) constructs by EdU incorporation assay followed by flow cytometry. dMFI: delta median EdU fluorescence intensity between EdU+ cells and EdU− cells. ( m ) Summary of (k) and (l). Constructs that dramatically decreased or increased cell proliferation were labeled in red in (g-i) and (k-m) and were used for experiments in (n-s). ( n-s ) The impact of manipulation of cell proliferation through KD/OE of cell cycle-related genes on iCM reprogramming. Reprogramming factors were introduced by lentiviral vectors instead of retroviral vectors to avoid retroviral infection bias of proliferating cells. CF were simultaneously transduced with lentiviral M/G/T ( n-p ) or inducible MGT (iMGT, q-s ) and lentiviral KD/OE constructs that dramatically decreased ( o, r ) or increased ( p, s ) cell proliferation. Percentages of αMHC-GFP+ and cTnT+ cells were quantified by flow cytometry. ( t-z ) The impact of large T transduction on iCM reprogramming. CFs were simultaneously transduced with reprogramming factors and lentiviral large T. After 10 days, αActinin+/cTnT+ cells were immunostained, imaged, and quantified by counting randomly selected 20× fields from multiple repeated experiments ( u-x ). Both percentages of positive cells per field ( v, x ) and numbers of + cells per field ( w ) were quantified. Percentages of cells showing sarcomere structure in αActinin+ cells were also quantified ( y-z ). The percentage of αActinin+ cells that show sarcomere structures decreased from 50% to 0% upon large T transduction and accelerated proliferation. ( u, y ) Representative images under 40× with hoechst nuclear staining. Scale bar = 100 μm. ( o-z ) Average ± SEM was shown. ( o-s ) n = 4 samples. ( v, w, z ) n = 20 images. ( x ) n = 10 images. ( b, c, w-z ) Two-sided student’s t test. ( o-s ) One-way ANOVA followed by Bonferroni correction (two-sided). Significance: * p

    Journal: Nature

    Article Title: Single Cell Transcriptomics Reconstructs Fate Conversion from Fibroblast to Cardiomyocyte

    doi: 10.1038/nature24454

    Figure Lengend Snippet: Inhibition of cell proliferation or cell cycle synchronization promotes iCM reprogramming Related to Fig. 1i-p . ( a ) Comparison of the ratio of CCA: CCI cells in the three treatment groups: uninfected, DsRed-infected, and M+G+T-infected. Chi-square test suggests that proliferation states were not significantly different among the treatment groups at day 3. ( b, c ) Knockdown (KD) efficiency of shRNAs ( b ) or overexpression (OE) levels ( c ) of cell cycle-related genes were determined by qRT-PCR on day 4 lentiviral transduced cells. shNT, non-targeting control shRNA. Average ± SD was shown. n = 3 samples. ( d-i ) Cell cycle staging of CF cells simultaneously transduced with reprogramming factors and shRNA ( e, g ) or OE ( f, h ) constructs by PI staining. ( e-f ) Flow cytometry histogram of PI staining intensity. ( g-i) Percentages of cells in G0/G1, S, or G2/M phases were calculated based on (e) and (f). ( i ) Summary of (g) and (h). ( j-m ) Measurement of DNA synthesis in CF cells simultaneously transduced with reprogramming factors and shRNA ( k ) or OE ( l ) constructs by EdU incorporation assay followed by flow cytometry. dMFI: delta median EdU fluorescence intensity between EdU+ cells and EdU− cells. ( m ) Summary of (k) and (l). Constructs that dramatically decreased or increased cell proliferation were labeled in red in (g-i) and (k-m) and were used for experiments in (n-s). ( n-s ) The impact of manipulation of cell proliferation through KD/OE of cell cycle-related genes on iCM reprogramming. Reprogramming factors were introduced by lentiviral vectors instead of retroviral vectors to avoid retroviral infection bias of proliferating cells. CF were simultaneously transduced with lentiviral M/G/T ( n-p ) or inducible MGT (iMGT, q-s ) and lentiviral KD/OE constructs that dramatically decreased ( o, r ) or increased ( p, s ) cell proliferation. Percentages of αMHC-GFP+ and cTnT+ cells were quantified by flow cytometry. ( t-z ) The impact of large T transduction on iCM reprogramming. CFs were simultaneously transduced with reprogramming factors and lentiviral large T. After 10 days, αActinin+/cTnT+ cells were immunostained, imaged, and quantified by counting randomly selected 20× fields from multiple repeated experiments ( u-x ). Both percentages of positive cells per field ( v, x ) and numbers of + cells per field ( w ) were quantified. Percentages of cells showing sarcomere structure in αActinin+ cells were also quantified ( y-z ). The percentage of αActinin+ cells that show sarcomere structures decreased from 50% to 0% upon large T transduction and accelerated proliferation. ( u, y ) Representative images under 40× with hoechst nuclear staining. Scale bar = 100 μm. ( o-z ) Average ± SEM was shown. ( o-s ) n = 4 samples. ( v, w, z ) n = 20 images. ( x ) n = 10 images. ( b, c, w-z ) Two-sided student’s t test. ( o-s ) One-way ANOVA followed by Bonferroni correction (two-sided). Significance: * p

    Article Snippet: The non-targeting shNT pLKO.1-scramble plasmid was described previously and all other shRNAs (pLKO.1 vector based, MISSION shRNA glycerol stock) were purchased from Sigma and their TRC numbers were listed in the .

    Techniques: Inhibition, Infection, Over Expression, Quantitative RT-PCR, shRNA, Transduction, Construct, Staining, Flow Cytometry, Cytometry, DNA Synthesis, Fluorescence, Labeling

    MUC1-C drives effectors of lineage plasticity. a RNA-seq was performed in triplicate on LNCaP-AI/tet-MUC1shRNA (left) and DU-145/tet-MUC1shRNA (right) cells treated with vehicle or 500 ng/ml DOX for 7 days. The datasets were analyzed with GSEA, using the Hallmark gene signature collection for the p53 Pathway. b RNA-seq was performed in triplicate on LNCaP-AI/tet-MUC1shRNA (left) and DU-145/tet-MUC1shRNA (right) cells treated with vehicle or 500 ng/ml DOX for 7 days. The datasets were analyzed with GSEA, using the Hallmark gene signature collection for E2F Targets. c and d . LNCaP cells expressing tet-MUC1-C were treated with vehicle or 500 ng/ml DOX for 7 days. MUC1-C, BRN2 and SOX2 mRNA levels were analyzed by qRT-PCR ( c ). The results (mean±SD of three determinations) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins ( d ). e and f LNCaP-AI cells stably expressing a tet-CshRNA or tet-MUC1shRNA were treated with vehicle or 500 ng/ml DOX for 7 days. SOX2 mRNA levels were analyzed by qRT-PCR ( e ). The results (mean±SD of five determinations) are expressed as relative mRNA levels compared to that obtained for DOX-treated LNCaP-AI/tet-CshRNA cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies ( f ). g Lysates from DU-145/tet-CshRNA and DU-145/tet-MUC1shRNA cells treated with vehicle or 500 ng/ml DOX for 7 days were immunoblotted with antibodies against the indicated proteins. h Lysates from DU-145/CshRNA and DU-145/BRN2shRNA cells were immunoblotted with antibodies against the indicated proteins. * p

    Journal: Nature Communications

    Article Title: MUC1-C regulates lineage plasticity driving progression to neuroendocrine prostate cancer

    doi: 10.1038/s41467-019-14219-6

    Figure Lengend Snippet: MUC1-C drives effectors of lineage plasticity. a RNA-seq was performed in triplicate on LNCaP-AI/tet-MUC1shRNA (left) and DU-145/tet-MUC1shRNA (right) cells treated with vehicle or 500 ng/ml DOX for 7 days. The datasets were analyzed with GSEA, using the Hallmark gene signature collection for the p53 Pathway. b RNA-seq was performed in triplicate on LNCaP-AI/tet-MUC1shRNA (left) and DU-145/tet-MUC1shRNA (right) cells treated with vehicle or 500 ng/ml DOX for 7 days. The datasets were analyzed with GSEA, using the Hallmark gene signature collection for E2F Targets. c and d . LNCaP cells expressing tet-MUC1-C were treated with vehicle or 500 ng/ml DOX for 7 days. MUC1-C, BRN2 and SOX2 mRNA levels were analyzed by qRT-PCR ( c ). The results (mean±SD of three determinations) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins ( d ). e and f LNCaP-AI cells stably expressing a tet-CshRNA or tet-MUC1shRNA were treated with vehicle or 500 ng/ml DOX for 7 days. SOX2 mRNA levels were analyzed by qRT-PCR ( e ). The results (mean±SD of five determinations) are expressed as relative mRNA levels compared to that obtained for DOX-treated LNCaP-AI/tet-CshRNA cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies ( f ). g Lysates from DU-145/tet-CshRNA and DU-145/tet-MUC1shRNA cells treated with vehicle or 500 ng/ml DOX for 7 days were immunoblotted with antibodies against the indicated proteins. h Lysates from DU-145/CshRNA and DU-145/BRN2shRNA cells were immunoblotted with antibodies against the indicated proteins. * p

    Article Snippet: BRN2shRNA (MISSION shRNA TRCN0000019330; Sigma) was inserted into the pLKO-puro vector.

    Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Stable Transfection

    Bioinformatic analysis of expression of deadenylases in Squamous Cell Lung Carcinoma (SCC). Microarray data retrieved from the Oncomine database. PARN ( p = 0.033) and NOC ( p = 0.048) are significantly overexpressed, while CNOT6L ( p = 0.0012), CNOT8 ( p

    Journal: Molecular Cancer

    Article Title: Poly(A)-specific ribonuclease and Nocturnin in squamous cell lung cancer: prognostic value and impact on gene expression

    doi: 10.1186/s12943-015-0457-3

    Figure Lengend Snippet: Bioinformatic analysis of expression of deadenylases in Squamous Cell Lung Carcinoma (SCC). Microarray data retrieved from the Oncomine database. PARN ( p = 0.033) and NOC ( p = 0.048) are significantly overexpressed, while CNOT6L ( p = 0.0012), CNOT8 ( p

    Article Snippet: The following shDNA plasmids were used to silence the respective deadenylases (MISSION® shRNA, SIGMA): PARN (NM_002582), NOCTURNIN (NM_012118), and non-targeting control (SHC016).

    Techniques: Expressing, Microarray

    Promoter CpG islands ( a ) and VEGF , FLT1 , and KDR pyrosequencing in 2 RCC lines ( b ). Closed bars , exon 1 region for each gene; arrows , translation start site; open bars , the region targeted for pyrosequencing; black segments within open bars , locations of sequencing primers for each gene

    Journal: Clinical Epigenetics

    Article Title: Decreased efficacy of drugs targeting the vascular endothelial growth factor pathway by the epigenetic silencing of FLT1 in renal cancer cells

    doi: 10.1186/s13148-015-0134-9

    Figure Lengend Snippet: Promoter CpG islands ( a ) and VEGF , FLT1 , and KDR pyrosequencing in 2 RCC lines ( b ). Closed bars , exon 1 region for each gene; arrows , translation start site; open bars , the region targeted for pyrosequencing; black segments within open bars , locations of sequencing primers for each gene

    Article Snippet: Effects of FLT1 or KDR knockdown on proliferation SNU482 cells, having low methylation of both FLT1 and KDR (Table ), were used for in vitro knockdown assays following lentiviral vector-mediated transduction of shRNAs for FLT1 (FLT1 MISSION shRNA Lentiviral Transduction Particles; TRCN0000194670; Sigma-Aldrich) or KDR (KDR MISSION shRNA Lentiviral Transduction Particles; TRCN0000199129; Sigma-Aldrich).

    Techniques: Sequencing

    FLT1 and KDR expression and the proliferation-inhibitory effects of sunitinib or axitinib after demethylation treatment. Promoter methylation and gene expression were evaluated after demethylation treatment in RCC lines. Changes in expression (RQ) and methylation (%) in FLT1 ( a ) and KDR ( b ) after demethylation using DAC, a demethylating agent. Proliferation of RCC lines following treatment with sunitinib or axitinib and/or DAC( c ). The error bars show standard errors

    Journal: Clinical Epigenetics

    Article Title: Decreased efficacy of drugs targeting the vascular endothelial growth factor pathway by the epigenetic silencing of FLT1 in renal cancer cells

    doi: 10.1186/s13148-015-0134-9

    Figure Lengend Snippet: FLT1 and KDR expression and the proliferation-inhibitory effects of sunitinib or axitinib after demethylation treatment. Promoter methylation and gene expression were evaluated after demethylation treatment in RCC lines. Changes in expression (RQ) and methylation (%) in FLT1 ( a ) and KDR ( b ) after demethylation using DAC, a demethylating agent. Proliferation of RCC lines following treatment with sunitinib or axitinib and/or DAC( c ). The error bars show standard errors

    Article Snippet: Effects of FLT1 or KDR knockdown on proliferation SNU482 cells, having low methylation of both FLT1 and KDR (Table ), were used for in vitro knockdown assays following lentiviral vector-mediated transduction of shRNAs for FLT1 (FLT1 MISSION shRNA Lentiviral Transduction Particles; TRCN0000194670; Sigma-Aldrich) or KDR (KDR MISSION shRNA Lentiviral Transduction Particles; TRCN0000199129; Sigma-Aldrich).

    Techniques: Expressing, Methylation

    Expression changes and anti-VEGF/VEGFR drug efficacies following knockdown of FLT1 or KDR in vitro. Expression of FLT1 ( a ) and KDR ( b ) in SNU482 renal cancer cells after transduction with an shRNA targeting FLT1 or KDR . The effects of bevacizumab, an anti-FLT1 peptide, an anti-KDR antibody, sunitinib, and axitinib on proliferation were evaluated in SNU482 cells transduced with lentiviral vectors expressing FLT1 or KDR or an empty lentiviral vector ( c ). The error bars show the standard errors

    Journal: Clinical Epigenetics

    Article Title: Decreased efficacy of drugs targeting the vascular endothelial growth factor pathway by the epigenetic silencing of FLT1 in renal cancer cells

    doi: 10.1186/s13148-015-0134-9

    Figure Lengend Snippet: Expression changes and anti-VEGF/VEGFR drug efficacies following knockdown of FLT1 or KDR in vitro. Expression of FLT1 ( a ) and KDR ( b ) in SNU482 renal cancer cells after transduction with an shRNA targeting FLT1 or KDR . The effects of bevacizumab, an anti-FLT1 peptide, an anti-KDR antibody, sunitinib, and axitinib on proliferation were evaluated in SNU482 cells transduced with lentiviral vectors expressing FLT1 or KDR or an empty lentiviral vector ( c ). The error bars show the standard errors

    Article Snippet: Effects of FLT1 or KDR knockdown on proliferation SNU482 cells, having low methylation of both FLT1 and KDR (Table ), were used for in vitro knockdown assays following lentiviral vector-mediated transduction of shRNAs for FLT1 (FLT1 MISSION shRNA Lentiviral Transduction Particles; TRCN0000194670; Sigma-Aldrich) or KDR (KDR MISSION shRNA Lentiviral Transduction Particles; TRCN0000199129; Sigma-Aldrich).

    Techniques: Expressing, In Vitro, Transduction, shRNA, Plasmid Preparation

    VEGF , FLT1 , and KDR methylation in responder and nonresponder patients with renal cancer receiving sunitinib. An analysis of VEGF , FLT1 , and KDR methylation statuses is shown for responders and nonresponders. NR nonresponder, R responder, * p

    Journal: Clinical Epigenetics

    Article Title: Decreased efficacy of drugs targeting the vascular endothelial growth factor pathway by the epigenetic silencing of FLT1 in renal cancer cells

    doi: 10.1186/s13148-015-0134-9

    Figure Lengend Snippet: VEGF , FLT1 , and KDR methylation in responder and nonresponder patients with renal cancer receiving sunitinib. An analysis of VEGF , FLT1 , and KDR methylation statuses is shown for responders and nonresponders. NR nonresponder, R responder, * p

    Article Snippet: Effects of FLT1 or KDR knockdown on proliferation SNU482 cells, having low methylation of both FLT1 and KDR (Table ), were used for in vitro knockdown assays following lentiviral vector-mediated transduction of shRNAs for FLT1 (FLT1 MISSION shRNA Lentiviral Transduction Particles; TRCN0000194670; Sigma-Aldrich) or KDR (KDR MISSION shRNA Lentiviral Transduction Particles; TRCN0000199129; Sigma-Aldrich).

    Techniques: Methylation

    Expression changes and anti-VEGF/VEGFR drug efficacies associated with FLT1 and KDR methylation changes. Analysis of gene expression of FLT1 ( a ) and KDR ( b ) in 13 RCC lines. Evaluation of the effects of bevacizumab, an anti-FLT1 peptide, an anti-KDR antibody, sunitinib, and axitinib on RCC line proliferation was classified according to the hypermethylation status of FLT1 and/or KDR ( c ). H460 cells and SNU1 cells were used as control cell lines that lacked or high methylation of either gene, respectively . The error bars show standard errors

    Journal: Clinical Epigenetics

    Article Title: Decreased efficacy of drugs targeting the vascular endothelial growth factor pathway by the epigenetic silencing of FLT1 in renal cancer cells

    doi: 10.1186/s13148-015-0134-9

    Figure Lengend Snippet: Expression changes and anti-VEGF/VEGFR drug efficacies associated with FLT1 and KDR methylation changes. Analysis of gene expression of FLT1 ( a ) and KDR ( b ) in 13 RCC lines. Evaluation of the effects of bevacizumab, an anti-FLT1 peptide, an anti-KDR antibody, sunitinib, and axitinib on RCC line proliferation was classified according to the hypermethylation status of FLT1 and/or KDR ( c ). H460 cells and SNU1 cells were used as control cell lines that lacked or high methylation of either gene, respectively . The error bars show standard errors

    Article Snippet: Effects of FLT1 or KDR knockdown on proliferation SNU482 cells, having low methylation of both FLT1 and KDR (Table ), were used for in vitro knockdown assays following lentiviral vector-mediated transduction of shRNAs for FLT1 (FLT1 MISSION shRNA Lentiviral Transduction Particles; TRCN0000194670; Sigma-Aldrich) or KDR (KDR MISSION shRNA Lentiviral Transduction Particles; TRCN0000199129; Sigma-Aldrich).

    Techniques: Expressing, Methylation

    Expression differences associated with promoter methylation changes of the VEGF , FLT1 , and KDR genes in TCGA. The query process was performed using the cBioPortal online tools ( www.cbioportal.org )

    Journal: Clinical Epigenetics

    Article Title: Decreased efficacy of drugs targeting the vascular endothelial growth factor pathway by the epigenetic silencing of FLT1 in renal cancer cells

    doi: 10.1186/s13148-015-0134-9

    Figure Lengend Snippet: Expression differences associated with promoter methylation changes of the VEGF , FLT1 , and KDR genes in TCGA. The query process was performed using the cBioPortal online tools ( www.cbioportal.org )

    Article Snippet: Effects of FLT1 or KDR knockdown on proliferation SNU482 cells, having low methylation of both FLT1 and KDR (Table ), were used for in vitro knockdown assays following lentiviral vector-mediated transduction of shRNAs for FLT1 (FLT1 MISSION shRNA Lentiviral Transduction Particles; TRCN0000194670; Sigma-Aldrich) or KDR (KDR MISSION shRNA Lentiviral Transduction Particles; TRCN0000199129; Sigma-Aldrich).

    Techniques: Expressing, Methylation

    VEGF , FLT1 , and KDR promoter methylation and expression differences between normal and cancer tissues. Normal , normal tissues; cancer , cancer tissues collected from eight renal cancer patients

    Journal: Clinical Epigenetics

    Article Title: Decreased efficacy of drugs targeting the vascular endothelial growth factor pathway by the epigenetic silencing of FLT1 in renal cancer cells

    doi: 10.1186/s13148-015-0134-9

    Figure Lengend Snippet: VEGF , FLT1 , and KDR promoter methylation and expression differences between normal and cancer tissues. Normal , normal tissues; cancer , cancer tissues collected from eight renal cancer patients

    Article Snippet: Effects of FLT1 or KDR knockdown on proliferation SNU482 cells, having low methylation of both FLT1 and KDR (Table ), were used for in vitro knockdown assays following lentiviral vector-mediated transduction of shRNAs for FLT1 (FLT1 MISSION shRNA Lentiviral Transduction Particles; TRCN0000194670; Sigma-Aldrich) or KDR (KDR MISSION shRNA Lentiviral Transduction Particles; TRCN0000199129; Sigma-Aldrich).

    Techniques: Methylation, Expressing

    MUC1-C suppresses CRB3 expression by a ZEB1-dependent mechanism A, BT-549 cells infected with lentiviruses to stably express a control scrambled shRNA (CshRNA) or a ZEB1shRNA were analyzed for CRB3 mRNA levels by qRT-PCR (left). The results are expressed as relative CRB3 mRNA levels (mean±SD of three determinations) as compared with that obtained for BT-549/CshRNA cells (assigned a value of 1). Lysates from the BT-549/CshRNA and BT-549/ZEB1shRNA cells were immunoblotted with the indicated antibodies (right). B, MDA-MB-231 cells stably expressing a control scrambled shRNA (CshRNA) or a ZEB1 shRNA were analyzed for CRB3 mRNA levels by qRT-PCR (left). The results are expressed as relative CRB3 mRNA levels (mean±SD of three determinations) as compared with that obtained for MDA-MB-231/CshRNA cells (assigned a value of 1). Lysates from the MDA-MB-231/CshRNA and MDA-MB-231/ZEB1shRNA cells were immunoblotted with the indicated antibodies (right). C, MCF-7/MUC1-C cells stably expressing a control scrambled shRNA (CshRNA) or a ZEB1 shRNA were analyzed for CRB3 mRNA levels by qRT-PCR (left). Lysates from MCF-7/MUC1-C/CshRNA and MCF-7/MUC1-C/ZEB1shRNA cells were immunoblotted with the indicated antibodies (right). D, Lysates from MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA (left) and from MDA-MB-231/CshRNA and MDA-MB-231/SNAIL1shRNA (right) cells were immunoblotted with the indicated antibodies. E, Lysates from MCF-7/vector and MCF-7/MUC1-C cells (left) were immunoblotted with the indicated antibodies. Lysates from MCF-7/MUC1-C cells infected with lentiviruses to stably express a control scrambled shRNA (CshRNA) or a SNAIL1shRNA were immunoblotted with the indicated antibodies (right).

    Journal: Molecular cancer research : MCR

    Article Title: MUC1-C Represses the Crumbs Complex Polarity Factor CRB3 and Downregulates the Hippo Pathway

    doi: 10.1158/1541-7786.MCR-16-0233

    Figure Lengend Snippet: MUC1-C suppresses CRB3 expression by a ZEB1-dependent mechanism A, BT-549 cells infected with lentiviruses to stably express a control scrambled shRNA (CshRNA) or a ZEB1shRNA were analyzed for CRB3 mRNA levels by qRT-PCR (left). The results are expressed as relative CRB3 mRNA levels (mean±SD of three determinations) as compared with that obtained for BT-549/CshRNA cells (assigned a value of 1). Lysates from the BT-549/CshRNA and BT-549/ZEB1shRNA cells were immunoblotted with the indicated antibodies (right). B, MDA-MB-231 cells stably expressing a control scrambled shRNA (CshRNA) or a ZEB1 shRNA were analyzed for CRB3 mRNA levels by qRT-PCR (left). The results are expressed as relative CRB3 mRNA levels (mean±SD of three determinations) as compared with that obtained for MDA-MB-231/CshRNA cells (assigned a value of 1). Lysates from the MDA-MB-231/CshRNA and MDA-MB-231/ZEB1shRNA cells were immunoblotted with the indicated antibodies (right). C, MCF-7/MUC1-C cells stably expressing a control scrambled shRNA (CshRNA) or a ZEB1 shRNA were analyzed for CRB3 mRNA levels by qRT-PCR (left). Lysates from MCF-7/MUC1-C/CshRNA and MCF-7/MUC1-C/ZEB1shRNA cells were immunoblotted with the indicated antibodies (right). D, Lysates from MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA (left) and from MDA-MB-231/CshRNA and MDA-MB-231/SNAIL1shRNA (right) cells were immunoblotted with the indicated antibodies. E, Lysates from MCF-7/vector and MCF-7/MUC1-C cells (left) were immunoblotted with the indicated antibodies. Lysates from MCF-7/MUC1-C cells infected with lentiviruses to stably express a control scrambled shRNA (CshRNA) or a SNAIL1shRNA were immunoblotted with the indicated antibodies (right).

    Article Snippet: MUC1shRNA (MISSION shRNA TRCN0000122938; Sigma) or a control scrambled CshRNA (Sigma) was inserted into the pLKO-tet-puro vector (Addgene, Plasmid #21915).

    Techniques: Expressing, Infection, Stable Transfection, shRNA, Quantitative RT-PCR, Multiple Displacement Amplification, Plasmid Preparation

    MUC1-C represses CRB3 promoter activation by a ZEB1-dependent mechanism A, Schema of CRB3 luciferase reporter plasmid. B, MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells (left), BT-549/CshRNA and BT-549/MUC1shRNA cells (middle) and MCF-7/Vector and MCF-7/MUC1-C cells (right) were transfected with the empty Luc vector or pCRB3-Luc. Cells were also transfected with the SV-40-Renilla-Luc plasmid as an internal control. Luciferase activity was measured at 48 h following transfection. The results are expressed as relative luciferase activity (mean±SD of three determinations) compared with that obtained from cells transfected with the empty Luc vector (assigned a value of 1). C (left), Soluble chromatin from the indicated MDA-MB-231 cells was precipitated with anti-ZEB1 or a control IgG. The final DNA precipitates were amplified by qPCR with pairs of primers for the ZEB1 binding region in CRB3 promoter region. Results are expressed as the relative fold enrichment (mean±SD of three determinations) compared with that obtained for the IgG control (assigned a value of 1). For re-ChIP analysis, soluble chromatin from MDA-MB-231 cells (right) was first precipitated with anti-ZEB1, then released and reimmunoprecipitated with anti-MUC1-C. The results are expressed as the relative fold enrichment (mean±SD of three determinations) compared with that obtained with the IgG control (assigned a value of 1). D, MDA-MB-231 cells were treated with 5 μM GO-203 or CP-2 each day for 3 days. Soluble chromatin was precipitated with anti-ZEB1 or a control IgG (left). For re-ChIP analysis, complexes were released and re-immunoprecipitated with anti-MUC1-C (right). Results are expressed as the relative fold enrichment (mean±SD of three determinations) compared with that obtained for the IgG control (assigned a value of 1). E, Soluble chromatin from MCF-7/vector and MCF-7/MUC1-C cells was precipitated with anti-ZEB1 or a control IgG (left). For re-ChIP analysis, complexes were released and re-immunoprecipitated with anti-MUC1-C (right). Results are expressed as the relative fold enrichment (mean±SD of three determinations) compared with that obtained for the IgG control (assigned a value of 1).

    Journal: Molecular cancer research : MCR

    Article Title: MUC1-C Represses the Crumbs Complex Polarity Factor CRB3 and Downregulates the Hippo Pathway

    doi: 10.1158/1541-7786.MCR-16-0233

    Figure Lengend Snippet: MUC1-C represses CRB3 promoter activation by a ZEB1-dependent mechanism A, Schema of CRB3 luciferase reporter plasmid. B, MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells (left), BT-549/CshRNA and BT-549/MUC1shRNA cells (middle) and MCF-7/Vector and MCF-7/MUC1-C cells (right) were transfected with the empty Luc vector or pCRB3-Luc. Cells were also transfected with the SV-40-Renilla-Luc plasmid as an internal control. Luciferase activity was measured at 48 h following transfection. The results are expressed as relative luciferase activity (mean±SD of three determinations) compared with that obtained from cells transfected with the empty Luc vector (assigned a value of 1). C (left), Soluble chromatin from the indicated MDA-MB-231 cells was precipitated with anti-ZEB1 or a control IgG. The final DNA precipitates were amplified by qPCR with pairs of primers for the ZEB1 binding region in CRB3 promoter region. Results are expressed as the relative fold enrichment (mean±SD of three determinations) compared with that obtained for the IgG control (assigned a value of 1). For re-ChIP analysis, soluble chromatin from MDA-MB-231 cells (right) was first precipitated with anti-ZEB1, then released and reimmunoprecipitated with anti-MUC1-C. The results are expressed as the relative fold enrichment (mean±SD of three determinations) compared with that obtained with the IgG control (assigned a value of 1). D, MDA-MB-231 cells were treated with 5 μM GO-203 or CP-2 each day for 3 days. Soluble chromatin was precipitated with anti-ZEB1 or a control IgG (left). For re-ChIP analysis, complexes were released and re-immunoprecipitated with anti-MUC1-C (right). Results are expressed as the relative fold enrichment (mean±SD of three determinations) compared with that obtained for the IgG control (assigned a value of 1). E, Soluble chromatin from MCF-7/vector and MCF-7/MUC1-C cells was precipitated with anti-ZEB1 or a control IgG (left). For re-ChIP analysis, complexes were released and re-immunoprecipitated with anti-MUC1-C (right). Results are expressed as the relative fold enrichment (mean±SD of three determinations) compared with that obtained for the IgG control (assigned a value of 1).

    Article Snippet: MUC1shRNA (MISSION shRNA TRCN0000122938; Sigma) or a control scrambled CshRNA (Sigma) was inserted into the pLKO-tet-puro vector (Addgene, Plasmid #21915).

    Techniques: Activation Assay, Luciferase, Plasmid Preparation, Multiple Displacement Amplification, Transfection, Activity Assay, Amplification, Real-time Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation

    MUC1-C-mediated CRB3 repression increases nuclear accumulation of YAP A, Lysates from MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells (left), BT-549/CshRNA and bt-549/MUC1shRNA (middle) and MCF-7/Vector and MCF-7/MUC1-C (right) were immunoblotted with the indicated antibodies. B, Cytosolic fraction from MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells (left), BT-549/CshRNA and BT-549/MUC1shRNA (middle) and MCF-7/Vector and MCF-7/MUC1-C (right) were immunoblotted with the indicated antibodies. C, Nuclear fraction lysates from MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells (left), BT-549/CshRNA and BT-549/MUC1shRNA (middle) and MCF-7/Vector and MCF-7/MUC1-C (right) were immunoblotted with the indicated antibodies. D, Nuclear fraction lysates from MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells (left), BT-549/CshRNA and BT-549/MUC1shRNA (middle) and MCF-7/Vector and MCF-7/MUC1-C (right) were immunoblotted with the indicated antibodies.

    Journal: Molecular cancer research : MCR

    Article Title: MUC1-C Represses the Crumbs Complex Polarity Factor CRB3 and Downregulates the Hippo Pathway

    doi: 10.1158/1541-7786.MCR-16-0233

    Figure Lengend Snippet: MUC1-C-mediated CRB3 repression increases nuclear accumulation of YAP A, Lysates from MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells (left), BT-549/CshRNA and bt-549/MUC1shRNA (middle) and MCF-7/Vector and MCF-7/MUC1-C (right) were immunoblotted with the indicated antibodies. B, Cytosolic fraction from MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells (left), BT-549/CshRNA and BT-549/MUC1shRNA (middle) and MCF-7/Vector and MCF-7/MUC1-C (right) were immunoblotted with the indicated antibodies. C, Nuclear fraction lysates from MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells (left), BT-549/CshRNA and BT-549/MUC1shRNA (middle) and MCF-7/Vector and MCF-7/MUC1-C (right) were immunoblotted with the indicated antibodies. D, Nuclear fraction lysates from MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells (left), BT-549/CshRNA and BT-549/MUC1shRNA (middle) and MCF-7/Vector and MCF-7/MUC1-C (right) were immunoblotted with the indicated antibodies.

    Article Snippet: MUC1shRNA (MISSION shRNA TRCN0000122938; Sigma) or a control scrambled CshRNA (Sigma) was inserted into the pLKO-tet-puro vector (Addgene, Plasmid #21915).

    Techniques: Multiple Displacement Amplification, Plasmid Preparation

    MUC1-C associates with YAP and β-catenin on the MYC promoter A, The indicated MDA-MB-231 cells were analyzed for CTGF and CYR61 mRNA levels by qRT-PCR. The results are expressed as relative mRNA levels (mean±SD of three determinations) as compared with that obtained for MDA-MB-231/CshRNA cells (assigned a value of 1). B, Soluble chromatin from the indicated MDA-MB-231 cells was precipitated with anti-β-catenin or a control IgG (left). The final DNA precipitates were amplified by qPCR with pairs of primers for the β-catenin binding region in the MYC promoter region. Results are expressed as the relative fold enrichment (mean±SD of three determinations) compared with that obtained for the IgG control (assigned a value of 1). For separate re-ChIP analysis, soluble chromatin from MDA-MB-231 cells was first precipitated with anti-β-catenin, then released and reimmunoprecipitated with anti-MUC1-C (middle) or anti-YAP (right). The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). C, MDA-MB-231 cells were treated with 5 μM GO-203 or CP-2 each day for 3 days. Soluble chromatin was precipitated with anti-β-catenin or a control IgG (left). For separate re-ChIP analysis, soluble chromatin from treated MDA-MB-231 cells was first precipitated with anti-β-catenin, then released and reimmunoprecipitated with anti-MUC1-C (middle) or anti-YAP (right). The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). D, MDA-MB-231 cells treated with 5 μM GO-203 or CP-2 each day for 3 days were analyzed for MYC mRNA by qRT-PCR (left). The results are expressed as relative MYC mRNA levels (mean±SD of three determinations) as compared with that obtained for cells treated with CP2 peptide (assigned a value of 1). Lysates from MDA-MB-231 cells treated with 5 μM GO-203 or CP-2 were immunoblotted with the indicated antibodies (right). E, MDA-MB-231 cells stably expressing a control scrambled shRNA (CshRNA) or a MUC1 shRNA were analyzed for MYC mRNA levels by qRT-PCR (left). The results are expressed as relative MYC mRNA levels (mean±SD of three determinations) as compared with that obtained for MDA-MB-231/CshRNA cells (assigned a value of 1). Lysates from the MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells were immunoblotted with the indicated antibodies (right). F, MDA-MB-231 cells were transiently transfected with a YAPsiRNA or a control siRNA (CsiRNA). Lysates were immunoblotted with the indicated antibodies.

    Journal: Molecular cancer research : MCR

    Article Title: MUC1-C Represses the Crumbs Complex Polarity Factor CRB3 and Downregulates the Hippo Pathway

    doi: 10.1158/1541-7786.MCR-16-0233

    Figure Lengend Snippet: MUC1-C associates with YAP and β-catenin on the MYC promoter A, The indicated MDA-MB-231 cells were analyzed for CTGF and CYR61 mRNA levels by qRT-PCR. The results are expressed as relative mRNA levels (mean±SD of three determinations) as compared with that obtained for MDA-MB-231/CshRNA cells (assigned a value of 1). B, Soluble chromatin from the indicated MDA-MB-231 cells was precipitated with anti-β-catenin or a control IgG (left). The final DNA precipitates were amplified by qPCR with pairs of primers for the β-catenin binding region in the MYC promoter region. Results are expressed as the relative fold enrichment (mean±SD of three determinations) compared with that obtained for the IgG control (assigned a value of 1). For separate re-ChIP analysis, soluble chromatin from MDA-MB-231 cells was first precipitated with anti-β-catenin, then released and reimmunoprecipitated with anti-MUC1-C (middle) or anti-YAP (right). The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). C, MDA-MB-231 cells were treated with 5 μM GO-203 or CP-2 each day for 3 days. Soluble chromatin was precipitated with anti-β-catenin or a control IgG (left). For separate re-ChIP analysis, soluble chromatin from treated MDA-MB-231 cells was first precipitated with anti-β-catenin, then released and reimmunoprecipitated with anti-MUC1-C (middle) or anti-YAP (right). The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). D, MDA-MB-231 cells treated with 5 μM GO-203 or CP-2 each day for 3 days were analyzed for MYC mRNA by qRT-PCR (left). The results are expressed as relative MYC mRNA levels (mean±SD of three determinations) as compared with that obtained for cells treated with CP2 peptide (assigned a value of 1). Lysates from MDA-MB-231 cells treated with 5 μM GO-203 or CP-2 were immunoblotted with the indicated antibodies (right). E, MDA-MB-231 cells stably expressing a control scrambled shRNA (CshRNA) or a MUC1 shRNA were analyzed for MYC mRNA levels by qRT-PCR (left). The results are expressed as relative MYC mRNA levels (mean±SD of three determinations) as compared with that obtained for MDA-MB-231/CshRNA cells (assigned a value of 1). Lysates from the MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells were immunoblotted with the indicated antibodies (right). F, MDA-MB-231 cells were transiently transfected with a YAPsiRNA or a control siRNA (CsiRNA). Lysates were immunoblotted with the indicated antibodies.

    Article Snippet: MUC1shRNA (MISSION shRNA TRCN0000122938; Sigma) or a control scrambled CshRNA (Sigma) was inserted into the pLKO-tet-puro vector (Addgene, Plasmid #21915).

    Techniques: Multiple Displacement Amplification, Quantitative RT-PCR, Amplification, Real-time Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Stable Transfection, Expressing, shRNA, Transfection

    MUC1-C downregulates CRB3 expression A, BT-549 cells infected with lentiviruses to stably express a control scrambled shRNA (CshRNA) or a MUC1shRNA were analyzed for MUC1, CRB3, HUGL2, PATJ and CDC42 mRNA levels by qRT-PCR (left). The results are expressed as relative mRNA levels (mean±SD of three determinations) as compared with that obtained for BT-549/CshRNA cells (assigned a value of 1). Lysates from the BT-549/CshRNA and BT-549/MUC1shRNA cells were immunoblotted with the indicated antibodies (right). B, MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells were analyzed for MUC1, CRB3, HUGL2, PATJ and CDC42 mRNA levels by qRT-PCR (left). The results are expressed as relative mRNA levels (mean±SD of three determinations) as compared with that obtained for MDA-MB-231/CshRNA cells (assigned a value of 1). Lysates from the MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells were immunoblotted with the indicated antibodies (right). C, BT-20 cells stably expressing an empty vector or MUC1-C were analyzed for CRB3 mRNA levels by qRT-PCR (left). The results are expressed as relative CRB3 mRNA levels (mean±SD of three determinations) as compared to that obtained for BT-20/vector cells (assigned a value of 1). Lysates from the BT-20/vector and BT-20/MUC1-C cells were immunoblotted with the indicated antibodies (right). D, Schematic showing the MUC1-C subunit and the amino acid (aa) sequence of the cytoplasmic domain (CD). ED, extracellular domain; TM, transmembrane domain. The CQC motif is necessary and sufficient for MUC1-C homodimerization. D-amino acid sequences are shown for GO-203 and CP-2. E, BT-549 cells were treated with 5 μM GO-203 or CP-2 each day for 3 days and then analyzed for CRB3 mRNA levels by qRT-PCR (left). Lysates from BT-549 cells treated as above were immunoblotted with the indicated antibodies (right). F, MDA-MB-231 cells were treated with 5 μM GO-203 or CP-2 each day for 3 days and then analyzed for CRB3 mRNA levels by qRT-PCR (left), while lysates from similarly treated cells were immunoblotted with the indicated antibodies (right).

    Journal: Molecular cancer research : MCR

    Article Title: MUC1-C Represses the Crumbs Complex Polarity Factor CRB3 and Downregulates the Hippo Pathway

    doi: 10.1158/1541-7786.MCR-16-0233

    Figure Lengend Snippet: MUC1-C downregulates CRB3 expression A, BT-549 cells infected with lentiviruses to stably express a control scrambled shRNA (CshRNA) or a MUC1shRNA were analyzed for MUC1, CRB3, HUGL2, PATJ and CDC42 mRNA levels by qRT-PCR (left). The results are expressed as relative mRNA levels (mean±SD of three determinations) as compared with that obtained for BT-549/CshRNA cells (assigned a value of 1). Lysates from the BT-549/CshRNA and BT-549/MUC1shRNA cells were immunoblotted with the indicated antibodies (right). B, MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells were analyzed for MUC1, CRB3, HUGL2, PATJ and CDC42 mRNA levels by qRT-PCR (left). The results are expressed as relative mRNA levels (mean±SD of three determinations) as compared with that obtained for MDA-MB-231/CshRNA cells (assigned a value of 1). Lysates from the MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells were immunoblotted with the indicated antibodies (right). C, BT-20 cells stably expressing an empty vector or MUC1-C were analyzed for CRB3 mRNA levels by qRT-PCR (left). The results are expressed as relative CRB3 mRNA levels (mean±SD of three determinations) as compared to that obtained for BT-20/vector cells (assigned a value of 1). Lysates from the BT-20/vector and BT-20/MUC1-C cells were immunoblotted with the indicated antibodies (right). D, Schematic showing the MUC1-C subunit and the amino acid (aa) sequence of the cytoplasmic domain (CD). ED, extracellular domain; TM, transmembrane domain. The CQC motif is necessary and sufficient for MUC1-C homodimerization. D-amino acid sequences are shown for GO-203 and CP-2. E, BT-549 cells were treated with 5 μM GO-203 or CP-2 each day for 3 days and then analyzed for CRB3 mRNA levels by qRT-PCR (left). Lysates from BT-549 cells treated as above were immunoblotted with the indicated antibodies (right). F, MDA-MB-231 cells were treated with 5 μM GO-203 or CP-2 each day for 3 days and then analyzed for CRB3 mRNA levels by qRT-PCR (left), while lysates from similarly treated cells were immunoblotted with the indicated antibodies (right).

    Article Snippet: MUC1shRNA (MISSION shRNA TRCN0000122938; Sigma) or a control scrambled CshRNA (Sigma) was inserted into the pLKO-tet-puro vector (Addgene, Plasmid #21915).

    Techniques: Expressing, Infection, Stable Transfection, shRNA, Quantitative RT-PCR, Multiple Displacement Amplification, Plasmid Preparation, Sequencing

    HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular cytokine staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.

    Journal: EBioMedicine

    Article Title: What Is the most Important for Elite Control: Genetic Background of Patient, Genetic Background of Partner, both or neither? Description of Complete Natural History within a Couple of MSM

    doi: 10.1016/j.ebiom.2017.12.003

    Figure Lengend Snippet: HIV infection and T-cell responses of PBMC from the case patient. (a) In vitro susceptibility to HIV infection in 2009. 9 CD4 + T-cells of the case patient or healthy donors (HD) were PHA-activated and infected in vitro with 100 ng p24 Gag of HIV YU2b (R5-tropic), HIV NL4–3 (× 4-tropic), and HIV KON a primary isolate of the same clade than CRF02_AG isolate. Viral replication was monitored by p24 Gag ELISA during 16 days post-infection (p.i.). (ni = non infected). (b) Ex vivo IFN-γ-ELISpot assay was performed in November 2009 using PBMC and lymph node (LN) cells loaded with 18 HIV-1 pools of 15-mers peptides overlapping by 11 amino acids. Eleven pools covered the three HIV type 1 Gag proteins: three pools for p17 Gag (1–55, 45–99, and 89–143), five pools for p24 Gag (133–187, 177–231, 221–275, 265–319, and 309–363), three pools for the small Gag proteins (Smp Gag) (p2/p7/p1/p6) (353–407, 397–451, and 441–512), four pools corresponding to poly-epitopic RT regions (293–352, 157–187, 177–216, and 4–52), and three pools corresponding to poly-epitopic Nef regions (181–206, 65–107, and 97–147). All results are expressed as specific IFN-γ-producing cells after subtracting the number of SFCs observed with cells alone, without stimulation. The IFN-γ producing HIV-specific CD8 + T cells were first investigated in an ELISpot assay detecting in 2009 significant responses to p17Gag (1–55) in PBMC and lymph node cells (110 SFC/10 6 cells) and to p24Gag (265–319) in PBMC alone (680 SFC/10 6 cells. (c) PBMC obtained in November 2009 were stimulated with the p17Gag 1 to 55 pool or with the p24 Gag 265 to 319 pool that elicited an IFN-γ-ELISpot production. The percentages of CD3 + CD8 + T cells producing IFN-γ, TNF-α or IL-2 were analyzed by intracellular cytokine staining and flow cytometry. The percentages of activated cells not subjected to peptide stimulation were subtracted. The immune-dominant CMV-derived HLA-B*07-restricted epitope (pp65 417 TPRVTGGGAM 426 ), and staphylococcal enterotoxin B (SEB) were used as positive controls. Cells alone served as negative control.

    Article Snippet: Poly-functional NK was performed to simultaneously detect degranulation (anti-CD107a mAb, BD Biosciences) and cytokine production (intracellular expression IFN-γ (BD Biosciences) and TNF-α (E-Biosciences) ( ).

    Techniques: Infection, In Vitro, Enzyme-linked Immunosorbent Assay, Ex Vivo, Enzyme-linked Immunospot, Staining, Flow Cytometry, Cytometry, Derivative Assay, Negative Control

    Poly-functional analysis of NK cells. (4) Poly-functional analysis of NK cells was performed to simultaneously detect degranulation (anti-CD107a mAb, BD Biosciences) and cytokine production (intracellular expression IFN-γ (BD Biosciences) and TNF-α ( E -Biosciences) controls . Cell-surface expression of CD107a and intracellular expression of TNF-α and IFN-γ were assessed on PBMC from the case report (in November 2010 and May 2016) or his partner (in August 2011) in the absence of target (Alone), and in the presence of MHC class-I negative K562 cells or CD20 + RAJI cells treated by 1 μg/mL of anti-CD20 mAb (αCD20; Rituximab, Roche), or an Isotype control (Ig Ctl). Effector and target are used at a 1/1 ratio. The values were analyzed with a Boolean gate algorithm (FlowJo; Tree Star). Data are presented as pie charts created with Pestle and Spice software. Colored arcs represent the frequencies of cells producing CD107a, TNF-α and IFN-γ. Pie fractions represent cells performing 0, 1, 2, or 3 functions simultaneously.

    Journal: EBioMedicine

    Article Title: What Is the most Important for Elite Control: Genetic Background of Patient, Genetic Background of Partner, both or neither? Description of Complete Natural History within a Couple of MSM

    doi: 10.1016/j.ebiom.2017.12.003

    Figure Lengend Snippet: Poly-functional analysis of NK cells. (4) Poly-functional analysis of NK cells was performed to simultaneously detect degranulation (anti-CD107a mAb, BD Biosciences) and cytokine production (intracellular expression IFN-γ (BD Biosciences) and TNF-α ( E -Biosciences) controls . Cell-surface expression of CD107a and intracellular expression of TNF-α and IFN-γ were assessed on PBMC from the case report (in November 2010 and May 2016) or his partner (in August 2011) in the absence of target (Alone), and in the presence of MHC class-I negative K562 cells or CD20 + RAJI cells treated by 1 μg/mL of anti-CD20 mAb (αCD20; Rituximab, Roche), or an Isotype control (Ig Ctl). Effector and target are used at a 1/1 ratio. The values were analyzed with a Boolean gate algorithm (FlowJo; Tree Star). Data are presented as pie charts created with Pestle and Spice software. Colored arcs represent the frequencies of cells producing CD107a, TNF-α and IFN-γ. Pie fractions represent cells performing 0, 1, 2, or 3 functions simultaneously.

    Article Snippet: Poly-functional NK was performed to simultaneously detect degranulation (anti-CD107a mAb, BD Biosciences) and cytokine production (intracellular expression IFN-γ (BD Biosciences) and TNF-α (E-Biosciences) ( ).

    Techniques: Functional Assay, Expressing, CTL Assay, Software

    A ) PEDF activates Wnt signaling in hMSCs. hMSCs were treated with Wnt3a (50 ng/ml) and PEDF (500 ng/ml) and immunoblotted for phosphorylated LRP6. B ) hMSCs were treated with IWP-2 (2 μM) for 24 and 48 h and then challenged with PEDF (500 ng/ml

    Journal: The FASEB Journal

    Article Title: Determination of mesenchymal stem cell fate by pigment epithelium-derived factor (PEDF) results in increased adiposity and reduced bone mineral content

    doi: 10.1096/fj.13-232900

    Figure Lengend Snippet: A ) PEDF activates Wnt signaling in hMSCs. hMSCs were treated with Wnt3a (50 ng/ml) and PEDF (500 ng/ml) and immunoblotted for phosphorylated LRP6. B ) hMSCs were treated with IWP-2 (2 μM) for 24 and 48 h and then challenged with PEDF (500 ng/ml

    Article Snippet: The inhibitor of Wnt production (IWP-2; Tocris Bioscience, Minneapolis, MN, USA; 2 μM × 24–48 h) was used to block production of endogenous Wnt proteins ( , ).

    Techniques: