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  • 99
    Thermo Fisher dmem
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    Thermo Fisher dulbecco s modified eagle s medium dmem
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    Thermo Fisher dmem medium
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    Thermo Fisher glucose dmem
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    Corning Life Sciences dmem
    Multipotent progenitor and NSC-like profile of LAM and <t>angiomyolipoma.</t> a Hematoxylin and eosin (H E) of the lung sections from sporadic LAM; GFAP or NSE expression in LAM. Arrows indicate LAM and arrowheads normal bronchial mucosa (upper) or normal pleura (lower images). b Sporadic angiomyolipomas (upper left and upper middle) and adjacent normal kidney (upper right) or TSC-associated (lower images) angiomyolipomas, stained with H E or for NSE, GFAP, NS-tubulin, or nestin (arrows indicate angiomyolipoma and arrowheads kidney). c (i) GFAP, (ii) NSE and nestin expression in (i) TSC-associated and sporadic (ii) angiomyolipomas (T) from patients (P) vs. normal kidney (NK) by western immunoblotting. d (i) Nestin and peripherin expression in LAM (first left) and co-expression analysis by digital pathology; red indicates peripherin (second), brown nestin (third image), and yellow indicates co-expression of both markers (markup, right fifth panel: details within the area marked by the corresponding insert in fourth panel, black arrows). (ii) Quantification for data shown in d (i) by Aperio Digital Pathology; the percentage of area with co-expression of peripherin and nestin in LAM from 9 patients. Error bars are defined as means + s.e.m. e (i) FACS of cells grown in <t>DMEM</t> medium. Percentages of 621–101 and CRL4004 (angiomyolipomas-derived) cells expressing nestin alone or co-expressing: melan-A, GFAP, SMA, and NS-tubulin; (ii) Nestin, GFAP, and NS-tubulin expression in 621–101 (grown in DMEM or IIA complete medium), CRL4004 angiomyolipoma cells, angiomyolipoma tumor, and corresponding normal kidney by western immunoblotting. Error bars are defined as means + s.e.m. f q(RT)-PCR of SOX10 , SOX9 , ID3 , NESTIN , c-Kit , DCT , and S100A1 mRNA levels relative to 18S in 621–101, CRL4004, angiomyolipoma tumor, and corresponding normal kidney. Data represent means (±s.e.m.). Data from nine LAM and eleven angiomyolipomas ( a – d ). Scale bar: 50 µm. * P ≤ 0.05, t- test. Data are representative of three to six ( e (i)) independent experiments
    Dmem, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 4969 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mediatech dmem
    Promoter-associated proteins after reversal of the AAR stress <t>HepG2</t> cells were cultured in <t>DMEM</t> ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse promoter binding of RNA Pol II ( A ) or ATF4 ( B ). A non-specific IgG was used as a negative control ( C ). The data are plotted as the ratio to input DNA. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.
    Dmem, supplied by Mediatech, used in various techniques. Bioz Stars score: 99/100, based on 4400 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dulbecco s modified eagle medium dmem
    Promoter-associated proteins after reversal of the AAR stress <t>HepG2</t> cells were cultured in <t>DMEM</t> ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse promoter binding of RNA Pol II ( A ) or ATF4 ( B ). A non-specific IgG was used as a negative control ( C ). The data are plotted as the ratio to input DNA. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.
    Dulbecco S Modified Eagle Medium Dmem, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3071 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cellgro dmem
    Promoter-associated proteins after reversal of the AAR stress <t>HepG2</t> cells were cultured in <t>DMEM</t> ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse promoter binding of RNA Pol II ( A ) or ATF4 ( B ). A non-specific IgG was used as a negative control ( C ). The data are plotted as the ratio to input DNA. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.
    Dmem, supplied by Cellgro, used in various techniques. Bioz Stars score: 92/100, based on 4080 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dmem f 12 medium
    Promoter-associated proteins after reversal of the AAR stress <t>HepG2</t> cells were cultured in <t>DMEM</t> ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse promoter binding of RNA Pol II ( A ) or ATF4 ( B ). A non-specific IgG was used as a negative control ( C ). The data are plotted as the ratio to input DNA. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.
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    Thermo Fisher knockout dmem
    Promoter-associated proteins after reversal of the AAR stress <t>HepG2</t> cells were cultured in <t>DMEM</t> ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse promoter binding of RNA Pol II ( A ) or ATF4 ( B ). A non-specific IgG was used as a negative control ( C ). The data are plotted as the ratio to input DNA. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.
    Knockout Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dmem media
    Promoter-associated proteins after reversal of the AAR stress <t>HepG2</t> cells were cultured in <t>DMEM</t> ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse promoter binding of RNA Pol II ( A ) or ATF4 ( B ). A non-specific IgG was used as a negative control ( C ). The data are plotted as the ratio to input DNA. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.
    Dmem Media, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5412 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher glutamax
    Promoter-associated proteins after reversal of the AAR stress <t>HepG2</t> cells were cultured in <t>DMEM</t> ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse promoter binding of RNA Pol II ( A ) or ATF4 ( B ). A non-specific IgG was used as a negative control ( C ). The data are plotted as the ratio to input DNA. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.
    Glutamax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53776 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Multipotent progenitor and NSC-like profile of LAM and angiomyolipoma. a Hematoxylin and eosin (H E) of the lung sections from sporadic LAM; GFAP or NSE expression in LAM. Arrows indicate LAM and arrowheads normal bronchial mucosa (upper) or normal pleura (lower images). b Sporadic angiomyolipomas (upper left and upper middle) and adjacent normal kidney (upper right) or TSC-associated (lower images) angiomyolipomas, stained with H E or for NSE, GFAP, NS-tubulin, or nestin (arrows indicate angiomyolipoma and arrowheads kidney). c (i) GFAP, (ii) NSE and nestin expression in (i) TSC-associated and sporadic (ii) angiomyolipomas (T) from patients (P) vs. normal kidney (NK) by western immunoblotting. d (i) Nestin and peripherin expression in LAM (first left) and co-expression analysis by digital pathology; red indicates peripherin (second), brown nestin (third image), and yellow indicates co-expression of both markers (markup, right fifth panel: details within the area marked by the corresponding insert in fourth panel, black arrows). (ii) Quantification for data shown in d (i) by Aperio Digital Pathology; the percentage of area with co-expression of peripherin and nestin in LAM from 9 patients. Error bars are defined as means + s.e.m. e (i) FACS of cells grown in DMEM medium. Percentages of 621–101 and CRL4004 (angiomyolipomas-derived) cells expressing nestin alone or co-expressing: melan-A, GFAP, SMA, and NS-tubulin; (ii) Nestin, GFAP, and NS-tubulin expression in 621–101 (grown in DMEM or IIA complete medium), CRL4004 angiomyolipoma cells, angiomyolipoma tumor, and corresponding normal kidney by western immunoblotting. Error bars are defined as means + s.e.m. f q(RT)-PCR of SOX10 , SOX9 , ID3 , NESTIN , c-Kit , DCT , and S100A1 mRNA levels relative to 18S in 621–101, CRL4004, angiomyolipoma tumor, and corresponding normal kidney. Data represent means (±s.e.m.). Data from nine LAM and eleven angiomyolipomas ( a – d ). Scale bar: 50 µm. * P ≤ 0.05, t- test. Data are representative of three to six ( e (i)) independent experiments

    Journal: Nature Communications

    Article Title: Notch transactivates Rheb to maintain the multipotency of TSC-null cells

    doi: 10.1038/s41467-017-01845-1

    Figure Lengend Snippet: Multipotent progenitor and NSC-like profile of LAM and angiomyolipoma. a Hematoxylin and eosin (H E) of the lung sections from sporadic LAM; GFAP or NSE expression in LAM. Arrows indicate LAM and arrowheads normal bronchial mucosa (upper) or normal pleura (lower images). b Sporadic angiomyolipomas (upper left and upper middle) and adjacent normal kidney (upper right) or TSC-associated (lower images) angiomyolipomas, stained with H E or for NSE, GFAP, NS-tubulin, or nestin (arrows indicate angiomyolipoma and arrowheads kidney). c (i) GFAP, (ii) NSE and nestin expression in (i) TSC-associated and sporadic (ii) angiomyolipomas (T) from patients (P) vs. normal kidney (NK) by western immunoblotting. d (i) Nestin and peripherin expression in LAM (first left) and co-expression analysis by digital pathology; red indicates peripherin (second), brown nestin (third image), and yellow indicates co-expression of both markers (markup, right fifth panel: details within the area marked by the corresponding insert in fourth panel, black arrows). (ii) Quantification for data shown in d (i) by Aperio Digital Pathology; the percentage of area with co-expression of peripherin and nestin in LAM from 9 patients. Error bars are defined as means + s.e.m. e (i) FACS of cells grown in DMEM medium. Percentages of 621–101 and CRL4004 (angiomyolipomas-derived) cells expressing nestin alone or co-expressing: melan-A, GFAP, SMA, and NS-tubulin; (ii) Nestin, GFAP, and NS-tubulin expression in 621–101 (grown in DMEM or IIA complete medium), CRL4004 angiomyolipoma cells, angiomyolipoma tumor, and corresponding normal kidney by western immunoblotting. Error bars are defined as means + s.e.m. f q(RT)-PCR of SOX10 , SOX9 , ID3 , NESTIN , c-Kit , DCT , and S100A1 mRNA levels relative to 18S in 621–101, CRL4004, angiomyolipoma tumor, and corresponding normal kidney. Data represent means (±s.e.m.). Data from nine LAM and eleven angiomyolipomas ( a – d ). Scale bar: 50 µm. * P ≤ 0.05, t- test. Data are representative of three to six ( e (i)) independent experiments

    Article Snippet: Cells Angiomyolipoma-derived cells : 621–101 with inactivation of both TSC2 alleles (from Dr. Elizabeth Henske), CRL4004 (ATCC) with a 5 bp deletion in TSC2 exon 33 from a sporadic angiomyolipoma were cultured in DMEM (high glucose) media (Corning) supplemented with 10% fetal bovine serum (FBS) or heat-inactivated FBS, penicillin/streptomycin, and glutamine .

    Techniques: Laser Capture Microdissection, Expressing, Staining, Western Blot, FACS, Derivative Assay, Polymerase Chain Reaction

    Notch1 directly activates the Rheb promoter. a q(RT)-PCR of (i, iii) RHEB and (ii, iv) HES1 relative to GAPDH in synchronized (i, ii) 621–101 and (iii, iv) CRL4004 angiomyolipoma cells in DMEM. The statistical significance of RHEB and HES1 expression at different time points was determined relative to time 0. b Expression of Rheb and Hes1 in synchronized (i) 621–101 and (ii) CRL4004 angiomyolipoma cells in DMEM by western immunoblotting. Numeric values represent densitometry analysis of the expression of Rheb relative to expression of Gapdh. c (i) Rheb promoter: triangles indicate potential RBPJ binding sites with a reverse (1, 2) and a forward (3, 4) orientation. (ii–v) Binding of Notch1 to (ii, iv) the potential RBPJ binding site 1, NRE2, NRE3, and the potential RBPJ binding site 4 of RHEB or (iii, v) HES1 promoter in non-synchronized (ii, iii) 621–101 or (iv, v) CRL4004 cells grown in DMEM by ChIP-qPCR; binding is shown as fold enrichment over the IgG (dotted line). d (i) Wild-type Rheb-luciferase (RHEB), RHEB-RBPJ1- (RBPJ1), RHEB-NRE2- (NRE2), RHEB-NRE3- (NRE3), or RHEB- RBPJ4- (RBPJ4) mutant-luciferase or control-luciferase (control) promoters activity in HeLa cells. (ii) Expression of N1ICD, MAML1, Hes1, Rheb, and phospho-S6K in 293T, HeLa, CRL4004, and H1299 cells. (iii) Expression of cleaved Notch1 (Val1744), Notch1, Hes1, Rheb in 621–101, CRL4004, and H1299 cells depleted of Notch1 using shRNA by western blotting or (iv) q(RT)-PCR. Numeric values represent densitometry analysis of the expression of Rheb relative to expression of Gapdh. (iv) NOTCH1 , RHEB , and HES1 relative to GAPDH in 621–101 cells depleted of Notch1 using shRNA. e Binding of Notch1 to the (i) potential RBPJ1 site, (ii) NRE2, and (iii) NRE3 within Rheb or (iv) Hes1 promoter in synchronized 621–101 cells in DMEM by ChIP-qPCR as in ( c (ii, iii)). Data represent means ± s.e.m. Error bars are defined as means + s.e.m. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, t -test. Data are representative of three ( a (iii, iv), b , d (i–iv)), four ( e ), and six ( a (i, ii), c (ii, iii)) independent experiments

    Journal: Nature Communications

    Article Title: Notch transactivates Rheb to maintain the multipotency of TSC-null cells

    doi: 10.1038/s41467-017-01845-1

    Figure Lengend Snippet: Notch1 directly activates the Rheb promoter. a q(RT)-PCR of (i, iii) RHEB and (ii, iv) HES1 relative to GAPDH in synchronized (i, ii) 621–101 and (iii, iv) CRL4004 angiomyolipoma cells in DMEM. The statistical significance of RHEB and HES1 expression at different time points was determined relative to time 0. b Expression of Rheb and Hes1 in synchronized (i) 621–101 and (ii) CRL4004 angiomyolipoma cells in DMEM by western immunoblotting. Numeric values represent densitometry analysis of the expression of Rheb relative to expression of Gapdh. c (i) Rheb promoter: triangles indicate potential RBPJ binding sites with a reverse (1, 2) and a forward (3, 4) orientation. (ii–v) Binding of Notch1 to (ii, iv) the potential RBPJ binding site 1, NRE2, NRE3, and the potential RBPJ binding site 4 of RHEB or (iii, v) HES1 promoter in non-synchronized (ii, iii) 621–101 or (iv, v) CRL4004 cells grown in DMEM by ChIP-qPCR; binding is shown as fold enrichment over the IgG (dotted line). d (i) Wild-type Rheb-luciferase (RHEB), RHEB-RBPJ1- (RBPJ1), RHEB-NRE2- (NRE2), RHEB-NRE3- (NRE3), or RHEB- RBPJ4- (RBPJ4) mutant-luciferase or control-luciferase (control) promoters activity in HeLa cells. (ii) Expression of N1ICD, MAML1, Hes1, Rheb, and phospho-S6K in 293T, HeLa, CRL4004, and H1299 cells. (iii) Expression of cleaved Notch1 (Val1744), Notch1, Hes1, Rheb in 621–101, CRL4004, and H1299 cells depleted of Notch1 using shRNA by western blotting or (iv) q(RT)-PCR. Numeric values represent densitometry analysis of the expression of Rheb relative to expression of Gapdh. (iv) NOTCH1 , RHEB , and HES1 relative to GAPDH in 621–101 cells depleted of Notch1 using shRNA. e Binding of Notch1 to the (i) potential RBPJ1 site, (ii) NRE2, and (iii) NRE3 within Rheb or (iv) Hes1 promoter in synchronized 621–101 cells in DMEM by ChIP-qPCR as in ( c (ii, iii)). Data represent means ± s.e.m. Error bars are defined as means + s.e.m. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, t -test. Data are representative of three ( a (iii, iv), b , d (i–iv)), four ( e ), and six ( a (i, ii), c (ii, iii)) independent experiments

    Article Snippet: Cells Angiomyolipoma-derived cells : 621–101 with inactivation of both TSC2 alleles (from Dr. Elizabeth Henske), CRL4004 (ATCC) with a 5 bp deletion in TSC2 exon 33 from a sporadic angiomyolipoma were cultured in DMEM (high glucose) media (Corning) supplemented with 10% fetal bovine serum (FBS) or heat-inactivated FBS, penicillin/streptomycin, and glutamine .

    Techniques: Polymerase Chain Reaction, Expressing, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Luciferase, Mutagenesis, Activity Assay, shRNA

    Suppression of Rheb and Hes1 oscillation associates with neuronal differentiation of angiomyolipoma cells. a q(RT)-PCR of (i) RHEB and (ii) HES1 relative to GAPDH in synchronized 621–101 cells in N-medium (released with chicken embryo extract (red lines) or serum (green lines)) vs. DMEM (released with serum (black lines)) (data from DMEM are also used in Fig. 2a (i–ii )). The statistical significance of RHEB and HES1 expression in DMEM vs. N-medium was determined between corresponding time points. b Binding of Notch1 to the (i) NRE2, NRE3, and (ii) potential RBPJ1 site within the Rheb or (iii) Hes1 promoter, in synchronized 621–101 cells in N-medium vs. DMEM at day 2 by ChIP-qPCR (data from DMEM are also used in Fig. 2e (i–iv )). Asterisks represent the statistical significance of mean of time points in DMEM vs. N-medium, two-way ANOVA. c q(RT)-PCR of NOTCH1 in 621–101 cells in N-medium at day 2, relative to DMEM (control) after normalization to GAPDH . d Western blot of Notch1, Hes5, and cleaved Notch1 (Val1744) in 621–101 cells in N-medium vs. DMEM. e Percentage of 621–101 cells expressing phospho-S6 in N-medium by FACS. f q(RT)-PCR of RHEB in 621–101 cells in N-medium at day 2, relative to DMEM (control) after normalization to GAPDH . g Western blot of Notch1, phospho-S6K, Hes5, Rheb, and cleaved Notch1 (Val1744) in CRL4004 cells in N-medium vs. DMEM. h Percentage of CRL4004 cells expressing phospho-S6 grown in N-medium by FACS. i q(RT)-PCR of ID1 , ID3 , and SOX9 in 621–101 cells in N-medium at day 2, relative to DMEM (control) after normalization to GAPDH . Data represent means ± s.e.m. Error bars are defined as means + s.e.m. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, t -test ( a , c , e , f , h , i ), two-way ANOVA ( b ). Data are representative of two, n = 2 ( i for SOX9 ); three ( g ); three, n = 2 ( a , b (iii)); three, n = 3 ( h ); three, n = 4 ( b (i, ii)); four ( d ), four, n = 3 ( f ); four, n = 4 ( c , e , i for ID1 , ID3 )

    Journal: Nature Communications

    Article Title: Notch transactivates Rheb to maintain the multipotency of TSC-null cells

    doi: 10.1038/s41467-017-01845-1

    Figure Lengend Snippet: Suppression of Rheb and Hes1 oscillation associates with neuronal differentiation of angiomyolipoma cells. a q(RT)-PCR of (i) RHEB and (ii) HES1 relative to GAPDH in synchronized 621–101 cells in N-medium (released with chicken embryo extract (red lines) or serum (green lines)) vs. DMEM (released with serum (black lines)) (data from DMEM are also used in Fig. 2a (i–ii )). The statistical significance of RHEB and HES1 expression in DMEM vs. N-medium was determined between corresponding time points. b Binding of Notch1 to the (i) NRE2, NRE3, and (ii) potential RBPJ1 site within the Rheb or (iii) Hes1 promoter, in synchronized 621–101 cells in N-medium vs. DMEM at day 2 by ChIP-qPCR (data from DMEM are also used in Fig. 2e (i–iv )). Asterisks represent the statistical significance of mean of time points in DMEM vs. N-medium, two-way ANOVA. c q(RT)-PCR of NOTCH1 in 621–101 cells in N-medium at day 2, relative to DMEM (control) after normalization to GAPDH . d Western blot of Notch1, Hes5, and cleaved Notch1 (Val1744) in 621–101 cells in N-medium vs. DMEM. e Percentage of 621–101 cells expressing phospho-S6 in N-medium by FACS. f q(RT)-PCR of RHEB in 621–101 cells in N-medium at day 2, relative to DMEM (control) after normalization to GAPDH . g Western blot of Notch1, phospho-S6K, Hes5, Rheb, and cleaved Notch1 (Val1744) in CRL4004 cells in N-medium vs. DMEM. h Percentage of CRL4004 cells expressing phospho-S6 grown in N-medium by FACS. i q(RT)-PCR of ID1 , ID3 , and SOX9 in 621–101 cells in N-medium at day 2, relative to DMEM (control) after normalization to GAPDH . Data represent means ± s.e.m. Error bars are defined as means + s.e.m. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, t -test ( a , c , e , f , h , i ), two-way ANOVA ( b ). Data are representative of two, n = 2 ( i for SOX9 ); three ( g ); three, n = 2 ( a , b (iii)); three, n = 3 ( h ); three, n = 4 ( b (i, ii)); four ( d ), four, n = 3 ( f ); four, n = 4 ( c , e , i for ID1 , ID3 )

    Article Snippet: Cells Angiomyolipoma-derived cells : 621–101 with inactivation of both TSC2 alleles (from Dr. Elizabeth Henske), CRL4004 (ATCC) with a 5 bp deletion in TSC2 exon 33 from a sporadic angiomyolipoma were cultured in DMEM (high glucose) media (Corning) supplemented with 10% fetal bovine serum (FBS) or heat-inactivated FBS, penicillin/streptomycin, and glutamine .

    Techniques: Polymerase Chain Reaction, Expressing, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Western Blot, FACS

    Neuronal differentiation of angiomyolipoma cells. a , d Brightfield images of a 621–101 and d CRL4004 cells in DMEM or N-medium. b , e Nestin and NS-tubulin in b 621–101 and e CRL4004 sphere-like structures in N-medium at day 4 and day 7 by immunofluorescence. c , f FACS of c 621–101 and f CRL4004 cells in N-medium. c , f Representative histograms and graphs showing percentages of cells in N-medium expressing ( c (i), f (i)) nestin only and ( c (ii), f (ii)) NS-tubulin only. c (iii), f (iii) Ki-67 in ( c (iii)) 621–101 and ( f (iii)) CRL4004 cells in N-medium vs. DMEM at days 4 and 7. c (iv), f (iv) q(RT)-PCR of Ngn1 , DCT , and c-Kit mRNA relative to GAPDH in c (iv) 621–101 or f (iv) CRL4004 cells in N-medium vs. DMEM at day 4. Data represent means ± s.e.m. Error bars are defined as means + s.e.m. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 by t -test. Data are representative of two ( b , e ), three, n = 3 ( f (i–iv), c (iv)), four ( d ); n = 4 ( c (i–iii)), and seven ( a ) independent experiments

    Journal: Nature Communications

    Article Title: Notch transactivates Rheb to maintain the multipotency of TSC-null cells

    doi: 10.1038/s41467-017-01845-1

    Figure Lengend Snippet: Neuronal differentiation of angiomyolipoma cells. a , d Brightfield images of a 621–101 and d CRL4004 cells in DMEM or N-medium. b , e Nestin and NS-tubulin in b 621–101 and e CRL4004 sphere-like structures in N-medium at day 4 and day 7 by immunofluorescence. c , f FACS of c 621–101 and f CRL4004 cells in N-medium. c , f Representative histograms and graphs showing percentages of cells in N-medium expressing ( c (i), f (i)) nestin only and ( c (ii), f (ii)) NS-tubulin only. c (iii), f (iii) Ki-67 in ( c (iii)) 621–101 and ( f (iii)) CRL4004 cells in N-medium vs. DMEM at days 4 and 7. c (iv), f (iv) q(RT)-PCR of Ngn1 , DCT , and c-Kit mRNA relative to GAPDH in c (iv) 621–101 or f (iv) CRL4004 cells in N-medium vs. DMEM at day 4. Data represent means ± s.e.m. Error bars are defined as means + s.e.m. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 by t -test. Data are representative of two ( b , e ), three, n = 3 ( f (i–iv), c (iv)), four ( d ); n = 4 ( c (i–iii)), and seven ( a ) independent experiments

    Article Snippet: Cells Angiomyolipoma-derived cells : 621–101 with inactivation of both TSC2 alleles (from Dr. Elizabeth Henske), CRL4004 (ATCC) with a 5 bp deletion in TSC2 exon 33 from a sporadic angiomyolipoma were cultured in DMEM (high glucose) media (Corning) supplemented with 10% fetal bovine serum (FBS) or heat-inactivated FBS, penicillin/streptomycin, and glutamine .

    Techniques: Immunofluorescence, FACS, Expressing, Polymerase Chain Reaction

    Promoter-associated proteins after reversal of the AAR stress HepG2 cells were cultured in DMEM ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse promoter binding of RNA Pol II ( A ) or ATF4 ( B ). A non-specific IgG was used as a negative control ( C ). The data are plotted as the ratio to input DNA. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.

    Journal: The Biochemical journal

    Article Title: Dynamic changes in genomic histone association and modification during activation of the ASNS and ATF3 genes by amino acid limitation

    doi: 10.1042/BJ20120958

    Figure Lengend Snippet: Promoter-associated proteins after reversal of the AAR stress HepG2 cells were cultured in DMEM ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse promoter binding of RNA Pol II ( A ) or ATF4 ( B ). A non-specific IgG was used as a negative control ( C ). The data are plotted as the ratio to input DNA. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.

    Article Snippet: HepG2 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium, pH 7.4; Mediatech), supplemented with 1× non-essential amino acids, 2 mM glutamine, 100 μ g/ml streptomycin sulfate, 100 units/ml penicillin G, 0.25 μ g/ml amphotericin B and 10 % (v/v) FBS (fetal bovine serum).

    Techniques: Cell Culture, Chromatin Immunoprecipitation, Binding Assay, Negative Control

    Analysis of ASNS transcriptional activity in response to a second round of AAR activation HepG2 cells were cultured in DMEM ± 2 mM HisOH for 0–4 h to activate the AAR. A batch of cells incubated with HisOH for 4 h was then transferred to DMEM lacking HisOH for 2 h to reverse the AAR induction. After a 2 h period in DMEM alone, one-half of the cells were transferred to DMEM + 2 mM HisOH for an additional 4 h to provide a second round of AAR activation. These changes in culture medium are summarized below the data using broken lines to designate DMEM and a solid lines to designate DMEM + HisOH. At the indicated time points, total RNA was isolated and qPCR was performed to analyse the ASNS transcriptional activity and steady-state mRNA levels of GAPDH . The data for ASNS transcriptional activity were calculated as the ratio to the GAPDH control and then plotted relative to the DMEM value at zero time. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.

    Journal: The Biochemical journal

    Article Title: Dynamic changes in genomic histone association and modification during activation of the ASNS and ATF3 genes by amino acid limitation

    doi: 10.1042/BJ20120958

    Figure Lengend Snippet: Analysis of ASNS transcriptional activity in response to a second round of AAR activation HepG2 cells were cultured in DMEM ± 2 mM HisOH for 0–4 h to activate the AAR. A batch of cells incubated with HisOH for 4 h was then transferred to DMEM lacking HisOH for 2 h to reverse the AAR induction. After a 2 h period in DMEM alone, one-half of the cells were transferred to DMEM + 2 mM HisOH for an additional 4 h to provide a second round of AAR activation. These changes in culture medium are summarized below the data using broken lines to designate DMEM and a solid lines to designate DMEM + HisOH. At the indicated time points, total RNA was isolated and qPCR was performed to analyse the ASNS transcriptional activity and steady-state mRNA levels of GAPDH . The data for ASNS transcriptional activity were calculated as the ratio to the GAPDH control and then plotted relative to the DMEM value at zero time. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.

    Article Snippet: HepG2 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium, pH 7.4; Mediatech), supplemented with 1× non-essential amino acids, 2 mM glutamine, 100 μ g/ml streptomycin sulfate, 100 units/ml penicillin G, 0.25 μ g/ml amphotericin B and 10 % (v/v) FBS (fetal bovine serum).

    Techniques: Activity Assay, Activation Assay, Cell Culture, Incubation, Isolation, Real-time Polymerase Chain Reaction

    H3K4 trimethylation after reversal of the AAR stress HepG2 cells were cultured in DMEM ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse association of histone H3K4me3 at both the promoter region ( A ) and at the + 1 kb region ( B ). The absolute abundance of the H3K4me3 mark is illustrated as the ratio to input DNA (left-hand panels) and in the right-hand panels, the values after normalization for nucleosome abundance are shown as the ratio of H3K4me3/total H3. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.

    Journal: The Biochemical journal

    Article Title: Dynamic changes in genomic histone association and modification during activation of the ASNS and ATF3 genes by amino acid limitation

    doi: 10.1042/BJ20120958

    Figure Lengend Snippet: H3K4 trimethylation after reversal of the AAR stress HepG2 cells were cultured in DMEM ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse association of histone H3K4me3 at both the promoter region ( A ) and at the + 1 kb region ( B ). The absolute abundance of the H3K4me3 mark is illustrated as the ratio to input DNA (left-hand panels) and in the right-hand panels, the values after normalization for nucleosome abundance are shown as the ratio of H3K4me3/total H3. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.

    Article Snippet: HepG2 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium, pH 7.4; Mediatech), supplemented with 1× non-essential amino acids, 2 mM glutamine, 100 μ g/ml streptomycin sulfate, 100 units/ml penicillin G, 0.25 μ g/ml amphotericin B and 10 % (v/v) FBS (fetal bovine serum).

    Techniques: Cell Culture, Chromatin Immunoprecipitation

    Acetylation of histone H4 after reversal of the AAR Stress HepG2 cells were cultured in DMEM ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse association of total histone H3 ( A ) or pan-H4Ac ( B ). In ( C ), the ratio of H4Ac/total H3 is illustrated as the ‘Normalized signal’ for both genes. The data are plotted as the ratio to input DNA. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.

    Journal: The Biochemical journal

    Article Title: Dynamic changes in genomic histone association and modification during activation of the ASNS and ATF3 genes by amino acid limitation

    doi: 10.1042/BJ20120958

    Figure Lengend Snippet: Acetylation of histone H4 after reversal of the AAR Stress HepG2 cells were cultured in DMEM ± 2 mM HisOH for the indicated times. ChIP analysis of the ASNS and ATF3 genes was performed to analyse association of total histone H3 ( A ) or pan-H4Ac ( B ). In ( C ), the ratio of H4Ac/total H3 is illustrated as the ‘Normalized signal’ for both genes. The data are plotted as the ratio to input DNA. The hash mark following the 4 h HisOH data point indicates the 10 min period of HisOH washout. Values are means ± S.D. for at least three samples and, where not shown, the error bars are contained within the symbol. * P ≤ 0.05 relative to the DMEM control for the same time point.

    Article Snippet: HepG2 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium, pH 7.4; Mediatech), supplemented with 1× non-essential amino acids, 2 mM glutamine, 100 μ g/ml streptomycin sulfate, 100 units/ml penicillin G, 0.25 μ g/ml amphotericin B and 10 % (v/v) FBS (fetal bovine serum).

    Techniques: Cell Culture, Chromatin Immunoprecipitation

    Identification of AAR-inducible histone modifications at the ASNS gene ( A ) Location of the amino-acid-responsive ATF4-binding CARE site. Arrows show the primer pairs used to scan the gene by ChIP analysis. ( B ) HepG2 cells were cultured in DMEM ± 2 mM HisOH for 4 h and then the binding of ATF4 and the relative abundance of histone modifications (H4Ac and H3K36me3) at the promoter and exon 7 within the ASNS gene were analysed. ( C ) The H3K27me3 modification level at the proximal promoter regions of the ASNS and SFRP1 genes was analysed by ChIP in HepG2 cells cultured in DMEM ± 2 mM HisOH for 16 h. Within each ChIP experiment, a non-specific IgG was used as a negative control. Values, plotted as the ratio to input DNA, are means ± S.D. for at least three samples. * P ≤ 0.05 relative to the DMEM control.

    Journal: The Biochemical journal

    Article Title: Dynamic changes in genomic histone association and modification during activation of the ASNS and ATF3 genes by amino acid limitation

    doi: 10.1042/BJ20120958

    Figure Lengend Snippet: Identification of AAR-inducible histone modifications at the ASNS gene ( A ) Location of the amino-acid-responsive ATF4-binding CARE site. Arrows show the primer pairs used to scan the gene by ChIP analysis. ( B ) HepG2 cells were cultured in DMEM ± 2 mM HisOH for 4 h and then the binding of ATF4 and the relative abundance of histone modifications (H4Ac and H3K36me3) at the promoter and exon 7 within the ASNS gene were analysed. ( C ) The H3K27me3 modification level at the proximal promoter regions of the ASNS and SFRP1 genes was analysed by ChIP in HepG2 cells cultured in DMEM ± 2 mM HisOH for 16 h. Within each ChIP experiment, a non-specific IgG was used as a negative control. Values, plotted as the ratio to input DNA, are means ± S.D. for at least three samples. * P ≤ 0.05 relative to the DMEM control.

    Article Snippet: HepG2 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium, pH 7.4; Mediatech), supplemented with 1× non-essential amino acids, 2 mM glutamine, 100 μ g/ml streptomycin sulfate, 100 units/ml penicillin G, 0.25 μ g/ml amphotericin B and 10 % (v/v) FBS (fetal bovine serum).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Modification, Negative Control