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  • 99
    ATCC psv2 dhfr
    Psv2 Dhfr, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cho dhfr cells
    Role of the CEA splice variant in MEDI-565 mediated T cell activation and target cell killing. A, expression of full-length CEA and CEA splice variant proteins in <t>CHO</t> cells as determined by flow cytometry. Mouse IgG1, mouse IgG1 control antibody. B, CHO <t>DHFR-,</t> CHO FL, CHO SV and CHO FL+SV cells were tested for their susceptibility to be killed by CD3+ T cells from 3 individual donors in the presence of MEDI-565 at the indicated concentrations. EC 50 values listed indicate the mean value among 3 donors±standard error of the mean. p = 0.79 comparing cytotoxicity EC 50 values between CHO FL CEA and CHO FL+SV CEA cells. C, activation (increased cell surface CD25/IL-2R levels) of CD8+ T cells and D, activation of CD4+ T cells isolated from each of the 3 healthy donors was investigated concurrently with the cytotoxicity assays shown in panel B. p = 0.60 comparing CD8+ T cell activation EC 50 values between CHO FL CEA and CHO FL+SV CEA; p = 0.15 comparing CD4+ T cell activation EC 50 values between CHO FL CEA and CHO FL+SV CEA. MFI CD25-APC = mean fluorescence intensity of bound APC labeled, anti-human CD25 mAb. Experiment was repeated once with similar results.
    Cho Dhfr Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc dhfr
    Role of the CEA splice variant in MEDI-565 mediated T cell activation and target cell killing. A, expression of full-length CEA and CEA splice variant proteins in <t>CHO</t> cells as determined by flow cytometry. Mouse IgG1, mouse IgG1 control antibody. B, CHO <t>DHFR-,</t> CHO FL, CHO SV and CHO FL+SV cells were tested for their susceptibility to be killed by CD3+ T cells from 3 individual donors in the presence of MEDI-565 at the indicated concentrations. EC 50 values listed indicate the mean value among 3 donors±standard error of the mean. p = 0.79 comparing cytotoxicity EC 50 values between CHO FL CEA and CHO FL+SV CEA cells. C, activation (increased cell surface CD25/IL-2R levels) of CD8+ T cells and D, activation of CD4+ T cells isolated from each of the 3 healthy donors was investigated concurrently with the cytotoxicity assays shown in panel B. p = 0.60 comparing CD8+ T cell activation EC 50 values between CHO FL CEA and CHO FL+SV CEA; p = 0.15 comparing CD4+ T cell activation EC 50 values between CHO FL CEA and CHO FL+SV CEA. MFI CD25-APC = mean fluorescence intensity of bound APC labeled, anti-human CD25 mAb. Experiment was repeated once with similar results.
    Dhfr, supplied by Cell Signaling Technology Inc, 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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    94
    Addgene inc dhfr
    The drug-stabilizable Gal4 variants 1xDHFR22 and 2xDHFR22 function in vivo in the format of the widely used Gal4- UAS bipartite expression system for Drosophila . (a) Schematic representation of the constructs used to create drug-inducible Gal4 driver lines. 1xDHFR22 encodes the single <t>DHFR-DD</t> architecture with the DHFR variant 22, 28 as a fusion to the Gal4VP16 transcription factor. A nuclear localization signal (NLS) is added N-terminally and expression is driven by the eye-specific enhancer, glass multiple reporter ( GMR ). Similarly, 2xDHFR22 encodes the double architecture of <t>DHFR22-DD.</t> (b) A population of F 1 progenies from 1xDHFR22 and 2xDHFR22 genetic crosses with UAS-eGFP reporter line was allowed to feed on standard fly food supplemented with DMSO (mock-treatment) or various concentrations of TMP for 5-days. A negative control population was derived from the Curly wings phenotype resulting from a dominant CyO marker from the heterozygote 1xDHFR or 2xDHFR driver line. Samples of the population were imaged by fluorescence microscopy. Representative images of adult fly eyes display an increase in eGFP fluorescence intensity as a function of the inducer TMP. The upper, middle and lower panels display, respectively, F 1 progenies with genotypes 1xDHFR22;UAS-eGFP, 2xDHFR22;UAS-eGFP and CyO;UAS-eGFP . Scale bar: 1 mm. (c) Quantification of eGFP fluorescence intensity in the Drosophila adult eyes either mock-treated with DMSO or with various concentrations of TMP. Data are presented as the mean fluorescence detected per eye. The statistical significance resulting from a one-way ANOVA and Tukey’s post hoc test is summarized with asterisk marks representing the level of significance (n.s.= P -value > 0.05, * = P -value ≤ 0.05, *** = P -value ≤ 0. 001, and **** = P -value ≤ 0.0001) on the indicated data set. The error bars represent the standard deviation over the mean across the biological replicates ( n = 8–76 individual eyes per dose).
    Dhfr, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cbs  (ATCC)
    93
    ATCC cbs
    The drug-stabilizable Gal4 variants 1xDHFR22 and 2xDHFR22 function in vivo in the format of the widely used Gal4- UAS bipartite expression system for Drosophila . (a) Schematic representation of the constructs used to create drug-inducible Gal4 driver lines. 1xDHFR22 encodes the single <t>DHFR-DD</t> architecture with the DHFR variant 22, 28 as a fusion to the Gal4VP16 transcription factor. A nuclear localization signal (NLS) is added N-terminally and expression is driven by the eye-specific enhancer, glass multiple reporter ( GMR ). Similarly, 2xDHFR22 encodes the double architecture of <t>DHFR22-DD.</t> (b) A population of F 1 progenies from 1xDHFR22 and 2xDHFR22 genetic crosses with UAS-eGFP reporter line was allowed to feed on standard fly food supplemented with DMSO (mock-treatment) or various concentrations of TMP for 5-days. A negative control population was derived from the Curly wings phenotype resulting from a dominant CyO marker from the heterozygote 1xDHFR or 2xDHFR driver line. Samples of the population were imaged by fluorescence microscopy. Representative images of adult fly eyes display an increase in eGFP fluorescence intensity as a function of the inducer TMP. The upper, middle and lower panels display, respectively, F 1 progenies with genotypes 1xDHFR22;UAS-eGFP, 2xDHFR22;UAS-eGFP and CyO;UAS-eGFP . Scale bar: 1 mm. (c) Quantification of eGFP fluorescence intensity in the Drosophila adult eyes either mock-treated with DMSO or with various concentrations of TMP. Data are presented as the mean fluorescence detected per eye. The statistical significance resulting from a one-way ANOVA and Tukey’s post hoc test is summarized with asterisk marks representing the level of significance (n.s.= P -value > 0.05, * = P -value ≤ 0.05, *** = P -value ≤ 0. 001, and **** = P -value ≤ 0.0001) on the indicated data set. The error bars represent the standard deviation over the mean across the biological replicates ( n = 8–76 individual eyes per dose).
    Cbs, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti dhfr antibody
    Phenotypes of L5Jcs5 and L5Jcs11 mutants . (A) Light micrographs of E7.5 WT and L5Jcs5/L5Jcs5 littermates taken at same magnification (8×). (B) Western blots of liver protein probed with <t>anti-TYMS,</t> <t>DHFR,</t> and beta actin. Each lane contains protein from separate animals. (C) Light micrographs of whole mount E7.5 WT and L5Jcs11/L5Jcs11 embryos, plus representative images of blastocyst outgrowths from the indicated genotypes. The mutant embryo is magnified 1.5 X compared to the WT. Notice that there is no evidence of growth of the embryo proper. ICM = Inner cell mass. Tr = trophectoderm.
    Anti Dhfr Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore variant dhfrs
    Phenotypes of L5Jcs5 and L5Jcs11 mutants . (A) Light micrographs of E7.5 WT and L5Jcs5/L5Jcs5 littermates taken at same magnification (8×). (B) Western blots of liver protein probed with <t>anti-TYMS,</t> <t>DHFR,</t> and beta actin. Each lane contains protein from separate animals. (C) Light micrographs of whole mount E7.5 WT and L5Jcs11/L5Jcs11 embryos, plus representative images of blastocyst outgrowths from the indicated genotypes. The mutant embryo is magnified 1.5 X compared to the WT. Notice that there is no evidence of growth of the embryo proper. ICM = Inner cell mass. Tr = trophectoderm.
    Variant Dhfrs, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore dhfr assays
    Phenotypes of L5Jcs5 and L5Jcs11 mutants . (A) Light micrographs of E7.5 WT and L5Jcs5/L5Jcs5 littermates taken at same magnification (8×). (B) Western blots of liver protein probed with <t>anti-TYMS,</t> <t>DHFR,</t> and beta actin. Each lane contains protein from separate animals. (C) Light micrographs of whole mount E7.5 WT and L5Jcs11/L5Jcs11 embryos, plus representative images of blastocyst outgrowths from the indicated genotypes. The mutant embryo is magnified 1.5 X compared to the WT. Notice that there is no evidence of growth of the embryo proper. ICM = Inner cell mass. Tr = trophectoderm.
    Dhfr Assays, supplied by Millipore, 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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    84
    Horizon Discovery sirna against dhfr
    MiR-24 regulates cell proliferation by regulating <t>DHFR</t> levels, independent of p53 status. (A–C) miR-24 over-expression down-regulated DHFR protein levels in three cancer cell lines independent of p53 function; in colon cancer HCT 116 (wt-p53) (A), osteosarcoma cell line U2-OS (wt-p53) (B), and HCT-116 (null-p53) cancer cell lines (C). Oligofectamine alone (oligo) and non-specific miRNA (neg) were used as the negative controls. <t>siRNA</t> specific against DHFR (siDHFR) was the positive control. (D–E) The levels of DHFR mRNA in HCT-116 and U-2 OS cells were determined by real time qRT-PCR analysis, GAPDH was used as an internal standard for normalization (data are shown as mean±SD. *, P
    Sirna Against Dhfr, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 84/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Proteintech rabbit dhfr polyclonal
    MiR-24 regulates cell proliferation by regulating <t>DHFR</t> levels, independent of p53 status. (A–C) miR-24 over-expression down-regulated DHFR protein levels in three cancer cell lines independent of p53 function; in colon cancer HCT 116 (wt-p53) (A), osteosarcoma cell line U2-OS (wt-p53) (B), and HCT-116 (null-p53) cancer cell lines (C). Oligofectamine alone (oligo) and non-specific miRNA (neg) were used as the negative controls. <t>siRNA</t> specific against DHFR (siDHFR) was the positive control. (D–E) The levels of DHFR mRNA in HCT-116 and U-2 OS cells were determined by real time qRT-PCR analysis, GAPDH was used as an internal standard for normalization (data are shown as mean±SD. *, P
    Rabbit Dhfr Polyclonal, supplied by Proteintech, 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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    dhfr  (Abcam)
    99
    Abcam dhfr
    Folate promotes oligodendrocyte maturation depending on <t>DHFR</t> activation in vitro . ( A ) Rat cortical oligodendrocyte precursors (OPCs) from pups at P2 isolated and cultured with different concentrations of folate (Ctrl: folate 4 μg/ml, FA-Low: folate 0.02 μg/ml) for 3 days stained with <t>Olig2,</t> CNP and MBP antibodies. ( B ) Quantification of the percentage of CNP + or MBP + cells among Olig2 + cells in ( A ) are shown in ( B ). Data represents the mean ± S.D. (n = 5, * p
    Dhfr, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson anti dhfr
    SMSs overexpression and 2OHOA effect on Proliferation and Differentiation in U118 cells. <t>DHFR</t> levels were determined by immunoblotting in U118 cells ( A ) overexpressing SMS1 or SMS2 (Tet-On Inducible Espression System) 72 h after doxycycline administration (1 µg/mL to induce overexpression), exposure to 2OHOA (200 µM) or both, or ( B ) transiently transfected with siRNA against SMS1 or SMS2 for 48 h, after exposure to 2OHOA (200 µM) for 24 h or both. N-Cad levels were determined by immunoblotting in U118 cells ( C ) overexpressing SMS1 or SMS2 (Tet-On Inducible Espression System) 72 h after doxycycline administration (1 µg/mL to induce overexpression), exposure to 2OHOA (200 µM) or both, or ( D ) transiently transfected with siRNA against SMS1 or SMS2 for 48 h, after exposure to 2OHOA (200 µM) for 24 h or both. The values in every lane were normalized to tubulin content and normalized values in treated cells were referred to those of control (untreated and not overexpressing) cells. SMS1 and SMS2 overexpression was measured using an anti-V5 antibody, and the downregulation after siRNA treatment was measured by RTqPCR ( F , G ). ( E ) <t>N-Cadherin</t> immunofluorescence (red, confocal microscopy) in U118 cells overexpressing SMS1 or SMS2 for 72 h. SMS1 ( F ) and SMS2 ( G ) mRNA level after 48-h treatment with a non-specific siRNA (Control), siRNA against the SMSs (siSMS1 and siSMS2) for 48 h, and SMSs overexpression (SMS1 and SMS2) for 72 h. ( H ) AXIN2 expression in U118 cells incubated with siSMS1 or siSMS2 for 48 h, overexpressing SMS1 or SMS2 (+SMS1/2), or treated with 2OHOA for 72 h, with respect to untreated cells (Control, 100%). ( I ) β-Catenin immunofluorescence (green) in U118 cells overexpressing SMS1 or SMS2 for 72 h. Nuclei were labeled with DAPI. Representative micrographies (single confocal planes) are shown. Scale bar, 5 µm. All bars represent mean ± s.e.m. values from 2–8 independent experiments; The differences were analyzed using a Student’s t -test or ANOVA; *, p
    Anti Dhfr, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dhfr  (ATCC)
    99
    ATCC dhfr
    Structure based sequence alighnment of <t>DHFR-TS</t> and stereo view of DHFR/NADPH/WR99210 complex inhibitor binding site. A. A structure based sequence alignment of the DHFR-TS enzymes from P. falciparum (P. fal), P. gallinaceum (P. gal), A. thaliana (A. tha), Medicago truncatula (M. tru), Theileria annulata (T. ann) and T. <t>gondii</t> (T. gon). The sequence numbering for the P. falciparum and T. gondii is given above and below the alignment, respectively. The secondary structure elements for P. falciparum DHFR-TS are given above the alignment with blue cylinders and red arrows representing α-helices and β-sheets, respectively. Residues which display sequence conservation across all species are highlighted by a black box with reverse type. Those residues which are involved in binding NADPH, pyrimethamine and WR99210 are highlighted by a red, blue and green box below the alignment, respectively, with those residues which bind both inhibitors and/or NADPH displayed with multiple colored boxes. B. A stereo view of the P. falciparum DHFR/NADPH/WR99210 complex inhibitor binding site with the closely related inhibitor JPC-2067-B modeled. Those residues which form close interactions with the inhibitor are labeled and shown in a stick format, colored yellow, red, blue and orange for carbon, oxygen, nitrogen and sulfur, respectively. The modeled JPC-2067-B inhibitor and NADPH cofactor are colored purple, blue, red, orange, cyan and green for carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, respectively. In addition His27, in T. gondii DHFR, which replaces Cys50 in P. falciparum DHFR, is also shown in a stick format (colored orange for carbon and blue for nitrogen) to demonstrate its close proximity to the modeled JPC-2067-B inhibitor.
    Dhfr, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher dhfr
    Phenotypes of L5Jcs5 and L5Jcs11 mutants . (A) Light micrographs of E7.5 WT and L5Jcs5/L5Jcs5 littermates taken at same magnification (8×). (B) Western blots of liver protein probed with <t>anti-TYMS,</t> <t>DHFR,</t> and beta actin. Each lane contains protein from separate animals. (C) Light micrographs of whole mount E7.5 WT and L5Jcs11/L5Jcs11 embryos, plus representative images of blastocyst outgrowths from the indicated genotypes. The mutant embryo is magnified 1.5 X compared to the WT. Notice that there is no evidence of growth of the embryo proper. ICM = Inner cell mass. Tr = trophectoderm.
    Dhfr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cosmo Genetech Co dhfr
    Effect of <t>ALDH1L1</t> over expression on induction of NADH and ATP production ( A – C ) EKVX and H23 cells were transfected with plasmid expressing ALDH1L1 for 24 h and incubated with siRNA of <t>DHFR</t> for 24 h. ( D – F ) EKVX and H23 cells were transfected with plasmid expressing DHFR for 24 h and incubated with siRNA of ALDH1L1 for 24 h. Effect of DHFR siRNA or ALDH1L1 siRNA on NADH/NAD+ (A, D) and ATP (B, E) was analysed. Immunoblot analysis was performed to confirm DHFR expression, ALDH1L1 knockdown (C, F). Data are representative of the mean and standard deviation three independent experiments. * p
    Dhfr, supplied by Cosmo Genetech Co, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Santa Cruz Biotechnology dhfr
    Effect of <t>ALDH1L1</t> over expression on induction of NADH and ATP production ( A – C ) EKVX and H23 cells were transfected with plasmid expressing ALDH1L1 for 24 h and incubated with siRNA of <t>DHFR</t> for 24 h. ( D – F ) EKVX and H23 cells were transfected with plasmid expressing DHFR for 24 h and incubated with siRNA of ALDH1L1 for 24 h. Effect of DHFR siRNA or ALDH1L1 siRNA on NADH/NAD+ (A, D) and ATP (B, E) was analysed. Immunoblot analysis was performed to confirm DHFR expression, ALDH1L1 knockdown (C, F). Data are representative of the mean and standard deviation three independent experiments. * p
    Dhfr, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Unigene dhfr
    Evidence for selective and combinatorial gene control of a subset of E2F target genes. ( A ) Schematic of E2F and/or TFE3 target promoters assayed in the ChIP assays. I: promoter sequences containing both E2F elements and E Box elements that cluster around the TSS (designated with an arrow); II: promoter sequences containing E2F elements and E Box elements that do not group around the TSS; III: E2F target promoter sequences that contain E2F elements but lack E Box elements; IV: promoter sequences that contain only E Box elements but not E2F elements. ( B ). Chromatin was immunoprecipitated with antibodies to either E2F1, E2F3, or TFE3, decrosslinked, and DNA released from the immunoprecipitates was used for PCR analysis to measure the presence of target promoter sequences as discussed above. The arrows indicate the position of the promoter PCR product. ( C ) ChIP assays for interactions of E2F1, E2F3, or TFE3 with mouse TK-1, p180, <t>DHFR,</t> and cyclin E promoters. ( D ) ChIP assays for interactions of E2F1, E2F3, or TFE3 with mouse E2F1, E2F2, and <t>p19ARF</t> promoters in vivo . ( E ) ChIP assays for interactions of E2F1, E2F3, or TFE3 with mouse SMAD7 and Tyrp1 promoters in vivo .
    Dhfr, supplied by Unigene, used in various techniques. Bioz Stars score: 86/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Addgene inc pu6 sag1 dhfr
    Evidence for selective and combinatorial gene control of a subset of E2F target genes. ( A ) Schematic of E2F and/or TFE3 target promoters assayed in the ChIP assays. I: promoter sequences containing both E2F elements and E Box elements that cluster around the TSS (designated with an arrow); II: promoter sequences containing E2F elements and E Box elements that do not group around the TSS; III: E2F target promoter sequences that contain E2F elements but lack E Box elements; IV: promoter sequences that contain only E Box elements but not E2F elements. ( B ). Chromatin was immunoprecipitated with antibodies to either E2F1, E2F3, or TFE3, decrosslinked, and DNA released from the immunoprecipitates was used for PCR analysis to measure the presence of target promoter sequences as discussed above. The arrows indicate the position of the promoter PCR product. ( C ) ChIP assays for interactions of E2F1, E2F3, or TFE3 with mouse TK-1, p180, <t>DHFR,</t> and cyclin E promoters. ( D ) ChIP assays for interactions of E2F1, E2F3, or TFE3 with mouse E2F1, E2F2, and <t>p19ARF</t> promoters in vivo . ( E ) ChIP assays for interactions of E2F1, E2F3, or TFE3 with mouse SMAD7 and Tyrp1 promoters in vivo .
    Pu6 Sag1 Dhfr, supplied by Addgene inc, 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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    91
    Horizon Discovery dhfr
    Western immunoblot analysis of p53, p21 and Bax expression in HCT-116 (wt-p53) cells (lane 1, vehicle control; lane 2, non specific control; lane 3, <t>DHFR</t> <t>siRNA</t> positive control; lane 4, 100 nM miR-192 ). α-tubulin was used as a protein loading
    Dhfr, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Parenta Pharmaceuticals dhfr
    <t>Dhfr</t> bioactivation is essential for folate-mediated CNS regeneration. Using the <t>SCRM</t> model of afferent spinal regeneration in rats, i.p. MTX was used to inhibit Dhfr, thus preventing the conversion of FA into the bioactive form tetrahydrofolate, as demonstrated in the Dhfr activity assay ( A ) ( n = 5 [uninjured]; n = 5 [injured]; n = 5 [injured and exposed to MTX]; Student’s t test; mean ± SEM; * P = 0.006), with only a small change in protein levels on Western immunoblot ( C , D ). MTX suppressed the regeneration of spinal axons into a nerve graft to below baseline levels ( B ) ( n = 10 [untreated controls]; n = 7 [FA]; n = 7 [MTX]; n = 6 [MTX and FA]). 1-way ANOVA with Bonferroni’s correction; mean ± SEM; * P
    Dhfr, supplied by Parenta Pharmaceuticals, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dhfr  (X-link)
    85
    X-link dhfr
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    FIR radiation induces tetrahydrobiopterin (BH 4 ) synthesis in vivo. Rats were exposed with or without FIR-emitting sericite board for 7 days. (a) Lung endothelial cells were isolated from control and FIR group rats using CD31 and CD45 beads. Endothelial biopterin and BH 4 levels were quantified by HPLC analysis. (b) GCH1, <t>PTS,</t> SPR, and <t>DHFR</t> protein expression in aorta tissues. β -actin is shown as a loading control. Protein levels were quantified by densitometric analysis. Data are shown as the mean ± SEM of three independent experiments. ∗ P
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    FIR radiation induces tetrahydrobiopterin (BH 4 ) synthesis in vivo. Rats were exposed with or without FIR-emitting sericite board for 7 days. (a) Lung endothelial cells were isolated from control and FIR group rats using CD31 and CD45 beads. Endothelial biopterin and BH 4 levels were quantified by HPLC analysis. (b) GCH1, <t>PTS,</t> SPR, and <t>DHFR</t> protein expression in aorta tissues. β -actin is shown as a loading control. Protein levels were quantified by densitometric analysis. Data are shown as the mean ± SEM of three independent experiments. ∗ P
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    Effect of dihydrofolate reductase ( <t>DHFR</t> ) silencing on FdUrd cytotoxicity. At 48 h after treatment of DLD-1 and HCT116 cells with control or DHFR siRNA, cells were treated with FdUrd for 72 h, and cell viability was determined. Silencing of DHFR reduced the efficacy of FdUrd at 0.1–10 μM in DLD-1 cells and at 0.1–3 μM in HCT116 cells. All data are expressed as the mean ± SD. * p
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    Effect of dihydrofolate reductase ( <t>DHFR</t> ) silencing on FdUrd cytotoxicity. At 48 h after treatment of DLD-1 and HCT116 cells with control or DHFR siRNA, cells were treated with FdUrd for 72 h, and cell viability was determined. Silencing of DHFR reduced the efficacy of FdUrd at 0.1–10 μM in DLD-1 cells and at 0.1–3 μM in HCT116 cells. All data are expressed as the mean ± SD. * p
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    Effect of dihydrofolate reductase ( <t>DHFR</t> ) silencing on FdUrd cytotoxicity. At 48 h after treatment of DLD-1 and HCT116 cells with control or DHFR siRNA, cells were treated with FdUrd for 72 h, and cell viability was determined. Silencing of DHFR reduced the efficacy of FdUrd at 0.1–10 μM in DLD-1 cells and at 0.1–3 μM in HCT116 cells. All data are expressed as the mean ± SD. * p
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    Effect of dihydrofolate reductase ( <t>DHFR</t> ) silencing on FdUrd cytotoxicity. At 48 h after treatment of DLD-1 and HCT116 cells with control or DHFR siRNA, cells were treated with FdUrd for 72 h, and cell viability was determined. Silencing of DHFR reduced the efficacy of FdUrd at 0.1–10 μM in DLD-1 cells and at 0.1–3 μM in HCT116 cells. All data are expressed as the mean ± SD. * p
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    Effect of dihydrofolate reductase ( <t>DHFR</t> ) silencing on FdUrd cytotoxicity. At 48 h after treatment of DLD-1 and HCT116 cells with control or DHFR siRNA, cells were treated with FdUrd for 72 h, and cell viability was determined. Silencing of DHFR reduced the efficacy of FdUrd at 0.1–10 μM in DLD-1 cells and at 0.1–3 μM in HCT116 cells. All data are expressed as the mean ± SD. * p
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    Zapalog can induce multiple rounds of dimerization. a, (below) HeLa cell expressing mitochondrial Tom20-mCherry-FKBP and <t>YFP-DHFR-Myc</t> incubated with 10μM CCCP for 6hrs to fragment mitochondria, then 2μM zapalog for 5min, which has caused YFP-DHFR-Myc to localize to outer mitochondrial membranes. (above) A blown-up image of the area defined below with a yellow square within which two mitochondrion-containing 2μm × 2μm ROIs (dotted white squares) were defined and imaged at high temporal resolution. In the time series to the right, one of the ROIs, (+) was periodically illuminated with a 405nm laser (~300nW) for 500msec, the other (−) was not. Each flash of blue light causes photolysis of zapalog and immediate reversal of the dimerization in ROI (+) but not (−). Each instance of YFP-DHFR-Myc removal from a mitochondrion is followed by rapid re-dimerization (~30sec) due to influx of unlysed zapalog from outside of the ROI, which outcompetes the lysed zapalog fragments bound to FKBP and DHFR. a’, schematic illustration of repeated fluorophore translocation mediated by zapalog. a”, quantification of normalized YFP signal within discrete mitochondrion-containing ROIs (2µm × 2µm) that were illuminated by 405nm laser, and nearby ROIs that were not. (n=7 cells / 3 independent repeats, center value = mean, error bars represent SE, source data in Sup. Table 1 ) b, (below) COS7 cell expressing peroxisomal PEX3-mRFP- FKBP and YFP-DHFR-Myc incubated with 2uM zapalog for 5min, which has caused YFP-DHFR-Myc to localize to peroxisomal membranes. (above) Two peroxisome-containing 6μm × 6μm ROIs were defined and imaged at high temporal resolution. In the time series to its right, one of the ROIs, (+) was illuminated with a 405nm laser (10%) for 500msec every 40sec, the other (−) was not. Each flash of blue light causes photolysis of zapalog and immediate reversal of the dimerization in ROI (+) but not (−). Each instance of YFP-DHFR-Myc removal from peroxisomes is followed by rapid re-dimerization (~30sec) due to influx of unlysed zapalog from outside of the ROI, which outcompetes the lysed zapalog fragments bound to FKBP and DHFR. b’, schematic illustration of repeated fluorophore translocation mediated by zapalog. b”, quantification of normalized YFP signal within peroxisome-containing ROIs (5µm × 5µm) that were illuminated by 405nm laser, and nearby ROIs that were not. (n=4 cells / 2 independent repeats, center value = mean, error bars represent SE, source data in Sup. Table 1 )
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    Image Search Results


    Role of the CEA splice variant in MEDI-565 mediated T cell activation and target cell killing. A, expression of full-length CEA and CEA splice variant proteins in CHO cells as determined by flow cytometry. Mouse IgG1, mouse IgG1 control antibody. B, CHO DHFR-, CHO FL, CHO SV and CHO FL+SV cells were tested for their susceptibility to be killed by CD3+ T cells from 3 individual donors in the presence of MEDI-565 at the indicated concentrations. EC 50 values listed indicate the mean value among 3 donors±standard error of the mean. p = 0.79 comparing cytotoxicity EC 50 values between CHO FL CEA and CHO FL+SV CEA cells. C, activation (increased cell surface CD25/IL-2R levels) of CD8+ T cells and D, activation of CD4+ T cells isolated from each of the 3 healthy donors was investigated concurrently with the cytotoxicity assays shown in panel B. p = 0.60 comparing CD8+ T cell activation EC 50 values between CHO FL CEA and CHO FL+SV CEA; p = 0.15 comparing CD4+ T cell activation EC 50 values between CHO FL CEA and CHO FL+SV CEA. MFI CD25-APC = mean fluorescence intensity of bound APC labeled, anti-human CD25 mAb. Experiment was repeated once with similar results.

    Journal: PLoS ONE

    Article Title: The CEA/CD3-Bispecific Antibody MEDI-565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA

    doi: 10.1371/journal.pone.0036412

    Figure Lengend Snippet: Role of the CEA splice variant in MEDI-565 mediated T cell activation and target cell killing. A, expression of full-length CEA and CEA splice variant proteins in CHO cells as determined by flow cytometry. Mouse IgG1, mouse IgG1 control antibody. B, CHO DHFR-, CHO FL, CHO SV and CHO FL+SV cells were tested for their susceptibility to be killed by CD3+ T cells from 3 individual donors in the presence of MEDI-565 at the indicated concentrations. EC 50 values listed indicate the mean value among 3 donors±standard error of the mean. p = 0.79 comparing cytotoxicity EC 50 values between CHO FL CEA and CHO FL+SV CEA cells. C, activation (increased cell surface CD25/IL-2R levels) of CD8+ T cells and D, activation of CD4+ T cells isolated from each of the 3 healthy donors was investigated concurrently with the cytotoxicity assays shown in panel B. p = 0.60 comparing CD8+ T cell activation EC 50 values between CHO FL CEA and CHO FL+SV CEA; p = 0.15 comparing CD4+ T cell activation EC 50 values between CHO FL CEA and CHO FL+SV CEA. MFI CD25-APC = mean fluorescence intensity of bound APC labeled, anti-human CD25 mAb. Experiment was repeated once with similar results.

    Article Snippet: CHO cells expressing the CEA splice variant (CHO SV CEA) were created by infection of CHO DHFR- cells with the pCDH1-HCS1-EF1-Puro-CEA splice variant lentiviral vector, and selected by culturing in medium containing 5 µg/mL puromycin (EMD Chemicals Group, Merck KGaA, Darmstadt, Germany) for 72 hours.

    Techniques: Variant Assay, Activation Assay, Expressing, Flow Cytometry, Cytometry, Isolation, Fluorescence, Labeling

    The drug-stabilizable Gal4 variants 1xDHFR22 and 2xDHFR22 function in vivo in the format of the widely used Gal4- UAS bipartite expression system for Drosophila . (a) Schematic representation of the constructs used to create drug-inducible Gal4 driver lines. 1xDHFR22 encodes the single DHFR-DD architecture with the DHFR variant 22, 28 as a fusion to the Gal4VP16 transcription factor. A nuclear localization signal (NLS) is added N-terminally and expression is driven by the eye-specific enhancer, glass multiple reporter ( GMR ). Similarly, 2xDHFR22 encodes the double architecture of DHFR22-DD. (b) A population of F 1 progenies from 1xDHFR22 and 2xDHFR22 genetic crosses with UAS-eGFP reporter line was allowed to feed on standard fly food supplemented with DMSO (mock-treatment) or various concentrations of TMP for 5-days. A negative control population was derived from the Curly wings phenotype resulting from a dominant CyO marker from the heterozygote 1xDHFR or 2xDHFR driver line. Samples of the population were imaged by fluorescence microscopy. Representative images of adult fly eyes display an increase in eGFP fluorescence intensity as a function of the inducer TMP. The upper, middle and lower panels display, respectively, F 1 progenies with genotypes 1xDHFR22;UAS-eGFP, 2xDHFR22;UAS-eGFP and CyO;UAS-eGFP . Scale bar: 1 mm. (c) Quantification of eGFP fluorescence intensity in the Drosophila adult eyes either mock-treated with DMSO or with various concentrations of TMP. Data are presented as the mean fluorescence detected per eye. The statistical significance resulting from a one-way ANOVA and Tukey’s post hoc test is summarized with asterisk marks representing the level of significance (n.s.= P -value > 0.05, * = P -value ≤ 0.05, *** = P -value ≤ 0. 001, and **** = P -value ≤ 0.0001) on the indicated data set. The error bars represent the standard deviation over the mean across the biological replicates ( n = 8–76 individual eyes per dose).

    Journal: ACS Synthetic Biology

    Article Title: Drug-Inducible Control of Lethality Genes: A Low Background Destabilizing Domain Architecture Applied to the Gal4-UAS System in Drosophila

    doi: 10.1021/acssynbio.7b00302

    Figure Lengend Snippet: The drug-stabilizable Gal4 variants 1xDHFR22 and 2xDHFR22 function in vivo in the format of the widely used Gal4- UAS bipartite expression system for Drosophila . (a) Schematic representation of the constructs used to create drug-inducible Gal4 driver lines. 1xDHFR22 encodes the single DHFR-DD architecture with the DHFR variant 22, 28 as a fusion to the Gal4VP16 transcription factor. A nuclear localization signal (NLS) is added N-terminally and expression is driven by the eye-specific enhancer, glass multiple reporter ( GMR ). Similarly, 2xDHFR22 encodes the double architecture of DHFR22-DD. (b) A population of F 1 progenies from 1xDHFR22 and 2xDHFR22 genetic crosses with UAS-eGFP reporter line was allowed to feed on standard fly food supplemented with DMSO (mock-treatment) or various concentrations of TMP for 5-days. A negative control population was derived from the Curly wings phenotype resulting from a dominant CyO marker from the heterozygote 1xDHFR or 2xDHFR driver line. Samples of the population were imaged by fluorescence microscopy. Representative images of adult fly eyes display an increase in eGFP fluorescence intensity as a function of the inducer TMP. The upper, middle and lower panels display, respectively, F 1 progenies with genotypes 1xDHFR22;UAS-eGFP, 2xDHFR22;UAS-eGFP and CyO;UAS-eGFP . Scale bar: 1 mm. (c) Quantification of eGFP fluorescence intensity in the Drosophila adult eyes either mock-treated with DMSO or with various concentrations of TMP. Data are presented as the mean fluorescence detected per eye. The statistical significance resulting from a one-way ANOVA and Tukey’s post hoc test is summarized with asterisk marks representing the level of significance (n.s.= P -value > 0.05, * = P -value ≤ 0.05, *** = P -value ≤ 0. 001, and **** = P -value ≤ 0.0001) on the indicated data set. The error bars represent the standard deviation over the mean across the biological replicates ( n = 8–76 individual eyes per dose).

    Article Snippet: DNA Constructs The DHFR- and FKBP12-based destabilization domains used in this study were originally developed by the Wandless lab., , The following DD variants were used in this study: DHFR (Addgene plasmid #29325), DHFR07 (#47080), DHFR22 (#47076) and FKBP (#31763).

    Techniques: In Vivo, Expressing, Construct, Variant Assay, Negative Control, Derivative Assay, Marker, Fluorescence, Microscopy, Standard Deviation

    Phenotypes of L5Jcs5 and L5Jcs11 mutants . (A) Light micrographs of E7.5 WT and L5Jcs5/L5Jcs5 littermates taken at same magnification (8×). (B) Western blots of liver protein probed with anti-TYMS, DHFR, and beta actin. Each lane contains protein from separate animals. (C) Light micrographs of whole mount E7.5 WT and L5Jcs11/L5Jcs11 embryos, plus representative images of blastocyst outgrowths from the indicated genotypes. The mutant embryo is magnified 1.5 X compared to the WT. Notice that there is no evidence of growth of the embryo proper. ICM = Inner cell mass. Tr = trophectoderm.

    Journal: BMC Genetics

    Article Title: High resolution mapping and positional cloning of ENU-induced mutations in the Rw region of mouse chromosome 5

    doi: 10.1186/1471-2156-11-106

    Figure Lengend Snippet: Phenotypes of L5Jcs5 and L5Jcs11 mutants . (A) Light micrographs of E7.5 WT and L5Jcs5/L5Jcs5 littermates taken at same magnification (8×). (B) Western blots of liver protein probed with anti-TYMS, DHFR, and beta actin. Each lane contains protein from separate animals. (C) Light micrographs of whole mount E7.5 WT and L5Jcs11/L5Jcs11 embryos, plus representative images of blastocyst outgrowths from the indicated genotypes. The mutant embryo is magnified 1.5 X compared to the WT. Notice that there is no evidence of growth of the embryo proper. ICM = Inner cell mass. Tr = trophectoderm.

    Article Snippet: The membrane was incubated for two hours at room temperature in one of two primary antibodies: 1:1000 -anti-TYMS (Zymed) or 1:2000 anti-DHFR (Sigma).

    Techniques: Western Blot, Mutagenesis

    MiR-24 regulates cell proliferation by regulating DHFR levels, independent of p53 status. (A–C) miR-24 over-expression down-regulated DHFR protein levels in three cancer cell lines independent of p53 function; in colon cancer HCT 116 (wt-p53) (A), osteosarcoma cell line U2-OS (wt-p53) (B), and HCT-116 (null-p53) cancer cell lines (C). Oligofectamine alone (oligo) and non-specific miRNA (neg) were used as the negative controls. siRNA specific against DHFR (siDHFR) was the positive control. (D–E) The levels of DHFR mRNA in HCT-116 and U-2 OS cells were determined by real time qRT-PCR analysis, GAPDH was used as an internal standard for normalization (data are shown as mean±SD. *, P

    Journal: PLoS ONE

    Article Title: MiR-24 Tumor Suppressor Activity Is Regulated Independent of p53 and through a Target Site Polymorphism

    doi: 10.1371/journal.pone.0008445

    Figure Lengend Snippet: MiR-24 regulates cell proliferation by regulating DHFR levels, independent of p53 status. (A–C) miR-24 over-expression down-regulated DHFR protein levels in three cancer cell lines independent of p53 function; in colon cancer HCT 116 (wt-p53) (A), osteosarcoma cell line U2-OS (wt-p53) (B), and HCT-116 (null-p53) cancer cell lines (C). Oligofectamine alone (oligo) and non-specific miRNA (neg) were used as the negative controls. siRNA specific against DHFR (siDHFR) was the positive control. (D–E) The levels of DHFR mRNA in HCT-116 and U-2 OS cells were determined by real time qRT-PCR analysis, GAPDH was used as an internal standard for normalization (data are shown as mean±SD. *, P

    Article Snippet: Transfections of miR-24 and siRNAs RKO, HT-29, U2-OS, MG63, HCT-116 (wt-p53) and HCT-116 (null-p53) cells (2×105 ) were plated in six-well plates and transfected with 100 nM of either miR-24 or non-specific miRNA (Ambion) after 24 h by Oligofectamine (Invitrogen) according to the manufacturer's protocols. siRNA against DHFR was purchased from Dharmacon and transfected with Oligofectamine (Invitrogen) at a final concentration of 100 nM.

    Techniques: Over Expression, Positive Control, Quantitative RT-PCR

    Overexpression of miR-24 down regulated DHFR expression, reduced anchorage-dependent growth, and induced morphological changes resembling differentiation. (A) DHFR protein levels were down regulated upon over-expression of DHFR specific siRNA (siDHFR) (by three fold) and miR-24 (by six fold). (B) miR-24 over expression conferred morphological changes that resembled differentiated in a colorectal cancer cell line (HCT-116-wt-p53). The differentiated-like morphological changes were not observed in HCT-116 cells transfected with siDHFR and oligofectamine alone (Cont). (C) Due to a lack of well established differentiation markers, the morphological changes in miR-24 transfected cells were quantitated by light microscopy. Although miR-24 transfection resulted in approximately 70% reduction in cell proliferation, approximately 12% of the total transfected cells, and 38% of total surviving cells, showed a differentiation-like phenotype. (D) Of interest, siRNA specific to DHFR that down regulated DHFR levels in the cells by threefold also reduced anchorage-dependent growth of the HCT-116 colorectal cancer cell line, suggesting that DHFR levels in the cell are associated with cell proliferation (data are shown as mean±SD. *, P

    Journal: PLoS ONE

    Article Title: MiR-24 Tumor Suppressor Activity Is Regulated Independent of p53 and through a Target Site Polymorphism

    doi: 10.1371/journal.pone.0008445

    Figure Lengend Snippet: Overexpression of miR-24 down regulated DHFR expression, reduced anchorage-dependent growth, and induced morphological changes resembling differentiation. (A) DHFR protein levels were down regulated upon over-expression of DHFR specific siRNA (siDHFR) (by three fold) and miR-24 (by six fold). (B) miR-24 over expression conferred morphological changes that resembled differentiated in a colorectal cancer cell line (HCT-116-wt-p53). The differentiated-like morphological changes were not observed in HCT-116 cells transfected with siDHFR and oligofectamine alone (Cont). (C) Due to a lack of well established differentiation markers, the morphological changes in miR-24 transfected cells were quantitated by light microscopy. Although miR-24 transfection resulted in approximately 70% reduction in cell proliferation, approximately 12% of the total transfected cells, and 38% of total surviving cells, showed a differentiation-like phenotype. (D) Of interest, siRNA specific to DHFR that down regulated DHFR levels in the cells by threefold also reduced anchorage-dependent growth of the HCT-116 colorectal cancer cell line, suggesting that DHFR levels in the cell are associated with cell proliferation (data are shown as mean±SD. *, P

    Article Snippet: Transfections of miR-24 and siRNAs RKO, HT-29, U2-OS, MG63, HCT-116 (wt-p53) and HCT-116 (null-p53) cells (2×105 ) were plated in six-well plates and transfected with 100 nM of either miR-24 or non-specific miRNA (Ambion) after 24 h by Oligofectamine (Invitrogen) according to the manufacturer's protocols. siRNA against DHFR was purchased from Dharmacon and transfected with Oligofectamine (Invitrogen) at a final concentration of 100 nM.

    Techniques: Over Expression, Expressing, Transfection, Light Microscopy

    A loss-of-function miR-24 target site SNP contributes to cellular transformation. (A) The miR-24-TS-SNP (829C→T) expressing CHO-DG44 cells over-expressed DHFR [22] . (B–C) DHFR over expression in CHO-DG44 cells due a loss of miR-24 function results in an increase in colony forming ability and (D) show anchorage-independent growth in semi solid agar as compared to vector alone expressing cells (p-value > 0.05) (Soft agar colony-forming assay is abbreviated as SAC). (E) Transfection of a siRNA specific to DHFR reduced the ability of miR-24-TS-SNP expressing cells to form soft agar colonies by threefold. (F-H) miR-24-TS-SNP expression in a human cell line - MCF10A (G) and two rodent cell lines - NIH3T3 (H) and RK3 (see Table S1 ) resulted in more soft agar colony formation as compared to the cells expressing the vector alone and DHFR with wt 3′UTR. (I) DHFR overexpression due to the miR-24-TS-SNP renders NIH3T3 cells tumorigenic when transplanted in nude mice.

    Journal: PLoS ONE

    Article Title: MiR-24 Tumor Suppressor Activity Is Regulated Independent of p53 and through a Target Site Polymorphism

    doi: 10.1371/journal.pone.0008445

    Figure Lengend Snippet: A loss-of-function miR-24 target site SNP contributes to cellular transformation. (A) The miR-24-TS-SNP (829C→T) expressing CHO-DG44 cells over-expressed DHFR [22] . (B–C) DHFR over expression in CHO-DG44 cells due a loss of miR-24 function results in an increase in colony forming ability and (D) show anchorage-independent growth in semi solid agar as compared to vector alone expressing cells (p-value > 0.05) (Soft agar colony-forming assay is abbreviated as SAC). (E) Transfection of a siRNA specific to DHFR reduced the ability of miR-24-TS-SNP expressing cells to form soft agar colonies by threefold. (F-H) miR-24-TS-SNP expression in a human cell line - MCF10A (G) and two rodent cell lines - NIH3T3 (H) and RK3 (see Table S1 ) resulted in more soft agar colony formation as compared to the cells expressing the vector alone and DHFR with wt 3′UTR. (I) DHFR overexpression due to the miR-24-TS-SNP renders NIH3T3 cells tumorigenic when transplanted in nude mice.

    Article Snippet: Transfections of miR-24 and siRNAs RKO, HT-29, U2-OS, MG63, HCT-116 (wt-p53) and HCT-116 (null-p53) cells (2×105 ) were plated in six-well plates and transfected with 100 nM of either miR-24 or non-specific miRNA (Ambion) after 24 h by Oligofectamine (Invitrogen) according to the manufacturer's protocols. siRNA against DHFR was purchased from Dharmacon and transfected with Oligofectamine (Invitrogen) at a final concentration of 100 nM.

    Techniques: Transformation Assay, Expressing, Over Expression, Plasmid Preparation, Significance Assay, Transfection, Mouse Assay

    Folate promotes oligodendrocyte maturation depending on DHFR activation in vitro . ( A ) Rat cortical oligodendrocyte precursors (OPCs) from pups at P2 isolated and cultured with different concentrations of folate (Ctrl: folate 4 μg/ml, FA-Low: folate 0.02 μg/ml) for 3 days stained with Olig2, CNP and MBP antibodies. ( B ) Quantification of the percentage of CNP + or MBP + cells among Olig2 + cells in ( A ) are shown in ( B ). Data represents the mean ± S.D. (n = 5, * p

    Journal: Scientific Reports

    Article Title: Folate Metabolism Regulates Oligodendrocyte Survival and Differentiation by Modulating AMPKα Activity

    doi: 10.1038/s41598-017-01732-1

    Figure Lengend Snippet: Folate promotes oligodendrocyte maturation depending on DHFR activation in vitro . ( A ) Rat cortical oligodendrocyte precursors (OPCs) from pups at P2 isolated and cultured with different concentrations of folate (Ctrl: folate 4 μg/ml, FA-Low: folate 0.02 μg/ml) for 3 days stained with Olig2, CNP and MBP antibodies. ( B ) Quantification of the percentage of CNP + or MBP + cells among Olig2 + cells in ( A ) are shown in ( B ). Data represents the mean ± S.D. (n = 5, * p

    Article Snippet: The primary antibodies were as follows: DHFR (Abcam ab85056, 1:500), Olig2 (Millipore Ab9610, 1:5000), CC1 (Calbiochem OP80, 1:50), MBP (Covance SMI-94R, 1:5000), PDGFRα (BD Bioscience 558774, 1:500), Sox10 (R & D systems NL2864R, 1:200), PLP (Millipore Mab388, 1:2000), NeuN (Millipore Mab377, 1:1000), cleaved-Caspase 3 (Cell signal 9661L, 1:1000), BrdU (BD pharmingen 555627, 1:200).

    Techniques: Activation Assay, In Vitro, Isolation, Cell Culture, Staining

    AMPK activity is involved in DHFR regulation of oligodendrocyte development. ( A ) The spinal cord of wild type mice at P8 was co-labeled for p-AMPKα with PDGFRα and CC1. Arrows indicate co-labeled cells. ( B ) qRT-PCR analysis of Prkaa1 , Olig2 , Mbp , Cnp and Myrf genes expression in Oli-neu cells transfected with AMPKα1 and control plasmids for 48 h. Data represents the mean ± S.D. (n > 3, *p

    Journal: Scientific Reports

    Article Title: Folate Metabolism Regulates Oligodendrocyte Survival and Differentiation by Modulating AMPKα Activity

    doi: 10.1038/s41598-017-01732-1

    Figure Lengend Snippet: AMPK activity is involved in DHFR regulation of oligodendrocyte development. ( A ) The spinal cord of wild type mice at P8 was co-labeled for p-AMPKα with PDGFRα and CC1. Arrows indicate co-labeled cells. ( B ) qRT-PCR analysis of Prkaa1 , Olig2 , Mbp , Cnp and Myrf genes expression in Oli-neu cells transfected with AMPKα1 and control plasmids for 48 h. Data represents the mean ± S.D. (n > 3, *p

    Article Snippet: The primary antibodies were as follows: DHFR (Abcam ab85056, 1:500), Olig2 (Millipore Ab9610, 1:5000), CC1 (Calbiochem OP80, 1:50), MBP (Covance SMI-94R, 1:5000), PDGFRα (BD Bioscience 558774, 1:500), Sox10 (R & D systems NL2864R, 1:200), PLP (Millipore Mab388, 1:2000), NeuN (Millipore Mab377, 1:1000), cleaved-Caspase 3 (Cell signal 9661L, 1:1000), BrdU (BD pharmingen 555627, 1:200).

    Techniques: Activity Assay, Mouse Assay, Labeling, Quantitative RT-PCR, Expressing, Transfection

    DHFR inhibition causes oligodendrocyte death and differentiation restrain. ( A ) Immunostaining of Olig2, CC1 and PDGFRα antibodies on spinal cord (left) and corpus callosum of brain (right) from control and MTX (2 mg/kg, 4 mg/kg)-treated mice at P8. ( B ) Quantification of the proportion of CC1+ or PDGFRα+ cells among Olig2+ cells in both spinal cord and brain of control and MTX (2 mg/kg, 4 mg/kg)-treated mice at P8 in ( A ). Data represents the mean ± S.D. (n = 5, * p

    Journal: Scientific Reports

    Article Title: Folate Metabolism Regulates Oligodendrocyte Survival and Differentiation by Modulating AMPKα Activity

    doi: 10.1038/s41598-017-01732-1

    Figure Lengend Snippet: DHFR inhibition causes oligodendrocyte death and differentiation restrain. ( A ) Immunostaining of Olig2, CC1 and PDGFRα antibodies on spinal cord (left) and corpus callosum of brain (right) from control and MTX (2 mg/kg, 4 mg/kg)-treated mice at P8. ( B ) Quantification of the proportion of CC1+ or PDGFRα+ cells among Olig2+ cells in both spinal cord and brain of control and MTX (2 mg/kg, 4 mg/kg)-treated mice at P8 in ( A ). Data represents the mean ± S.D. (n = 5, * p

    Article Snippet: The primary antibodies were as follows: DHFR (Abcam ab85056, 1:500), Olig2 (Millipore Ab9610, 1:5000), CC1 (Calbiochem OP80, 1:50), MBP (Covance SMI-94R, 1:5000), PDGFRα (BD Bioscience 558774, 1:500), Sox10 (R & D systems NL2864R, 1:200), PLP (Millipore Mab388, 1:2000), NeuN (Millipore Mab377, 1:1000), cleaved-Caspase 3 (Cell signal 9661L, 1:1000), BrdU (BD pharmingen 555627, 1:200).

    Techniques: Inhibition, Immunostaining, Mouse Assay

    Methotrexate treatment selection for MSH2 deficient cells is via inhibition of folate synthesis Western blot analysis of DHFR siRNA silencing. Cells were transfected with either control siRNA or two different DHFR siRNAs. Protein lysates were immunoblotted and probed for DHFR and β-tubulin (loading control). Silencing of DHFR is selective for MSH2 deficient cells. Survival bar chart is shown of Hec59 + Chr2 and Hec59 transfected with siRNA oligonucleotides targeting DHFR. * p ≤ 0.0001 compared to the similarly transfected MSH2 proficient Hec59 + Chr2 cells (Student's t -test). Error bars represent standard errors of the mean. Folic acid rescues methotrexate selectivity in MSH2 deficient cells. Survival curves are shown of Hec59 and Hec59 + Chr2 cells under continuous exposure to a range of concentrations of methotrexate ± 100 µM folic acid for 14 days. Error bars represent standard errors of the mean. DHFR silencing causes an accumulation of 8-OHdG accumulation. Hec59 and Hec59 + Chr2 cells were transfected with control or DHFR siRNA and DNA analysed for 8-OHdG content as before. A bar chart showing relative levels of 8-OHdG in each cell line is shown. Assays were performed in triplicate. * p ≤ 0.0003 compared to the similarly transfected MSH2 proficient Hec59 + Chr2 cells (Student's t -test). Error bars represent standard errors of the mean.

    Journal: EMBO Molecular Medicine

    Article Title: Methotrexate induces oxidative DNA damage and is selectively lethal to tumour cells with defects in the DNA mismatch repair gene MSH2

    doi: 10.1002/emmm.200900040

    Figure Lengend Snippet: Methotrexate treatment selection for MSH2 deficient cells is via inhibition of folate synthesis Western blot analysis of DHFR siRNA silencing. Cells were transfected with either control siRNA or two different DHFR siRNAs. Protein lysates were immunoblotted and probed for DHFR and β-tubulin (loading control). Silencing of DHFR is selective for MSH2 deficient cells. Survival bar chart is shown of Hec59 + Chr2 and Hec59 transfected with siRNA oligonucleotides targeting DHFR. * p ≤ 0.0001 compared to the similarly transfected MSH2 proficient Hec59 + Chr2 cells (Student's t -test). Error bars represent standard errors of the mean. Folic acid rescues methotrexate selectivity in MSH2 deficient cells. Survival curves are shown of Hec59 and Hec59 + Chr2 cells under continuous exposure to a range of concentrations of methotrexate ± 100 µM folic acid for 14 days. Error bars represent standard errors of the mean. DHFR silencing causes an accumulation of 8-OHdG accumulation. Hec59 and Hec59 + Chr2 cells were transfected with control or DHFR siRNA and DNA analysed for 8-OHdG content as before. A bar chart showing relative levels of 8-OHdG in each cell line is shown. Assays were performed in triplicate. * p ≤ 0.0003 compared to the similarly transfected MSH2 proficient Hec59 + Chr2 cells (Student's t -test). Error bars represent standard errors of the mean.

    Article Snippet: Lysates were electrophoresed on Novex precast gels (Invitrogen, UK) and immunoblotted using the following antibodies: anti-MSH2 (Ab-1, Calbiochem, UK), anti-DHFR (ab49881, Abcam, UK), anti-PARP (Cell Signalling), anti-MYH (ab55551, Abcam), anti-OGG1 (Novus), anti-MTH1 (ab98–230, Abcam) and anti-β-tubulin (T4026, Sigma, UK).

    Techniques: Selection, Inhibition, Western Blot, Transfection

    Upregulation of DHFR and MDR1 expression in OS-TICs. (A) Total RNA was purified from parental and OS-TICs, and the elevated expression of DHFR and MDR1 genes in derived OS-TICs was detected by quantitative reverse transcription-polymerase chain reaction analysis. (B) Total proteins were prepared from parental or OS-TICs cells and analyzed by immunoblotting with anti-DHFR, anti-MDR1 or anti-GAPDH antibodies as indicated. The amount of GAPDH protein of different crude cell extracts was referred as loading control. (C) Parental and enriched OS-TICs from U2OS cells were stained with anti-MDR1 to detect the intracellular level of MDR1 proteins by immunofluorescence analysis. *P

    Journal: Oncology Letters

    Article Title: DHFR and MDR1 upregulation is associated with chemoresistance in osteosarcoma stem-like cells

    doi: 10.3892/ol.2017.6132

    Figure Lengend Snippet: Upregulation of DHFR and MDR1 expression in OS-TICs. (A) Total RNA was purified from parental and OS-TICs, and the elevated expression of DHFR and MDR1 genes in derived OS-TICs was detected by quantitative reverse transcription-polymerase chain reaction analysis. (B) Total proteins were prepared from parental or OS-TICs cells and analyzed by immunoblotting with anti-DHFR, anti-MDR1 or anti-GAPDH antibodies as indicated. The amount of GAPDH protein of different crude cell extracts was referred as loading control. (C) Parental and enriched OS-TICs from U2OS cells were stained with anti-MDR1 to detect the intracellular level of MDR1 proteins by immunofluorescence analysis. *P

    Article Snippet: The following primary antibodies were used: Anti-Oct-4 antibody (1:1,000; #2750; Cell Signaling Technology, Inc.), anti-Nanog antibody (1:500; #3850; Cell Signaling Technology, Inc.), anti-Nestin antibody (1:1,000; MAB5326; EMD Millipore), anti-GAPDH antibody (1:2,000; GTX627408; GeneTex, Inc., Irvine, CA, USA), anti-DHFR antibody (1:1,000; ab49881; Abcam, Cambridge, UK) and anti-MDR1 antibody (1:1,000; #13978; Cell Signaling Technology, Inc.).

    Techniques: Expressing, Purification, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Immunofluorescence

    SMSs overexpression and 2OHOA effect on Proliferation and Differentiation in U118 cells. DHFR levels were determined by immunoblotting in U118 cells ( A ) overexpressing SMS1 or SMS2 (Tet-On Inducible Espression System) 72 h after doxycycline administration (1 µg/mL to induce overexpression), exposure to 2OHOA (200 µM) or both, or ( B ) transiently transfected with siRNA against SMS1 or SMS2 for 48 h, after exposure to 2OHOA (200 µM) for 24 h or both. N-Cad levels were determined by immunoblotting in U118 cells ( C ) overexpressing SMS1 or SMS2 (Tet-On Inducible Espression System) 72 h after doxycycline administration (1 µg/mL to induce overexpression), exposure to 2OHOA (200 µM) or both, or ( D ) transiently transfected with siRNA against SMS1 or SMS2 for 48 h, after exposure to 2OHOA (200 µM) for 24 h or both. The values in every lane were normalized to tubulin content and normalized values in treated cells were referred to those of control (untreated and not overexpressing) cells. SMS1 and SMS2 overexpression was measured using an anti-V5 antibody, and the downregulation after siRNA treatment was measured by RTqPCR ( F , G ). ( E ) N-Cadherin immunofluorescence (red, confocal microscopy) in U118 cells overexpressing SMS1 or SMS2 for 72 h. SMS1 ( F ) and SMS2 ( G ) mRNA level after 48-h treatment with a non-specific siRNA (Control), siRNA against the SMSs (siSMS1 and siSMS2) for 48 h, and SMSs overexpression (SMS1 and SMS2) for 72 h. ( H ) AXIN2 expression in U118 cells incubated with siSMS1 or siSMS2 for 48 h, overexpressing SMS1 or SMS2 (+SMS1/2), or treated with 2OHOA for 72 h, with respect to untreated cells (Control, 100%). ( I ) β-Catenin immunofluorescence (green) in U118 cells overexpressing SMS1 or SMS2 for 72 h. Nuclei were labeled with DAPI. Representative micrographies (single confocal planes) are shown. Scale bar, 5 µm. All bars represent mean ± s.e.m. values from 2–8 independent experiments; The differences were analyzed using a Student’s t -test or ANOVA; *, p

    Journal: Cancers

    Article Title: The Opposing Contribution of SMS1 and SMS2 to Glioma Progression and Their Value in the Therapeutic Response to 2OHOA

    doi: 10.3390/cancers11010088

    Figure Lengend Snippet: SMSs overexpression and 2OHOA effect on Proliferation and Differentiation in U118 cells. DHFR levels were determined by immunoblotting in U118 cells ( A ) overexpressing SMS1 or SMS2 (Tet-On Inducible Espression System) 72 h after doxycycline administration (1 µg/mL to induce overexpression), exposure to 2OHOA (200 µM) or both, or ( B ) transiently transfected with siRNA against SMS1 or SMS2 for 48 h, after exposure to 2OHOA (200 µM) for 24 h or both. N-Cad levels were determined by immunoblotting in U118 cells ( C ) overexpressing SMS1 or SMS2 (Tet-On Inducible Espression System) 72 h after doxycycline administration (1 µg/mL to induce overexpression), exposure to 2OHOA (200 µM) or both, or ( D ) transiently transfected with siRNA against SMS1 or SMS2 for 48 h, after exposure to 2OHOA (200 µM) for 24 h or both. The values in every lane were normalized to tubulin content and normalized values in treated cells were referred to those of control (untreated and not overexpressing) cells. SMS1 and SMS2 overexpression was measured using an anti-V5 antibody, and the downregulation after siRNA treatment was measured by RTqPCR ( F , G ). ( E ) N-Cadherin immunofluorescence (red, confocal microscopy) in U118 cells overexpressing SMS1 or SMS2 for 72 h. SMS1 ( F ) and SMS2 ( G ) mRNA level after 48-h treatment with a non-specific siRNA (Control), siRNA against the SMSs (siSMS1 and siSMS2) for 48 h, and SMSs overexpression (SMS1 and SMS2) for 72 h. ( H ) AXIN2 expression in U118 cells incubated with siSMS1 or siSMS2 for 48 h, overexpressing SMS1 or SMS2 (+SMS1/2), or treated with 2OHOA for 72 h, with respect to untreated cells (Control, 100%). ( I ) β-Catenin immunofluorescence (green) in U118 cells overexpressing SMS1 or SMS2 for 72 h. Nuclei were labeled with DAPI. Representative micrographies (single confocal planes) are shown. Scale bar, 5 µm. All bars represent mean ± s.e.m. values from 2–8 independent experiments; The differences were analyzed using a Student’s t -test or ANOVA; *, p

    Article Snippet: The membranes were incubated overnight with primary antibodies diluted in fresh blocking solution: rabbit anti-SMS1 1:5000 or rabbit anti-SMS2 1:5000 from the above-described sera (ProteoGenix, Schiltigheim, France); mouse anti-N-Cadherin, anti-β-Catenin and anti-DHFR diluted 1:1000 (BD- Bioscience, Madrid, Spain); rabbit anti-LC3 and mouse anti-eIF2α diluted 1:1000 (Cell Signalling, Leiden, The Netherlands); mouse anti-V5 tag was diluted 1:5000 (Thermo Scientific, Barcelona, Spain).

    Techniques: Over Expression, Transfection, Immunofluorescence, Confocal Microscopy, Expressing, Incubation, Labeling

    Decreased dihydrofolate reductase (DHFR) protein expression in Bmal1-knockout (KO) endothelial cells Expression levels of GTP cyclochydrolase-1 (GTPCH-1) (first step limiting enzyme in de novo synthesis of tetrahydrobiopterin [BH4]) and DHFR (main enzyme used by endothelial cells in dihydrobiopterin [BH2] to tetrahydrobiopterin [BH4] recycling pathway) were analyzed by immunoblotting in mouse endothelial cell lysates. Western blotting revealed no significant change in GTPCH-1 levels ( A ) and a significant reduction in DHFR protein levels ( B ) in endothelial cells isolated from Bmal1-KO mouse aorta (each band shown is an individual endothelial cell culture plate from the representative n=5 per group; * P

    Journal: Circulation research

    Article Title: Increased Superoxide and Endothelial NO Synthase Uncoupling in Blood Vessels of Bmal1-Knockout Mice

    doi: 10.1161/CIRCRESAHA.111.261750

    Figure Lengend Snippet: Decreased dihydrofolate reductase (DHFR) protein expression in Bmal1-knockout (KO) endothelial cells Expression levels of GTP cyclochydrolase-1 (GTPCH-1) (first step limiting enzyme in de novo synthesis of tetrahydrobiopterin [BH4]) and DHFR (main enzyme used by endothelial cells in dihydrobiopterin [BH2] to tetrahydrobiopterin [BH4] recycling pathway) were analyzed by immunoblotting in mouse endothelial cell lysates. Western blotting revealed no significant change in GTPCH-1 levels ( A ) and a significant reduction in DHFR protein levels ( B ) in endothelial cells isolated from Bmal1-KO mouse aorta (each band shown is an individual endothelial cell culture plate from the representative n=5 per group; * P

    Article Snippet: DHFR (BD biosciences) and GTPCH-1 (Novus Biologicals) protein expression was detected with rabbit anti-mouse polyclonal antibodies, followed by enhanced chemiluminescence (enhanced chemiluminescence kit, Amersham).

    Techniques: Expressing, Knock-Out, Western Blot, Isolation, Cell Culture

    Dihydrofolate reductase (DHFR) and GTP cyclochydrolase-1 (GTPCH-1) exhibit a circadian rhythm that is abolished in vasculature of Bmal1-knockout (KO) mice GTPCH-1 and DHFR protein level expression oscillates in mouse aorta samples in wild-type (WT) animals with GTPCH-1 ( A ) peaking at 11 AM (Zeitgeber time [ZT] 4), whereas DHFR ( B ) peaks at 7 PM (ZT12) (n=3/group). In Bmal1-KO aortic samples, GTPCH-1 ( C ) and DHFR ( D ) protein oscillation was abolished (equal amounts of protein were loaded for WT and Bmal1-KO samples). E At the end of serum shock (ZT0), cells were lyzed beginning at ZT2 and at 6-hour intervals thereafter for 24 hours. Lysates were then immunoblotted for DHFR. DHFR protein expression displays a 24-hour circadian rhythm peaking at ZT14 as quantified by densitometry ( right , n=5 per time point; * P

    Journal: Circulation research

    Article Title: Increased Superoxide and Endothelial NO Synthase Uncoupling in Blood Vessels of Bmal1-Knockout Mice

    doi: 10.1161/CIRCRESAHA.111.261750

    Figure Lengend Snippet: Dihydrofolate reductase (DHFR) and GTP cyclochydrolase-1 (GTPCH-1) exhibit a circadian rhythm that is abolished in vasculature of Bmal1-knockout (KO) mice GTPCH-1 and DHFR protein level expression oscillates in mouse aorta samples in wild-type (WT) animals with GTPCH-1 ( A ) peaking at 11 AM (Zeitgeber time [ZT] 4), whereas DHFR ( B ) peaks at 7 PM (ZT12) (n=3/group). In Bmal1-KO aortic samples, GTPCH-1 ( C ) and DHFR ( D ) protein oscillation was abolished (equal amounts of protein were loaded for WT and Bmal1-KO samples). E At the end of serum shock (ZT0), cells were lyzed beginning at ZT2 and at 6-hour intervals thereafter for 24 hours. Lysates were then immunoblotted for DHFR. DHFR protein expression displays a 24-hour circadian rhythm peaking at ZT14 as quantified by densitometry ( right , n=5 per time point; * P

    Article Snippet: DHFR (BD biosciences) and GTPCH-1 (Novus Biologicals) protein expression was detected with rabbit anti-mouse polyclonal antibodies, followed by enhanced chemiluminescence (enhanced chemiluminescence kit, Amersham).

    Techniques: Knock-Out, Mouse Assay, Expressing

    Structure based sequence alighnment of DHFR-TS and stereo view of DHFR/NADPH/WR99210 complex inhibitor binding site. A. A structure based sequence alignment of the DHFR-TS enzymes from P. falciparum (P. fal), P. gallinaceum (P. gal), A. thaliana (A. tha), Medicago truncatula (M. tru), Theileria annulata (T. ann) and T. gondii (T. gon). The sequence numbering for the P. falciparum and T. gondii is given above and below the alignment, respectively. The secondary structure elements for P. falciparum DHFR-TS are given above the alignment with blue cylinders and red arrows representing α-helices and β-sheets, respectively. Residues which display sequence conservation across all species are highlighted by a black box with reverse type. Those residues which are involved in binding NADPH, pyrimethamine and WR99210 are highlighted by a red, blue and green box below the alignment, respectively, with those residues which bind both inhibitors and/or NADPH displayed with multiple colored boxes. B. A stereo view of the P. falciparum DHFR/NADPH/WR99210 complex inhibitor binding site with the closely related inhibitor JPC-2067-B modeled. Those residues which form close interactions with the inhibitor are labeled and shown in a stick format, colored yellow, red, blue and orange for carbon, oxygen, nitrogen and sulfur, respectively. The modeled JPC-2067-B inhibitor and NADPH cofactor are colored purple, blue, red, orange, cyan and green for carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, respectively. In addition His27, in T. gondii DHFR, which replaces Cys50 in P. falciparum DHFR, is also shown in a stick format (colored orange for carbon and blue for nitrogen) to demonstrate its close proximity to the modeled JPC-2067-B inhibitor.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Novel Triazine JPC-2067-B Inhibits Toxoplasma gondii In Vitro and In Vivo

    doi: 10.1371/journal.pntd.0000190

    Figure Lengend Snippet: Structure based sequence alighnment of DHFR-TS and stereo view of DHFR/NADPH/WR99210 complex inhibitor binding site. A. A structure based sequence alignment of the DHFR-TS enzymes from P. falciparum (P. fal), P. gallinaceum (P. gal), A. thaliana (A. tha), Medicago truncatula (M. tru), Theileria annulata (T. ann) and T. gondii (T. gon). The sequence numbering for the P. falciparum and T. gondii is given above and below the alignment, respectively. The secondary structure elements for P. falciparum DHFR-TS are given above the alignment with blue cylinders and red arrows representing α-helices and β-sheets, respectively. Residues which display sequence conservation across all species are highlighted by a black box with reverse type. Those residues which are involved in binding NADPH, pyrimethamine and WR99210 are highlighted by a red, blue and green box below the alignment, respectively, with those residues which bind both inhibitors and/or NADPH displayed with multiple colored boxes. B. A stereo view of the P. falciparum DHFR/NADPH/WR99210 complex inhibitor binding site with the closely related inhibitor JPC-2067-B modeled. Those residues which form close interactions with the inhibitor are labeled and shown in a stick format, colored yellow, red, blue and orange for carbon, oxygen, nitrogen and sulfur, respectively. The modeled JPC-2067-B inhibitor and NADPH cofactor are colored purple, blue, red, orange, cyan and green for carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, respectively. In addition His27, in T. gondii DHFR, which replaces Cys50 in P. falciparum DHFR, is also shown in a stick format (colored orange for carbon and blue for nitrogen) to demonstrate its close proximity to the modeled JPC-2067-B inhibitor.

    Article Snippet: DHFR from T. gondii was isolated directly from RH strain T. gondii grown in culture on Chinese hamster ovary cells lacking DHFR (CHO/dhfr- , American Type Culture Collection 3952 CL) .

    Techniques: Sequencing, Binding Assay, Labeling

    Functionally active integrin α V β 3 in cancer cells is required for ATX binding. (A) Flow cytometry detection of cell surface expression of integrin α V β 3 in parental CHO-dhfr + (dhfr + , left), CHO-β 3 WT (β3WT,

    Journal: Blood

    Article Title: Interaction of platelet-derived autotaxin with tumor integrin αVβ3 controls metastasis of breast cancer cells to bone

    doi: 10.1182/blood-2014-04-568683

    Figure Lengend Snippet: Functionally active integrin α V β 3 in cancer cells is required for ATX binding. (A) Flow cytometry detection of cell surface expression of integrin α V β 3 in parental CHO-dhfr + (dhfr + , left), CHO-β 3 WT (β3WT,

    Article Snippet: CHO-dhfr+ , CHOβ3WT, and CHOβ3-Δ744 cells and culture conditions were described previously., Human cell lines from breast cancer (MDA-MB-231, MCF-7, ZR7-51, BT474, SKBr3, Hs578T, and T47D), prostate cancer (PC-3 and DU145), and osteosarcoma (MG63) were obtained from and cultured as recommended by American Type Culture Collection (Gaithersburg, MD).

    Techniques: Binding Assay, Flow Cytometry, Cytometry, Expressing

    Inhibition of early steps of tumor cell bone colonization by circulating inactive ATX. (A) Detection of functionally active ATX and lysoPLD-deficient ATX-T209A expression in cell lysates and in conditioned media (CM) of CHO-dhfr + /ATX and CHO-dhfr + /ATX-T209A

    Journal: Blood

    Article Title: Interaction of platelet-derived autotaxin with tumor integrin αVβ3 controls metastasis of breast cancer cells to bone

    doi: 10.1182/blood-2014-04-568683

    Figure Lengend Snippet: Inhibition of early steps of tumor cell bone colonization by circulating inactive ATX. (A) Detection of functionally active ATX and lysoPLD-deficient ATX-T209A expression in cell lysates and in conditioned media (CM) of CHO-dhfr + /ATX and CHO-dhfr + /ATX-T209A

    Article Snippet: CHO-dhfr+ , CHOβ3WT, and CHOβ3-Δ744 cells and culture conditions were described previously., Human cell lines from breast cancer (MDA-MB-231, MCF-7, ZR7-51, BT474, SKBr3, Hs578T, and T47D), prostate cancer (PC-3 and DU145), and osteosarcoma (MG63) were obtained from and cultured as recommended by American Type Culture Collection (Gaithersburg, MD).

    Techniques: Inhibition, Expressing

    Role of the CEA splice variant in MEDI-565 mediated T cell activation and target cell killing. A, expression of full-length CEA and CEA splice variant proteins in CHO cells as determined by flow cytometry. Mouse IgG1, mouse IgG1 control antibody. B, CHO DHFR-, CHO FL, CHO SV and CHO FL+SV cells were tested for their susceptibility to be killed by CD3+ T cells from 3 individual donors in the presence of MEDI-565 at the indicated concentrations. EC 50 values listed indicate the mean value among 3 donors±standard error of the mean. p = 0.79 comparing cytotoxicity EC 50 values between CHO FL CEA and CHO FL+SV CEA cells. C, activation (increased cell surface CD25/IL-2R levels) of CD8+ T cells and D, activation of CD4+ T cells isolated from each of the 3 healthy donors was investigated concurrently with the cytotoxicity assays shown in panel B. p = 0.60 comparing CD8+ T cell activation EC 50 values between CHO FL CEA and CHO FL+SV CEA; p = 0.15 comparing CD4+ T cell activation EC 50 values between CHO FL CEA and CHO FL+SV CEA. MFI CD25-APC = mean fluorescence intensity of bound APC labeled, anti-human CD25 mAb. Experiment was repeated once with similar results.

    Journal: PLoS ONE

    Article Title: The CEA/CD3-Bispecific Antibody MEDI-565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA

    doi: 10.1371/journal.pone.0036412

    Figure Lengend Snippet: Role of the CEA splice variant in MEDI-565 mediated T cell activation and target cell killing. A, expression of full-length CEA and CEA splice variant proteins in CHO cells as determined by flow cytometry. Mouse IgG1, mouse IgG1 control antibody. B, CHO DHFR-, CHO FL, CHO SV and CHO FL+SV cells were tested for their susceptibility to be killed by CD3+ T cells from 3 individual donors in the presence of MEDI-565 at the indicated concentrations. EC 50 values listed indicate the mean value among 3 donors±standard error of the mean. p = 0.79 comparing cytotoxicity EC 50 values between CHO FL CEA and CHO FL+SV CEA cells. C, activation (increased cell surface CD25/IL-2R levels) of CD8+ T cells and D, activation of CD4+ T cells isolated from each of the 3 healthy donors was investigated concurrently with the cytotoxicity assays shown in panel B. p = 0.60 comparing CD8+ T cell activation EC 50 values between CHO FL CEA and CHO FL+SV CEA; p = 0.15 comparing CD4+ T cell activation EC 50 values between CHO FL CEA and CHO FL+SV CEA. MFI CD25-APC = mean fluorescence intensity of bound APC labeled, anti-human CD25 mAb. Experiment was repeated once with similar results.

    Article Snippet: Dihydrofolate reductase deficient (DHFR-) Chinese hamster ovary (CHO) cells or CHO DHFR- cells (American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 media (Invitrogen) containing 10% fetal bovine serum (FBS; Invitrogen) in a humidified cell culture incubator at 37°C and 5% CO2 .

    Techniques: Variant Assay, Activation Assay, Expressing, Flow Cytometry, Cytometry, Isolation, Fluorescence, Labeling

    Map of pFab CMV dhfr vector for expression of full-length IgG1 in CHO cells and structure of the IgG1 light-chain and heavy-chain DNA inserts. (A) Locations of the various genes present in the expression vector. LC, light-chain DNA; pA, poly(A) addition

    Journal: Journal of Virology

    Article Title: Identification of Chimpanzee Fab Fragments by Repertoire Cloning and Production of a Full-Length Humanized Immunoglobulin G1 Antibody That Is Highly Efficient for Neutralization of Dengue Type 4 Virus

    doi: 10.1128/JVI.78.9.4665-4674.2004

    Figure Lengend Snippet: Map of pFab CMV dhfr vector for expression of full-length IgG1 in CHO cells and structure of the IgG1 light-chain and heavy-chain DNA inserts. (A) Locations of the various genes present in the expression vector. LC, light-chain DNA; pA, poly(A) addition

    Article Snippet: CHO dhfr− ( duk− ) cells were purchased from the American Type Culture Collection.

    Techniques: Plasmid Preparation, Expressing

    Titer and specific productivity comparison of a BAC derived recombinant CHO-S cell line producing gp140 (CN54) and an already existing recombinant plasmid derived CHO-DUKX-B11 cell line .

    Journal: BMC Proceedings

    Article Title: Powerful expression in Chinese Hamster Ovary cells using bacterial artificial chromosomes: parameters influencing productivity

    doi: 10.1186/1753-6561-7-S6-P25

    Figure Lengend Snippet: Titer and specific productivity comparison of a BAC derived recombinant CHO-S cell line producing gp140 (CN54) and an already existing recombinant plasmid derived CHO-DUKX-B11 cell line .

    Article Snippet: Methods Cell culture: CHO-DUKX-B11 (ATCC-CRL-9096) and CHO-DG44 (life technologies) were serum-free cultivated in spinner flasks.

    Techniques: BAC Assay, Derivative Assay, Recombinant, Plasmid Preparation

    Expression of cytokines and HBcAg (HBc) from mono- and bicistronic mRNAs. ( A ) Schematic diagram of mono- and bicistronic vector constructs (pCI based) used to measure protein expression after transient transfection into different cell lines. P CMV in the open arrow marks the CMV promoter. Cyt indicates the cytokine gene for either IFNγ, GM-CSF or Il-7. IRES P represents the poliovirus-derived IRES element. ( B ) Determination of IFNγ and GM-CSF in the supernatant of the indicated cells by ELISA. The indicated plasmids were transfected into the cell lines LMH, C2C12 and BHK21 and cytokine concentrations were determined after a transient expression period of 48 h. ( C ) Detection of HBcAg (HBc) in the lysates of LMH cells by western blot analysis. HBcAg was expressed either monocistronically (lane 1), as a first (lanes 5–7) or as a second (lanes 2–4) cistron.

    Journal: Nucleic Acids Research

    Article Title: Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs

    doi:

    Figure Lengend Snippet: Expression of cytokines and HBcAg (HBc) from mono- and bicistronic mRNAs. ( A ) Schematic diagram of mono- and bicistronic vector constructs (pCI based) used to measure protein expression after transient transfection into different cell lines. P CMV in the open arrow marks the CMV promoter. Cyt indicates the cytokine gene for either IFNγ, GM-CSF or Il-7. IRES P represents the poliovirus-derived IRES element. ( B ) Determination of IFNγ and GM-CSF in the supernatant of the indicated cells by ELISA. The indicated plasmids were transfected into the cell lines LMH, C2C12 and BHK21 and cytokine concentrations were determined after a transient expression period of 48 h. ( C ) Detection of HBcAg (HBc) in the lysates of LMH cells by western blot analysis. HBcAg was expressed either monocistronically (lane 1), as a first (lanes 5–7) or as a second (lanes 2–4) cistron.

    Article Snippet: Murine C2C12 cells (ATCC no. CRL-9096) were cultured in RPMI 1640 (Gibco BRL) supplemented with 5% FCS, antibiotics and glutamine.

    Techniques: Expressing, Plasmid Preparation, Construct, Transfection, Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot

    Phenotypes of L5Jcs5 and L5Jcs11 mutants . (A) Light micrographs of E7.5 WT and L5Jcs5/L5Jcs5 littermates taken at same magnification (8×). (B) Western blots of liver protein probed with anti-TYMS, DHFR, and beta actin. Each lane contains protein from separate animals. (C) Light micrographs of whole mount E7.5 WT and L5Jcs11/L5Jcs11 embryos, plus representative images of blastocyst outgrowths from the indicated genotypes. The mutant embryo is magnified 1.5 X compared to the WT. Notice that there is no evidence of growth of the embryo proper. ICM = Inner cell mass. Tr = trophectoderm.

    Journal: BMC Genetics

    Article Title: High resolution mapping and positional cloning of ENU-induced mutations in the Rw region of mouse chromosome 5

    doi: 10.1186/1471-2156-11-106

    Figure Lengend Snippet: Phenotypes of L5Jcs5 and L5Jcs11 mutants . (A) Light micrographs of E7.5 WT and L5Jcs5/L5Jcs5 littermates taken at same magnification (8×). (B) Western blots of liver protein probed with anti-TYMS, DHFR, and beta actin. Each lane contains protein from separate animals. (C) Light micrographs of whole mount E7.5 WT and L5Jcs11/L5Jcs11 embryos, plus representative images of blastocyst outgrowths from the indicated genotypes. The mutant embryo is magnified 1.5 X compared to the WT. Notice that there is no evidence of growth of the embryo proper. ICM = Inner cell mass. Tr = trophectoderm.

    Article Snippet: After four washes (phosphate buffered saline with 0.1% Tween-20) of 10 minutes each, the membranes were incubated for 1 hour in 1:10,000 HRP-conjugated goat anti-mouse antibody for TYMS or 1:20,000 HRP-conjugated goat anti-rabbit for DHFR (Pierce).

    Techniques: Western Blot, Mutagenesis

    Effect of ALDH1L1 over expression on induction of NADH and ATP production ( A – C ) EKVX and H23 cells were transfected with plasmid expressing ALDH1L1 for 24 h and incubated with siRNA of DHFR for 24 h. ( D – F ) EKVX and H23 cells were transfected with plasmid expressing DHFR for 24 h and incubated with siRNA of ALDH1L1 for 24 h. Effect of DHFR siRNA or ALDH1L1 siRNA on NADH/NAD+ (A, D) and ATP (B, E) was analysed. Immunoblot analysis was performed to confirm DHFR expression, ALDH1L1 knockdown (C, F). Data are representative of the mean and standard deviation three independent experiments. * p

    Journal: Oncotarget

    Article Title: Aldehyde dehydrogenase inhibition combined with phenformin treatment reversed NSCLC through ATP depletion

    doi: 10.18632/oncotarget.10354

    Figure Lengend Snippet: Effect of ALDH1L1 over expression on induction of NADH and ATP production ( A – C ) EKVX and H23 cells were transfected with plasmid expressing ALDH1L1 for 24 h and incubated with siRNA of DHFR for 24 h. ( D – F ) EKVX and H23 cells were transfected with plasmid expressing DHFR for 24 h and incubated with siRNA of ALDH1L1 for 24 h. Effect of DHFR siRNA or ALDH1L1 siRNA on NADH/NAD+ (A, D) and ATP (B, E) was analysed. Immunoblot analysis was performed to confirm DHFR expression, ALDH1L1 knockdown (C, F). Data are representative of the mean and standard deviation three independent experiments. * p

    Article Snippet: For ALDH overexpression, p3x FLAG-CMV-ALDH isoform constructs individually expressing ALDH1L1 and DHFR were produced by Cosmogenetech (Seoul, KOREA).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Incubation, Standard Deviation

    Evidence for selective and combinatorial gene control of a subset of E2F target genes. ( A ) Schematic of E2F and/or TFE3 target promoters assayed in the ChIP assays. I: promoter sequences containing both E2F elements and E Box elements that cluster around the TSS (designated with an arrow); II: promoter sequences containing E2F elements and E Box elements that do not group around the TSS; III: E2F target promoter sequences that contain E2F elements but lack E Box elements; IV: promoter sequences that contain only E Box elements but not E2F elements. ( B ). Chromatin was immunoprecipitated with antibodies to either E2F1, E2F3, or TFE3, decrosslinked, and DNA released from the immunoprecipitates was used for PCR analysis to measure the presence of target promoter sequences as discussed above. The arrows indicate the position of the promoter PCR product. ( C ) ChIP assays for interactions of E2F1, E2F3, or TFE3 with mouse TK-1, p180, DHFR, and cyclin E promoters. ( D ) ChIP assays for interactions of E2F1, E2F3, or TFE3 with mouse E2F1, E2F2, and p19ARF promoters in vivo . ( E ) ChIP assays for interactions of E2F1, E2F3, or TFE3 with mouse SMAD7 and Tyrp1 promoters in vivo .

    Journal: The EMBO Journal

    Article Title: Combinatorial gene control involving E2F and E Box family members

    doi: 10.1038/sj.emboj.7600134

    Figure Lengend Snippet: Evidence for selective and combinatorial gene control of a subset of E2F target genes. ( A ) Schematic of E2F and/or TFE3 target promoters assayed in the ChIP assays. I: promoter sequences containing both E2F elements and E Box elements that cluster around the TSS (designated with an arrow); II: promoter sequences containing E2F elements and E Box elements that do not group around the TSS; III: E2F target promoter sequences that contain E2F elements but lack E Box elements; IV: promoter sequences that contain only E Box elements but not E2F elements. ( B ). Chromatin was immunoprecipitated with antibodies to either E2F1, E2F3, or TFE3, decrosslinked, and DNA released from the immunoprecipitates was used for PCR analysis to measure the presence of target promoter sequences as discussed above. The arrows indicate the position of the promoter PCR product. ( C ) ChIP assays for interactions of E2F1, E2F3, or TFE3 with mouse TK-1, p180, DHFR, and cyclin E promoters. ( D ) ChIP assays for interactions of E2F1, E2F3, or TFE3 with mouse E2F1, E2F2, and p19ARF promoters in vivo . ( E ) ChIP assays for interactions of E2F1, E2F3, or TFE3 with mouse SMAD7 and Tyrp1 promoters in vivo .

    Article Snippet: To amplify E2F- and/or TFE3-responsive promoter regions, the following primer sets were designed to promoter sequences between −500 to +50 for the following genes: p68 (Unigene ID: Mm.320), RR1 (Unigene ID: Mm.656), RR2 (Unigene ID: Mm.99), E2F2 (Unigene ID: Mm.100478), E2F1 (Unigene ID: Mm.18036), p19ARF (Unigene ID: Mm.4733), TK-1 (Unigene ID: Mm.2661), p180 (Unigene ID: Mm.1923), DHFR (Unigene ID: Mm.23695), SMAD7 (Unigene ID: Mm.34407), cyclin E (Unigene ID: Mm.16110), and TS (Unigene ID: Mm.5879).

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, In Vivo

    Western immunoblot analysis of p53, p21 and Bax expression in HCT-116 (wt-p53) cells (lane 1, vehicle control; lane 2, non specific control; lane 3, DHFR siRNA positive control; lane 4, 100 nM miR-192 ). α-tubulin was used as a protein loading

    Journal:

    Article Title: miR-192 Regulates Dihydrofolate Reductase and Cellular Proliferation Through the p53-miRNA Circuit

    doi: 10.1158/1078-0432.CCR-08-1422

    Figure Lengend Snippet: Western immunoblot analysis of p53, p21 and Bax expression in HCT-116 (wt-p53) cells (lane 1, vehicle control; lane 2, non specific control; lane 3, DHFR siRNA positive control; lane 4, 100 nM miR-192 ). α-tubulin was used as a protein loading

    Article Snippet: HCT-116 (wt-p53), HCT-116 (null-p53), RKO (wt-p53), and HT-29 cells (2×105 ) were plated in six-well plates, and transfected with 100 nM of either miR-192, miR-24 precursors or non-specific control miR (Ambion) after 24 h with Oligofectamine (Invitrogen) according to the manufacturer’s instructions. siRNA specific to DHFR (ON-TARGET plus SMARTpool L-008799-00-0010, human DHFR, ) was purchased from Dharmacon and transfected with Oligofectamine (Invitrogen) according to the manufacturer’s protocols at a final concentration of 100 nM. siRNA specific to DHFR was used as the positive control. miR-24 , a recently reported miRNA that also targets DHFR , was also used as a positive control.

    Techniques: Western Blot, Expressing, Positive Control

    Impact of miR-192 on cell proliferation with MTX treatment in HCT-116 (wt-p53) cells transfected with DHFR specific siRNA or miR-192 (lane 1, non-specific control miR ; lane 2, 100 nM non-specific control miR + 25 nM MTX; lane 3, 100 nM DHFR siRNA + 25

    Journal:

    Article Title: miR-192 Regulates Dihydrofolate Reductase and Cellular Proliferation Through the p53-miRNA Circuit

    doi: 10.1158/1078-0432.CCR-08-1422

    Figure Lengend Snippet: Impact of miR-192 on cell proliferation with MTX treatment in HCT-116 (wt-p53) cells transfected with DHFR specific siRNA or miR-192 (lane 1, non-specific control miR ; lane 2, 100 nM non-specific control miR + 25 nM MTX; lane 3, 100 nM DHFR siRNA + 25

    Article Snippet: HCT-116 (wt-p53), HCT-116 (null-p53), RKO (wt-p53), and HT-29 cells (2×105 ) were plated in six-well plates, and transfected with 100 nM of either miR-192, miR-24 precursors or non-specific control miR (Ambion) after 24 h with Oligofectamine (Invitrogen) according to the manufacturer’s instructions. siRNA specific to DHFR (ON-TARGET plus SMARTpool L-008799-00-0010, human DHFR, ) was purchased from Dharmacon and transfected with Oligofectamine (Invitrogen) according to the manufacturer’s protocols at a final concentration of 100 nM. siRNA specific to DHFR was used as the positive control. miR-24 , a recently reported miRNA that also targets DHFR , was also used as a positive control.

    Techniques: Transfection

    The miR-192 binding site at 3′-UTR of DHFR mRNA (A). Western immunoblot analysis of DHFR protein expression levels in HCT-116 (wt-p53) cells transfected with miR-192 (lane 1, vehicle control; lane 2, non specific-miR control; lane 3, DHFR siRNA

    Journal:

    Article Title: miR-192 Regulates Dihydrofolate Reductase and Cellular Proliferation Through the p53-miRNA Circuit

    doi: 10.1158/1078-0432.CCR-08-1422

    Figure Lengend Snippet: The miR-192 binding site at 3′-UTR of DHFR mRNA (A). Western immunoblot analysis of DHFR protein expression levels in HCT-116 (wt-p53) cells transfected with miR-192 (lane 1, vehicle control; lane 2, non specific-miR control; lane 3, DHFR siRNA

    Article Snippet: HCT-116 (wt-p53), HCT-116 (null-p53), RKO (wt-p53), and HT-29 cells (2×105 ) were plated in six-well plates, and transfected with 100 nM of either miR-192, miR-24 precursors or non-specific control miR (Ambion) after 24 h with Oligofectamine (Invitrogen) according to the manufacturer’s instructions. siRNA specific to DHFR (ON-TARGET plus SMARTpool L-008799-00-0010, human DHFR, ) was purchased from Dharmacon and transfected with Oligofectamine (Invitrogen) according to the manufacturer’s protocols at a final concentration of 100 nM. siRNA specific to DHFR was used as the positive control. miR-24 , a recently reported miRNA that also targets DHFR , was also used as a positive control.

    Techniques: Binding Assay, Western Blot, Expressing, Transfection

    Dhfr bioactivation is essential for folate-mediated CNS regeneration. Using the SCRM model of afferent spinal regeneration in rats, i.p. MTX was used to inhibit Dhfr, thus preventing the conversion of FA into the bioactive form tetrahydrofolate, as demonstrated in the Dhfr activity assay ( A ) ( n = 5 [uninjured]; n = 5 [injured]; n = 5 [injured and exposed to MTX]; Student’s t test; mean ± SEM; * P = 0.006), with only a small change in protein levels on Western immunoblot ( C , D ). MTX suppressed the regeneration of spinal axons into a nerve graft to below baseline levels ( B ) ( n = 10 [untreated controls]; n = 7 [FA]; n = 7 [MTX]; n = 6 [MTX and FA]). 1-way ANOVA with Bonferroni’s correction; mean ± SEM; * P

    Journal: The Journal of Clinical Investigation

    Article Title: Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation

    doi: 10.1172/JCI40000

    Figure Lengend Snippet: Dhfr bioactivation is essential for folate-mediated CNS regeneration. Using the SCRM model of afferent spinal regeneration in rats, i.p. MTX was used to inhibit Dhfr, thus preventing the conversion of FA into the bioactive form tetrahydrofolate, as demonstrated in the Dhfr activity assay ( A ) ( n = 5 [uninjured]; n = 5 [injured]; n = 5 [injured and exposed to MTX]; Student’s t test; mean ± SEM; * P = 0.006), with only a small change in protein levels on Western immunoblot ( C , D ). MTX suppressed the regeneration of spinal axons into a nerve graft to below baseline levels ( B ) ( n = 10 [untreated controls]; n = 7 [FA]; n = 7 [MTX]; n = 6 [MTX and FA]). 1-way ANOVA with Bonferroni’s correction; mean ± SEM; * P

    Article Snippet: Surgery was performed as detailed above (SCRM) to test regeneration in response to inhibitors of Dhfr (MTX; Parenta Pharmaceuticals) and MS (nitrous oxide; Material Distribution Service, University of Wisconsin).

    Techniques: Activity Assay, Western Blot

    FIR radiation induces tetrahydrobiopterin (BH 4 ) synthesis in vivo. Rats were exposed with or without FIR-emitting sericite board for 7 days. (a) Lung endothelial cells were isolated from control and FIR group rats using CD31 and CD45 beads. Endothelial biopterin and BH 4 levels were quantified by HPLC analysis. (b) GCH1, PTS, SPR, and DHFR protein expression in aorta tissues. β -actin is shown as a loading control. Protein levels were quantified by densitometric analysis. Data are shown as the mean ± SEM of three independent experiments. ∗ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Far-Infrared-Emitting Sericite Board Upregulates Endothelial Nitric Oxide Synthase Activity through Increasing Biosynthesis of Tetrahydrobiopterin in Endothelial Cells

    doi: 10.1155/2019/1813282

    Figure Lengend Snippet: FIR radiation induces tetrahydrobiopterin (BH 4 ) synthesis in vivo. Rats were exposed with or without FIR-emitting sericite board for 7 days. (a) Lung endothelial cells were isolated from control and FIR group rats using CD31 and CD45 beads. Endothelial biopterin and BH 4 levels were quantified by HPLC analysis. (b) GCH1, PTS, SPR, and DHFR protein expression in aorta tissues. β -actin is shown as a loading control. Protein levels were quantified by densitometric analysis. Data are shown as the mean ± SEM of three independent experiments. ∗ P

    Article Snippet: Antibodies and Immunoblotting The following antibodies were used: anti-Akt (Cell Signaling Technology, Danvers, MA, USA), antiphospho Akt (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-β -actin (Sigma-Aldrich, St. Louis, MO, USA), anti-eNOS (Santa Cruz Biotechnology), antiphospho eNOS (S1177) (Cell Signaling Technology), antivascular cell adhesion molecule (VCAM)-1 (Santa Cruz Biotechnology), anti-intercellular adhesion molecule (ICAM)-1 (Santa Cruz Biotechnology), anti-GCH1 (Santa Cruz Biotechnology), anti-6-pyruvoyl tetrahydrobiopterin synthase (PTS) (Santa Cruz Biotechnology), antisepiapterin reductase (SPR) (Santa Cruz Biotechnology), and anti-DHFR (Santa Cruz Biotechnology).

    Techniques: In Vivo, Isolation, High Performance Liquid Chromatography, SPR Assay, Expressing

    FIR radiation induces BH 4 synthesis in HUVECs. HUVECs were cultured with or without FIR-emitting sericite board for 48 hours. (a) Schematic model summarizing the mechanism of FIR-induced improvement of vascular function. (b) Intracellular levels of total biopterin and BH 4 . (c) The protein expression of GCH1, PTS, SPR, and DHFR. β -actin is shown as a loading control. (d) Protein levels were quantified by densitometric analysis. Data are shown as the mean ± SEM of three independent experiments. ∗ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Far-Infrared-Emitting Sericite Board Upregulates Endothelial Nitric Oxide Synthase Activity through Increasing Biosynthesis of Tetrahydrobiopterin in Endothelial Cells

    doi: 10.1155/2019/1813282

    Figure Lengend Snippet: FIR radiation induces BH 4 synthesis in HUVECs. HUVECs were cultured with or without FIR-emitting sericite board for 48 hours. (a) Schematic model summarizing the mechanism of FIR-induced improvement of vascular function. (b) Intracellular levels of total biopterin and BH 4 . (c) The protein expression of GCH1, PTS, SPR, and DHFR. β -actin is shown as a loading control. (d) Protein levels were quantified by densitometric analysis. Data are shown as the mean ± SEM of three independent experiments. ∗ P

    Article Snippet: Antibodies and Immunoblotting The following antibodies were used: anti-Akt (Cell Signaling Technology, Danvers, MA, USA), antiphospho Akt (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-β -actin (Sigma-Aldrich, St. Louis, MO, USA), anti-eNOS (Santa Cruz Biotechnology), antiphospho eNOS (S1177) (Cell Signaling Technology), antivascular cell adhesion molecule (VCAM)-1 (Santa Cruz Biotechnology), anti-intercellular adhesion molecule (ICAM)-1 (Santa Cruz Biotechnology), anti-GCH1 (Santa Cruz Biotechnology), anti-6-pyruvoyl tetrahydrobiopterin synthase (PTS) (Santa Cruz Biotechnology), antisepiapterin reductase (SPR) (Santa Cruz Biotechnology), and anti-DHFR (Santa Cruz Biotechnology).

    Techniques: Cell Culture, Expressing, SPR Assay

    Effect of dihydrofolate reductase ( DHFR ) silencing on FdUrd cytotoxicity. At 48 h after treatment of DLD-1 and HCT116 cells with control or DHFR siRNA, cells were treated with FdUrd for 72 h, and cell viability was determined. Silencing of DHFR reduced the efficacy of FdUrd at 0.1–10 μM in DLD-1 cells and at 0.1–3 μM in HCT116 cells. All data are expressed as the mean ± SD. * p

    Journal: PLoS ONE

    Article Title: Folic Acid-Metabolizing Enzymes Regulate the Antitumor Effect of 5-Fluoro-2′-Deoxyuridine in Colorectal Cancer Cell Lines

    doi: 10.1371/journal.pone.0163961

    Figure Lengend Snippet: Effect of dihydrofolate reductase ( DHFR ) silencing on FdUrd cytotoxicity. At 48 h after treatment of DLD-1 and HCT116 cells with control or DHFR siRNA, cells were treated with FdUrd for 72 h, and cell viability was determined. Silencing of DHFR reduced the efficacy of FdUrd at 0.1–10 μM in DLD-1 cells and at 0.1–3 μM in HCT116 cells. All data are expressed as the mean ± SD. * p

    Article Snippet: Probe IDs were Hs00195560_m1 for MTHFR , Hs01068263_m1 for MTHFD1 , Hs00894582_m1 for GART , Hs01124179_g1 for FOLR1 , Hs00758822_s1 for DHFR , and Hs00426586_m1 for TYMS .

    Techniques:

    Zapalog can induce multiple rounds of dimerization. a, (below) HeLa cell expressing mitochondrial Tom20-mCherry-FKBP and YFP-DHFR-Myc incubated with 10μM CCCP for 6hrs to fragment mitochondria, then 2μM zapalog for 5min, which has caused YFP-DHFR-Myc to localize to outer mitochondrial membranes. (above) A blown-up image of the area defined below with a yellow square within which two mitochondrion-containing 2μm × 2μm ROIs (dotted white squares) were defined and imaged at high temporal resolution. In the time series to the right, one of the ROIs, (+) was periodically illuminated with a 405nm laser (~300nW) for 500msec, the other (−) was not. Each flash of blue light causes photolysis of zapalog and immediate reversal of the dimerization in ROI (+) but not (−). Each instance of YFP-DHFR-Myc removal from a mitochondrion is followed by rapid re-dimerization (~30sec) due to influx of unlysed zapalog from outside of the ROI, which outcompetes the lysed zapalog fragments bound to FKBP and DHFR. a’, schematic illustration of repeated fluorophore translocation mediated by zapalog. a”, quantification of normalized YFP signal within discrete mitochondrion-containing ROIs (2µm × 2µm) that were illuminated by 405nm laser, and nearby ROIs that were not. (n=7 cells / 3 independent repeats, center value = mean, error bars represent SE, source data in Sup. Table 1 ) b, (below) COS7 cell expressing peroxisomal PEX3-mRFP- FKBP and YFP-DHFR-Myc incubated with 2uM zapalog for 5min, which has caused YFP-DHFR-Myc to localize to peroxisomal membranes. (above) Two peroxisome-containing 6μm × 6μm ROIs were defined and imaged at high temporal resolution. In the time series to its right, one of the ROIs, (+) was illuminated with a 405nm laser (10%) for 500msec every 40sec, the other (−) was not. Each flash of blue light causes photolysis of zapalog and immediate reversal of the dimerization in ROI (+) but not (−). Each instance of YFP-DHFR-Myc removal from peroxisomes is followed by rapid re-dimerization (~30sec) due to influx of unlysed zapalog from outside of the ROI, which outcompetes the lysed zapalog fragments bound to FKBP and DHFR. b’, schematic illustration of repeated fluorophore translocation mediated by zapalog. b”, quantification of normalized YFP signal within peroxisome-containing ROIs (5µm × 5µm) that were illuminated by 405nm laser, and nearby ROIs that were not. (n=4 cells / 2 independent repeats, center value = mean, error bars represent SE, source data in Sup. Table 1 )

    Journal: Nature cell biology

    Article Title: The light-sensitive dimerizer zapalog reveals distinct modes of immobilization for axonal mitochondria.

    doi: 10.1038/s41556-019-0317-2

    Figure Lengend Snippet: Zapalog can induce multiple rounds of dimerization. a, (below) HeLa cell expressing mitochondrial Tom20-mCherry-FKBP and YFP-DHFR-Myc incubated with 10μM CCCP for 6hrs to fragment mitochondria, then 2μM zapalog for 5min, which has caused YFP-DHFR-Myc to localize to outer mitochondrial membranes. (above) A blown-up image of the area defined below with a yellow square within which two mitochondrion-containing 2μm × 2μm ROIs (dotted white squares) were defined and imaged at high temporal resolution. In the time series to the right, one of the ROIs, (+) was periodically illuminated with a 405nm laser (~300nW) for 500msec, the other (−) was not. Each flash of blue light causes photolysis of zapalog and immediate reversal of the dimerization in ROI (+) but not (−). Each instance of YFP-DHFR-Myc removal from a mitochondrion is followed by rapid re-dimerization (~30sec) due to influx of unlysed zapalog from outside of the ROI, which outcompetes the lysed zapalog fragments bound to FKBP and DHFR. a’, schematic illustration of repeated fluorophore translocation mediated by zapalog. a”, quantification of normalized YFP signal within discrete mitochondrion-containing ROIs (2µm × 2µm) that were illuminated by 405nm laser, and nearby ROIs that were not. (n=7 cells / 3 independent repeats, center value = mean, error bars represent SE, source data in Sup. Table 1 ) b, (below) COS7 cell expressing peroxisomal PEX3-mRFP- FKBP and YFP-DHFR-Myc incubated with 2uM zapalog for 5min, which has caused YFP-DHFR-Myc to localize to peroxisomal membranes. (above) Two peroxisome-containing 6μm × 6μm ROIs were defined and imaged at high temporal resolution. In the time series to its right, one of the ROIs, (+) was illuminated with a 405nm laser (10%) for 500msec every 40sec, the other (−) was not. Each flash of blue light causes photolysis of zapalog and immediate reversal of the dimerization in ROI (+) but not (−). Each instance of YFP-DHFR-Myc removal from peroxisomes is followed by rapid re-dimerization (~30sec) due to influx of unlysed zapalog from outside of the ROI, which outcompetes the lysed zapalog fragments bound to FKBP and DHFR. b’, schematic illustration of repeated fluorophore translocation mediated by zapalog. b”, quantification of normalized YFP signal within peroxisome-containing ROIs (5µm × 5µm) that were illuminated by 405nm laser, and nearby ROIs that were not. (n=4 cells / 2 independent repeats, center value = mean, error bars represent SE, source data in Sup. Table 1 )

    Article Snippet: Repeated, localized fluorophore-translocation assay on non-networked mitochondria – 18hrs after HeLa cells were transfected with Tom20-mCherry-FKBP and YFP-DHFR-Myc, they were incubated for 6hrs in 10µM CCCP.

    Techniques: Expressing, Incubation, Translocation Assay

    Zapalog, a photocleavable heterodimerizer, can be used to reversibly translocate cytosolic YFP to mitochondria. a, Schematic illustration of zapalog function: 1. Two proteins of interest are tagged with DHFR and FKBP domains, 2. Addition of zapalog induces dimerization of the tagged proteins, 3. 405nm light photocleaves zapalog, causing rapid dissociation of the dimer, 4. Addition of uncleaved zapalog outcompetes photolyzed zapalog moieties, reestablishing dimerization. b, Chemical structure of zapalog before and after photolysis of the DANB moiety by 405nm light. Bottom - Schematic illustration of zapalog-induced translocation of YFP: zapalog attaches cytoplasmic YFP-DHFR-Myc to FKBP domains tethered to mitochondrial outer membranes. Exposure to 405nm light photocleaves zapalog, releasing the YFP-DHFR-Myc back to the cytoplasm. c, Time-lapsed imaging demonstrates full translocation of YFP-DHFR-Myc onto mitochondria in a COS7 cell, within ~1m after addition of 10μM zapalog to the medium. c’, Quantification of multiple experiments as in (c) using 10μM zapalog. The ratio of mitochondrial to cytoplasmic YFP-DHFR-Myc was derived from automated image analyses of the datasets, normalized, and average YFP intensity was calculated for each timepoint (n= 5 cells / 3 independent repeats, center value = mean, error bars represent SE, source data in Sup. Table 1 ) c”, From assays of YFP translocation to mitochondria, quantified as in (c’), a dose-response curve was generated by plotting time from 10% to 90% of full YFP-DHFR-Myc translocation for each concentration of zapalog administered. (n=98 cells, 6 independent repeats, center value = mean, error bars represent SD, source data in Sup. Table 1 ) d, Prior to the series of images shown, YFP-DHFR-Myc translocation to mitochondria was induced by zapalog, as in (c). After wash-out of free zapalog from HeLa cells, the YFP reporter was released from the mitochondria sequentially in each of the 3 cells by exposing the circled region to a brief (500ms) pulse of 405nm light. d’, Quantification of data shown in (d), demonstrating that photolysis of zapalog is rapid, complete, and spatially localizable. scale bars = 5µm

    Journal: Nature cell biology

    Article Title: The light-sensitive dimerizer zapalog reveals distinct modes of immobilization for axonal mitochondria.

    doi: 10.1038/s41556-019-0317-2

    Figure Lengend Snippet: Zapalog, a photocleavable heterodimerizer, can be used to reversibly translocate cytosolic YFP to mitochondria. a, Schematic illustration of zapalog function: 1. Two proteins of interest are tagged with DHFR and FKBP domains, 2. Addition of zapalog induces dimerization of the tagged proteins, 3. 405nm light photocleaves zapalog, causing rapid dissociation of the dimer, 4. Addition of uncleaved zapalog outcompetes photolyzed zapalog moieties, reestablishing dimerization. b, Chemical structure of zapalog before and after photolysis of the DANB moiety by 405nm light. Bottom - Schematic illustration of zapalog-induced translocation of YFP: zapalog attaches cytoplasmic YFP-DHFR-Myc to FKBP domains tethered to mitochondrial outer membranes. Exposure to 405nm light photocleaves zapalog, releasing the YFP-DHFR-Myc back to the cytoplasm. c, Time-lapsed imaging demonstrates full translocation of YFP-DHFR-Myc onto mitochondria in a COS7 cell, within ~1m after addition of 10μM zapalog to the medium. c’, Quantification of multiple experiments as in (c) using 10μM zapalog. The ratio of mitochondrial to cytoplasmic YFP-DHFR-Myc was derived from automated image analyses of the datasets, normalized, and average YFP intensity was calculated for each timepoint (n= 5 cells / 3 independent repeats, center value = mean, error bars represent SE, source data in Sup. Table 1 ) c”, From assays of YFP translocation to mitochondria, quantified as in (c’), a dose-response curve was generated by plotting time from 10% to 90% of full YFP-DHFR-Myc translocation for each concentration of zapalog administered. (n=98 cells, 6 independent repeats, center value = mean, error bars represent SD, source data in Sup. Table 1 ) d, Prior to the series of images shown, YFP-DHFR-Myc translocation to mitochondria was induced by zapalog, as in (c). After wash-out of free zapalog from HeLa cells, the YFP reporter was released from the mitochondria sequentially in each of the 3 cells by exposing the circled region to a brief (500ms) pulse of 405nm light. d’, Quantification of data shown in (d), demonstrating that photolysis of zapalog is rapid, complete, and spatially localizable. scale bars = 5µm

    Article Snippet: Repeated, localized fluorophore-translocation assay on non-networked mitochondria – 18hrs after HeLa cells were transfected with Tom20-mCherry-FKBP and YFP-DHFR-Myc, they were incubated for 6hrs in 10µM CCCP.

    Techniques: Translocation Assay, Imaging, Derivative Assay, Generated, Concentration Assay