dna Thermo Fisher Search Results


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  • 90
    Thermo Fisher dna analyzer
    Dna Analyzer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1098 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher deoxyribonucleic acid
    RT progression assay determining G-quartet formation in the Sp1 binding region. Cation-dependent pausing of reverse transcription at the guanine-rich elements in the U3 region was analyzed with <t>RNA</t> and <t>DNA</t> templates. A fragment of the RNA/DNA sequence of the Sp1 binding region is shown (top) with G-rich elements (shaded). Strong pauses of the RT near G-rich elements were observed in the presence of 50 mM of KCl, but not LiCl, indicating that these elements are involved in the formation of structure, which is stabilized by potassium ions but destabilized by lithium ions. This is indicative of a G-quadruplex. The cation-independent RT pauses are likely caused by hairpin structures. DNA primer, P; DNA marker, M; KCl, K + ; LiCl, Li + .
    Deoxyribonucleic Acid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2265 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hoechst deoxyribonucleic acid dye
    Diabetes-induced increase in inflammatory proteins in photoreceptor cells. There was no detection of iNOS in the photoreceptor region in the nondiabetic retina ( A ), but in diabetes, there were increased levels of iNOS in the photoreceptor region ( B ). There was no detection of COX2 in the photoreceptor region of the nondiabetic retina ( C ), but in diabetes, there were increased levels of COX2 in the photoreceptor region in diabetes ( D ). The nuclei were stained with <t>Hoechst</t> DNA dye ( blue ). Micrographs are representative of three or more animals per group.
    Hoechst Deoxyribonucleic Acid Dye, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher duo thermo fisher
    Diabetes-induced increase in inflammatory proteins in photoreceptor cells. There was no detection of iNOS in the photoreceptor region in the nondiabetic retina ( A ), but in diabetes, there were increased levels of iNOS in the photoreceptor region ( B ). There was no detection of COX2 in the photoreceptor region of the nondiabetic retina ( C ), but in diabetes, there were increased levels of COX2 in the photoreceptor region in diabetes ( D ). The nuclei were stained with <t>Hoechst</t> DNA dye ( blue ). Micrographs are representative of three or more animals per group.
    Duo Thermo Fisher, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ned thermo fisher labeled dna primer
    Diabetes-induced increase in inflammatory proteins in photoreceptor cells. There was no detection of iNOS in the photoreceptor region in the nondiabetic retina ( A ), but in diabetes, there were increased levels of iNOS in the photoreceptor region ( B ). There was no detection of COX2 in the photoreceptor region of the nondiabetic retina ( C ), but in diabetes, there were increased levels of COX2 in the photoreceptor region in diabetes ( D ). The nuclei were stained with <t>Hoechst</t> DNA dye ( blue ). Micrographs are representative of three or more animals per group.
    Ned Thermo Fisher Labeled Dna Primer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    Thermo Fisher saliva deoxyribonucleic acid
    Diabetes-induced increase in inflammatory proteins in photoreceptor cells. There was no detection of iNOS in the photoreceptor region in the nondiabetic retina ( A ), but in diabetes, there were increased levels of iNOS in the photoreceptor region ( B ). There was no detection of COX2 in the photoreceptor region of the nondiabetic retina ( C ), but in diabetes, there were increased levels of COX2 in the photoreceptor region in diabetes ( D ). The nuclei were stained with <t>Hoechst</t> DNA dye ( blue ). Micrographs are representative of three or more animals per group.
    Saliva Deoxyribonucleic Acid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher thermo fisher predesigned snp
    Diabetes-induced increase in inflammatory proteins in photoreceptor cells. There was no detection of iNOS in the photoreceptor region in the nondiabetic retina ( A ), but in diabetes, there were increased levels of iNOS in the photoreceptor region ( B ). There was no detection of COX2 in the photoreceptor region of the nondiabetic retina ( C ), but in diabetes, there were increased levels of COX2 in the photoreceptor region in diabetes ( D ). The nuclei were stained with <t>Hoechst</t> DNA dye ( blue ). Micrographs are representative of three or more animals per group.
    Thermo Fisher Predesigned Snp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher complementary deoxyribonucleic acid cdna
    Osteoclastogenesis in wild-type (B6) and Fas deficient (Fas −/−) mice 4 weeks after ovariectomy (OVX) Osteoclastogenesis in vivo was assessed by histomorphometric analysis of distal femoral sections from sham-operated (SH) and OVX B6 and Fas −/− mice stained histochemically for the activity of tartrate-resistant acid phosphatase (TRAP). Osteoclastogenesis in vitro was assessed in cultures prepared from bone marrow of SH and OVX B6 and Fas −/− mice. (A) Number of TRAP positive osteoclasts per millimeter bone perimeter in distal femoral metaphyseal sections (mean±SD, t-test, *p≤0.03 vs. SH mice). (B) Number of TRAP positive osteoclasts (mean±SD, t-test, *p≤0.02 vs. SH B6 mice) on day 6 of cell culture. (C) Gene expression pattern in osteoclastogenic cultures from SH and OVX B6 and Fas −/− mice. For each time point in each group, cells were cultured in quadruplicates and pooled for RNA isolation. Day 0 represents gene expression in freshly isolated bone marrow cells. Values were calculated according to the standard curve of gene expression in the calibrator sample <t>(cDNA</t> from osteoclastogenic cultures) and normalized to the expression of the gene for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time <t>PCR</t> reaction duplicates prepared from the same sample of the representative experiment. Csf1r, colony stimulating factor 1 receptor; Calcr, calcitonin receptor; Tnfrsf11a, tumor necrosis factor receptor superfamily member 11a, RANK.
    Complementary Deoxyribonucleic Acid Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 624 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher thermo fisher scientific open biosystems
    Osteoclastogenesis in wild-type (B6) and Fas deficient (Fas −/−) mice 4 weeks after ovariectomy (OVX) Osteoclastogenesis in vivo was assessed by histomorphometric analysis of distal femoral sections from sham-operated (SH) and OVX B6 and Fas −/− mice stained histochemically for the activity of tartrate-resistant acid phosphatase (TRAP). Osteoclastogenesis in vitro was assessed in cultures prepared from bone marrow of SH and OVX B6 and Fas −/− mice. (A) Number of TRAP positive osteoclasts per millimeter bone perimeter in distal femoral metaphyseal sections (mean±SD, t-test, *p≤0.03 vs. SH mice). (B) Number of TRAP positive osteoclasts (mean±SD, t-test, *p≤0.02 vs. SH B6 mice) on day 6 of cell culture. (C) Gene expression pattern in osteoclastogenic cultures from SH and OVX B6 and Fas −/− mice. For each time point in each group, cells were cultured in quadruplicates and pooled for RNA isolation. Day 0 represents gene expression in freshly isolated bone marrow cells. Values were calculated according to the standard curve of gene expression in the calibrator sample <t>(cDNA</t> from osteoclastogenic cultures) and normalized to the expression of the gene for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time <t>PCR</t> reaction duplicates prepared from the same sample of the representative experiment. Csf1r, colony stimulating factor 1 receptor; Calcr, calcitonin receptor; Tnfrsf11a, tumor necrosis factor receptor superfamily member 11a, RANK.
    Thermo Fisher Scientific Open Biosystems, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher genomic deoxyribonucleic acids dnas
    Osteoclastogenesis in wild-type (B6) and Fas deficient (Fas −/−) mice 4 weeks after ovariectomy (OVX) Osteoclastogenesis in vivo was assessed by histomorphometric analysis of distal femoral sections from sham-operated (SH) and OVX B6 and Fas −/− mice stained histochemically for the activity of tartrate-resistant acid phosphatase (TRAP). Osteoclastogenesis in vitro was assessed in cultures prepared from bone marrow of SH and OVX B6 and Fas −/− mice. (A) Number of TRAP positive osteoclasts per millimeter bone perimeter in distal femoral metaphyseal sections (mean±SD, t-test, *p≤0.03 vs. SH mice). (B) Number of TRAP positive osteoclasts (mean±SD, t-test, *p≤0.02 vs. SH B6 mice) on day 6 of cell culture. (C) Gene expression pattern in osteoclastogenic cultures from SH and OVX B6 and Fas −/− mice. For each time point in each group, cells were cultured in quadruplicates and pooled for RNA isolation. Day 0 represents gene expression in freshly isolated bone marrow cells. Values were calculated according to the standard curve of gene expression in the calibrator sample <t>(cDNA</t> from osteoclastogenic cultures) and normalized to the expression of the gene for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time <t>PCR</t> reaction duplicates prepared from the same sample of the representative experiment. Csf1r, colony stimulating factor 1 receptor; Calcr, calcitonin receptor; Tnfrsf11a, tumor necrosis factor receptor superfamily member 11a, RANK.
    Genomic Deoxyribonucleic Acids Dnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Thermo Fisher first strand complementary deoxyribonucleic acid kit
    Osteoclastogenesis in wild-type (B6) and Fas deficient (Fas −/−) mice 4 weeks after ovariectomy (OVX) Osteoclastogenesis in vivo was assessed by histomorphometric analysis of distal femoral sections from sham-operated (SH) and OVX B6 and Fas −/− mice stained histochemically for the activity of tartrate-resistant acid phosphatase (TRAP). Osteoclastogenesis in vitro was assessed in cultures prepared from bone marrow of SH and OVX B6 and Fas −/− mice. (A) Number of TRAP positive osteoclasts per millimeter bone perimeter in distal femoral metaphyseal sections (mean±SD, t-test, *p≤0.03 vs. SH mice). (B) Number of TRAP positive osteoclasts (mean±SD, t-test, *p≤0.02 vs. SH B6 mice) on day 6 of cell culture. (C) Gene expression pattern in osteoclastogenic cultures from SH and OVX B6 and Fas −/− mice. For each time point in each group, cells were cultured in quadruplicates and pooled for RNA isolation. Day 0 represents gene expression in freshly isolated bone marrow cells. Values were calculated according to the standard curve of gene expression in the calibrator sample <t>(cDNA</t> from osteoclastogenic cultures) and normalized to the expression of the gene for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time <t>PCR</t> reaction duplicates prepared from the same sample of the representative experiment. Csf1r, colony stimulating factor 1 receptor; Calcr, calcitonin receptor; Tnfrsf11a, tumor necrosis factor receptor superfamily member 11a, RANK.
    First Strand Complementary Deoxyribonucleic Acid Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher chain complementary deoxyribonucleic acid cdna
    Osteoclastogenesis in wild-type (B6) and Fas deficient (Fas −/−) mice 4 weeks after ovariectomy (OVX) Osteoclastogenesis in vivo was assessed by histomorphometric analysis of distal femoral sections from sham-operated (SH) and OVX B6 and Fas −/− mice stained histochemically for the activity of tartrate-resistant acid phosphatase (TRAP). Osteoclastogenesis in vitro was assessed in cultures prepared from bone marrow of SH and OVX B6 and Fas −/− mice. (A) Number of TRAP positive osteoclasts per millimeter bone perimeter in distal femoral metaphyseal sections (mean±SD, t-test, *p≤0.03 vs. SH mice). (B) Number of TRAP positive osteoclasts (mean±SD, t-test, *p≤0.02 vs. SH B6 mice) on day 6 of cell culture. (C) Gene expression pattern in osteoclastogenic cultures from SH and OVX B6 and Fas −/− mice. For each time point in each group, cells were cultured in quadruplicates and pooled for RNA isolation. Day 0 represents gene expression in freshly isolated bone marrow cells. Values were calculated according to the standard curve of gene expression in the calibrator sample <t>(cDNA</t> from osteoclastogenic cultures) and normalized to the expression of the gene for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time <t>PCR</t> reaction duplicates prepared from the same sample of the representative experiment. Csf1r, colony stimulating factor 1 receptor; Calcr, calcitonin receptor; Tnfrsf11a, tumor necrosis factor receptor superfamily member 11a, RANK.
    Chain Complementary Deoxyribonucleic Acid Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher data analysis genomic deoxyribonucleic acid dna
    Osteoclastogenesis in wild-type (B6) and Fas deficient (Fas −/−) mice 4 weeks after ovariectomy (OVX) Osteoclastogenesis in vivo was assessed by histomorphometric analysis of distal femoral sections from sham-operated (SH) and OVX B6 and Fas −/− mice stained histochemically for the activity of tartrate-resistant acid phosphatase (TRAP). Osteoclastogenesis in vitro was assessed in cultures prepared from bone marrow of SH and OVX B6 and Fas −/− mice. (A) Number of TRAP positive osteoclasts per millimeter bone perimeter in distal femoral metaphyseal sections (mean±SD, t-test, *p≤0.03 vs. SH mice). (B) Number of TRAP positive osteoclasts (mean±SD, t-test, *p≤0.02 vs. SH B6 mice) on day 6 of cell culture. (C) Gene expression pattern in osteoclastogenic cultures from SH and OVX B6 and Fas −/− mice. For each time point in each group, cells were cultured in quadruplicates and pooled for RNA isolation. Day 0 represents gene expression in freshly isolated bone marrow cells. Values were calculated according to the standard curve of gene expression in the calibrator sample <t>(cDNA</t> from osteoclastogenic cultures) and normalized to the expression of the gene for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time <t>PCR</t> reaction duplicates prepared from the same sample of the representative experiment. Csf1r, colony stimulating factor 1 receptor; Calcr, calcitonin receptor; Tnfrsf11a, tumor necrosis factor receptor superfamily member 11a, RANK.
    Data Analysis Genomic Deoxyribonucleic Acid Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Thermo Fisher high capacity complementary deoxyribonucleic acid reverse transcription kit
    Osteoclastogenesis in wild-type (B6) and Fas deficient (Fas −/−) mice 4 weeks after ovariectomy (OVX) Osteoclastogenesis in vivo was assessed by histomorphometric analysis of distal femoral sections from sham-operated (SH) and OVX B6 and Fas −/− mice stained histochemically for the activity of tartrate-resistant acid phosphatase (TRAP). Osteoclastogenesis in vitro was assessed in cultures prepared from bone marrow of SH and OVX B6 and Fas −/− mice. (A) Number of TRAP positive osteoclasts per millimeter bone perimeter in distal femoral metaphyseal sections (mean±SD, t-test, *p≤0.03 vs. SH mice). (B) Number of TRAP positive osteoclasts (mean±SD, t-test, *p≤0.02 vs. SH B6 mice) on day 6 of cell culture. (C) Gene expression pattern in osteoclastogenic cultures from SH and OVX B6 and Fas −/− mice. For each time point in each group, cells were cultured in quadruplicates and pooled for RNA isolation. Day 0 represents gene expression in freshly isolated bone marrow cells. Values were calculated according to the standard curve of gene expression in the calibrator sample <t>(cDNA</t> from osteoclastogenic cultures) and normalized to the expression of the gene for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time <t>PCR</t> reaction duplicates prepared from the same sample of the representative experiment. Csf1r, colony stimulating factor 1 receptor; Calcr, calcitonin receptor; Tnfrsf11a, tumor necrosis factor receptor superfamily member 11a, RANK.
    High Capacity Complementary Deoxyribonucleic Acid Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine 2000
    TK and CD gene expression in HTF cells. (A) The plasmid map showing the expression vector, pAcGFP1-Hyg, carrying the TK and CD genes. (B) The 1,617 bp fragment of the TK-CD gene was confirmed by restriction enzyme digestion of the plasmid with  Xho I and  Kpn I followed by 1% DNA gel electrophoresis. Lane M, DNA marker; lane 1, vector pAcGFP1-Hyg; and lane 2, pAcGFP1-Hyg-TK-CD. (C) Reverse transcription-polymerase chain reaction analysis of HFT cells transfected with pAcGFP1-Hyg-TK-CD. Lane M, DL2000 marker; lane 1, fragment of TK-CD (403 bp) and β-actin (561 bp) in the transfected cells; lane 2, untransfected; and lane 3, negative control groups. (D) TK and CD gene expression mediated either by Lipo or G5-PAMAM-D was accessed by fluorescence microscopy (magnification, ×100). TK, thymidine kinase; CD, cytosine deaminase; HTFs, human Tenon's capsule fibroblasts; G5-PAMAM-D, 5-polyamidoamine dendrimers; bp, base pairs; Lipo, Lipofectamine 2000; ctrl, control.
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 377302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher trizol
    Splicing to include exon 3a is a developmentally regulated event in skeletal muscle. ( A ) Microscopy of cell cultures before, during and after differentiation. C2C12 myoblasts were grown to 70% confluence and induced to differentiate in DMEM+10% horse serum. Partially differentiated cultures containing both myoblasts and myotubes were observed by 2 days post-differentiation. After 48 hours, medium was replaced with DMEM+2% horse serum and 10 µM Ara-C and cultured for an additional 4 days. ( B ) qPCR of <t>RNA</t> harvested in <t>Trizol</t> showed that concurrent with a decrease in normal Actg1 , splicing to generate alternative Actg1 increases during differentiation into myotubes. Expression of both the normal and alternative transcripts was normalized to Ppia and is presented as fold-difference compared to skeletal muscle. A two-tailed type 2 Student's T-test was used to compare expression differences between time points. For all time points compared, p
    Trizol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 250292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher turbo dna free kit
    SMC4 coregulates loci silenced by maintenance <t>DNA</t> methylation and histone modifications associated with heterochromatin. ( A ) SMC4 silences a significant subset of MET1 and DDM1 targets. Venn diagram describing the relationship between TEs derepressed in the smc4-1 , met1-3 , and ddm1-2 mutants. Asterisks denote statistically significant overlaps. ( B ) Venn diagram showing the overlap between TEs derepressed in the smc4-1 , met1-3 , and cmt3-11t mutants. Asterisks denote statistically significant overlaps. ( C ) Venn diagram describing the relationships between TEs derepressed in the smc4-1 , ddm1-2 , and cmt3-11t mutants. Asterisks denote statistically significant overlaps. ( D ) Overlap between TEs derepressed in smc4-1 and suvh4 suvh5 suvh6 triple mutants. Asterisks denote statistically significant overlaps. ( E ) ATXR5 and ATXR6 help silence a subset of TEs that also require MET1, DDM1, and SMC4. The Venn diagram compares the 100 TEs derepressed in an atxr5 atxr6 double mutant with the 169 TEs that represent the overlap between the set of TEs derepressed in met1 , ddm1 , and smc4-1 . ( F ) Genome browser snapshot of H3K9me2 enrichment, H3K27me1 enrichment, localization of SMC4-dependent TEs, and the centromeric position on chromosome 1 in wild-type Col-0. Raw counts of fragment pileup for the ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data are shown in the top two tracks. Vertical bars in the third and fourth tracks represent SMC4-dependent TEs or centromeric repeats, respectively. ( G ) RT–PCR verification of the derepression of four TEs ( AT4TE15030 , AT2TE15880 , AT2TE19625 , and 106B ) predicted from <t>RNA-seq</t> data to be silenced via the partnership of MET1, DDM1, ATXR5/6, and SMC4. Genotypes tested are indicated at the top of the figure. UBQ reactions served as loading controls. Reactions omitting reverse transcriptase (no RT) control for DNA contamination. ( H ) Hierarchical clustering of the 169 TEs coregulated by SMC4, MET1, and DDM1, with TE expression levels displayed as a heat map. Expression levels were determined as RNA-seq reads corresponding to the TEs, normalized to the total number of mapped reads per genotype.
    Turbo Dna Free Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 21205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ilk complementary deoxyribonucleic acid cdna adenovirus vector
    SMC4 coregulates loci silenced by maintenance <t>DNA</t> methylation and histone modifications associated with heterochromatin. ( A ) SMC4 silences a significant subset of MET1 and DDM1 targets. Venn diagram describing the relationship between TEs derepressed in the smc4-1 , met1-3 , and ddm1-2 mutants. Asterisks denote statistically significant overlaps. ( B ) Venn diagram showing the overlap between TEs derepressed in the smc4-1 , met1-3 , and cmt3-11t mutants. Asterisks denote statistically significant overlaps. ( C ) Venn diagram describing the relationships between TEs derepressed in the smc4-1 , ddm1-2 , and cmt3-11t mutants. Asterisks denote statistically significant overlaps. ( D ) Overlap between TEs derepressed in smc4-1 and suvh4 suvh5 suvh6 triple mutants. Asterisks denote statistically significant overlaps. ( E ) ATXR5 and ATXR6 help silence a subset of TEs that also require MET1, DDM1, and SMC4. The Venn diagram compares the 100 TEs derepressed in an atxr5 atxr6 double mutant with the 169 TEs that represent the overlap between the set of TEs derepressed in met1 , ddm1 , and smc4-1 . ( F ) Genome browser snapshot of H3K9me2 enrichment, H3K27me1 enrichment, localization of SMC4-dependent TEs, and the centromeric position on chromosome 1 in wild-type Col-0. Raw counts of fragment pileup for the ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data are shown in the top two tracks. Vertical bars in the third and fourth tracks represent SMC4-dependent TEs or centromeric repeats, respectively. ( G ) RT–PCR verification of the derepression of four TEs ( AT4TE15030 , AT2TE15880 , AT2TE19625 , and 106B ) predicted from <t>RNA-seq</t> data to be silenced via the partnership of MET1, DDM1, ATXR5/6, and SMC4. Genotypes tested are indicated at the top of the figure. UBQ reactions served as loading controls. Reactions omitting reverse transcriptase (no RT) control for DNA contamination. ( H ) Hierarchical clustering of the 169 TEs coregulated by SMC4, MET1, and DDM1, with TE expression levels displayed as a heat map. Expression levels were determined as RNA-seq reads corresponding to the TEs, normalized to the total number of mapped reads per genotype.
    Ilk Complementary Deoxyribonucleic Acid Cdna Adenovirus Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher high capacity complimentary deoxyribonucleic acid cdna archive kit
    SMC4 coregulates loci silenced by maintenance <t>DNA</t> methylation and histone modifications associated with heterochromatin. ( A ) SMC4 silences a significant subset of MET1 and DDM1 targets. Venn diagram describing the relationship between TEs derepressed in the smc4-1 , met1-3 , and ddm1-2 mutants. Asterisks denote statistically significant overlaps. ( B ) Venn diagram showing the overlap between TEs derepressed in the smc4-1 , met1-3 , and cmt3-11t mutants. Asterisks denote statistically significant overlaps. ( C ) Venn diagram describing the relationships between TEs derepressed in the smc4-1 , ddm1-2 , and cmt3-11t mutants. Asterisks denote statistically significant overlaps. ( D ) Overlap between TEs derepressed in smc4-1 and suvh4 suvh5 suvh6 triple mutants. Asterisks denote statistically significant overlaps. ( E ) ATXR5 and ATXR6 help silence a subset of TEs that also require MET1, DDM1, and SMC4. The Venn diagram compares the 100 TEs derepressed in an atxr5 atxr6 double mutant with the 169 TEs that represent the overlap between the set of TEs derepressed in met1 , ddm1 , and smc4-1 . ( F ) Genome browser snapshot of H3K9me2 enrichment, H3K27me1 enrichment, localization of SMC4-dependent TEs, and the centromeric position on chromosome 1 in wild-type Col-0. Raw counts of fragment pileup for the ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data are shown in the top two tracks. Vertical bars in the third and fourth tracks represent SMC4-dependent TEs or centromeric repeats, respectively. ( G ) RT–PCR verification of the derepression of four TEs ( AT4TE15030 , AT2TE15880 , AT2TE19625 , and 106B ) predicted from <t>RNA-seq</t> data to be silenced via the partnership of MET1, DDM1, ATXR5/6, and SMC4. Genotypes tested are indicated at the top of the figure. UBQ reactions served as loading controls. Reactions omitting reverse transcriptase (no RT) control for DNA contamination. ( H ) Hierarchical clustering of the 169 TEs coregulated by SMC4, MET1, and DDM1, with TE expression levels displayed as a heat map. Expression levels were determined as RNA-seq reads corresponding to the TEs, normalized to the total number of mapped reads per genotype.
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    Thermo Fisher dcm dna
    ( a ) TLC identification of 5hmC in A. mellifera. Single radiolabelled nucleotides derived from a variety of samples were separated by TLC on PEI cellulose. Samples obtained from single dNTPs were used as standards. Three genomic <t>DNA</t> preparations were digested to single nucleotides and immunoprecipitated with anti-5hmC antibodies. Purified nucleotides were radiolabelled and resolved by TLC. A spot representing 5hmC is present in drone testes DNA, whereas a much stronger spot can be seen for mouse brain known to be enriched in 5hmC (white arrows). No 5hmC spot can be found in λ phage (Dam − <t>Dcm</t> − ). All lanes are from the same TLC plate. ( b ) An image of a DNA dot-blot hybridized with an anti-5hmC antibody. For each sample, 1 and 2 µg of DNA were spotted on the membrane. A PCR product with dCTP substituted for d5hmCTP was used as control. ( c ) 5hmC quantification in various honeybee DNA samples. The data points were obtained using two methods; a densitometry scan of the dot-blots shown in ( b ), and a β-glucosyltransferase assay (see Material and methods). The resulting values were plotted as either fractions of total cytosines (top) or as a number of 5hmCs in a haploid genome (bottom). The overall correlation between two methods is 0.712. Q, W, D and E refer to queen, worker, drone and embryos, respectively.
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    Thermo Fisher edta
    Predicted domain organization and transglutaminase activity of TgpA protein. (A) Map of the predicted domains DUF3488 (PF11992) and TG (PF01841) along the primary sequence of the PA2873 gene product, called TgpA. The sequence of TgpA spanning aa 396 to 467 of the TG domain is highlighted and aligned to homologous functional TG domains of human coagulation Factor XIII, fish-derived transglutaminase (FTG) and WbmE protein from B. bronchiseptica . Conserved aminoacids of catalytic triad are indicated by an asterisk. (B) Colorimetric assay of transglutaminase activity of purified TgpA TG 180–544 domain by Transglutaminase Colorimetric Microassay Kit (TCM kit; Covalab). TCM kit uses immobilized N-carbobenzoxy(CBZ)-Gln-Gly as the amine acceptor and biotin-conjugated cadaverine as the amine donor. The indicated amounts of purified TgpA TG 180–544 (stock: 2.7 mg/ml, 95% purity) were incubated in 96-well microtiter plate coated with CBZ-Gln-Gly at <t>37°C</t> for 15 min with calcium, DTT and biotinylated cadaverine, both in the presence and the absence of <t>EDTA</t> supplied in the kit. As a reference for TGase activity, the indicated amounts of kit-included purified guinea pig TGase with specific activity of 0.1 U/mg were incubated under the same conditions. The wells were washed extensively and filled with streptavidin-labelled horseradish peroxidase (HRP) to assay the formation of immobilized γ-glutamyl-cadaverine-biotin by OD 450 measurement of HRP activity using H 2 O 2 as substrate and tetramethyl benzidine as electron acceptor (chromogen).
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    Recombinant CMV mutant lacking expression of pUL31-YFP exhibits an MOI-dependent replication defect. (A) UL31 was disrupted in CMV TB40/E (TB wt ) by replacing 1,200 bp of the UL31 ORF with the galK gene (UL31 del ) or insertion of galK into the first ATG of UL31 (UL31 in ). The locations of the first two ATG sequences are indicated. (B) MRC-5 fibroblasts were infected using TB GFP , UL31 del , or UL31 in virus at an MOI of 3. Viral titers from culture supernatants at 72 and 96 hpi were quantified using a TCID 50 assay. Data are from three biological replicates and two technical replicates, with error bars representing standard deviations from the means. (C) Whole-cell lysates were collected at 96 hpi and analyzed using Western blotting and the indicated antibodies. (D) A stop codon, TAG, was introduced into the second ATG of AD169 UL31 YFP , resulting in UL31 stop virus. (E) Fibroblasts were infected with UL31 YFP or UL31 stop virus at an MOI of 3. Total <t>RNA</t> was collected at 96 hpi and analyzed using qRT-PCR with primers to UL32 (pp150) and GAPDH. Data include three biological replicates and two technical replicates, with error bars representing standard deviations from the means. Whole-cell lysates were analyzed by Western blotting. (F) Fibroblasts were infected with UL31 YFP or UL31 stop virus at an MOI of 0.05 IU per cell. Whole-cell <t>DNA</t> was isolated at the indicated times, and relative viral genomes were determined by qPCR using primers for UL123 and GAPDH. Viral titers were determined from cell-free virus at 120 hpi. (G) Titers were also determined following infection at an MOI of 3. Data include three biological replicates and two technical replicates, with error bars representing standard deviations from the means.
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    Thermo Fisher quant it picrogreen double stranded deoxyribonucleic acid dna assay kit
    Recombinant CMV mutant lacking expression of pUL31-YFP exhibits an MOI-dependent replication defect. (A) UL31 was disrupted in CMV TB40/E (TB wt ) by replacing 1,200 bp of the UL31 ORF with the galK gene (UL31 del ) or insertion of galK into the first ATG of UL31 (UL31 in ). The locations of the first two ATG sequences are indicated. (B) MRC-5 fibroblasts were infected using TB GFP , UL31 del , or UL31 in virus at an MOI of 3. Viral titers from culture supernatants at 72 and 96 hpi were quantified using a TCID 50 assay. Data are from three biological replicates and two technical replicates, with error bars representing standard deviations from the means. (C) Whole-cell lysates were collected at 96 hpi and analyzed using Western blotting and the indicated antibodies. (D) A stop codon, TAG, was introduced into the second ATG of AD169 UL31 YFP , resulting in UL31 stop virus. (E) Fibroblasts were infected with UL31 YFP or UL31 stop virus at an MOI of 3. Total <t>RNA</t> was collected at 96 hpi and analyzed using qRT-PCR with primers to UL32 (pp150) and GAPDH. Data include three biological replicates and two technical replicates, with error bars representing standard deviations from the means. Whole-cell lysates were analyzed by Western blotting. (F) Fibroblasts were infected with UL31 YFP or UL31 stop virus at an MOI of 0.05 IU per cell. Whole-cell <t>DNA</t> was isolated at the indicated times, and relative viral genomes were determined by qPCR using primers for UL123 and GAPDH. Viral titers were determined from cell-free virus at 120 hpi. (G) Titers were also determined following infection at an MOI of 3. Data include three biological replicates and two technical replicates, with error bars representing standard deviations from the means.
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    Real-time one-step RT-PCR evaluation of cDNA priming strategies and the effect of unbalanced target concentrations . A) Real-time one-step RT-PCR evaluation of the effect of different RT primers on quantification of RNA expression in singleplex and in triplex. Reactions employed either an oligo(dT) 18 RT primer, a random decamer RT primer, or a combination of oligo(dT) 18 and random decamer RT primers for cDNA synthesis. In addition, each one-step RT-PCR protocol employed <t>Taq</t> <t>DNA</t> polymerase, M-MLV RT, 0.8 μg of human thymus total RNA, and CleanAmp™ Precision PCR primers. Reactions were performed in triplicate. B) Real-time one-step RT-PCR evaluation of triplex one-step RT-PCR amplifications using different custom prepared mixes containing three RNA standards in different ratios. The relative abundance for each mixture A through H is represented in the following format: (X:Y:Z), where the copies of the ABCA5 RNA standard is present at 10^X copies, the ABCA6 RNA standard is present at 10^Y copies, and the ABCA7 RNA standard is present at 10^Z copies. The observed copy number for each reaction, which was performed in triplicate, was obtained by extrapolation of the Cq to a standard curve for the ABCA5, ABCA6, and ABCA7 RNA standards. The resultant data for each RNA sample was normalized to ABCA7 and was plotted graphically.
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    Derivation of RelA −/− VECs, VSMCs, and MSCs from RelA −/− ESCs . (A) Flow cytometric analysis of WT and  RelA −/−  VECs with VEC-specific markers CD34 and CD201. IgG-FITC and IgG-PE were used as isotype controls. (B) Flow cytometric analysis of WT and  RelA −/−  VSMCs with VSMC-specific marker, CD140b. IgG-APC was used as an isotype control. (C) Flow cytometric analysis of WT and  RelA −/−  MSCs with MSC-specific markers, CD73, CD90 and CD105. IgG-FITC, IgG-PE and IgG-APC were used as isotype controls. (D) Immunostaining of WT and  RelA −/−  VECs with VEC-specific markers, vWF and CD31. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (E) Immunostaining of WT and  RelA −/−  VSMCs with VSMC-specific markers, SM22 and Calponin. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (F) Western blot analysis of RelA protein in WT and  RelA −/−  VECs, VSMCs and MSCs, respectively. β-Actin was used as a loading control. (G) Immunostaining of RelA in WT and  RelA −/−  VECs, VSMCs and MSCs under basal condition. DNA was labeled by Hoechst 33342. Scale bar, 10 μm
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    Derivation of RelA −/− VECs, VSMCs, and MSCs from RelA −/− ESCs . (A) Flow cytometric analysis of WT and  RelA −/−  VECs with VEC-specific markers CD34 and CD201. IgG-FITC and IgG-PE were used as isotype controls. (B) Flow cytometric analysis of WT and  RelA −/−  VSMCs with VSMC-specific marker, CD140b. IgG-APC was used as an isotype control. (C) Flow cytometric analysis of WT and  RelA −/−  MSCs with MSC-specific markers, CD73, CD90 and CD105. IgG-FITC, IgG-PE and IgG-APC were used as isotype controls. (D) Immunostaining of WT and  RelA −/−  VECs with VEC-specific markers, vWF and CD31. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (E) Immunostaining of WT and  RelA −/−  VSMCs with VSMC-specific markers, SM22 and Calponin. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (F) Western blot analysis of RelA protein in WT and  RelA −/−  VECs, VSMCs and MSCs, respectively. β-Actin was used as a loading control. (G) Immunostaining of RelA in WT and  RelA −/−  VECs, VSMCs and MSCs under basal condition. DNA was labeled by Hoechst 33342. Scale bar, 10 μm
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    Derivation of RelA −/− VECs, VSMCs, and MSCs from RelA −/− ESCs . (A) Flow cytometric analysis of WT and  RelA −/−  VECs with VEC-specific markers CD34 and CD201. IgG-FITC and IgG-PE were used as isotype controls. (B) Flow cytometric analysis of WT and  RelA −/−  VSMCs with VSMC-specific marker, CD140b. IgG-APC was used as an isotype control. (C) Flow cytometric analysis of WT and  RelA −/−  MSCs with MSC-specific markers, CD73, CD90 and CD105. IgG-FITC, IgG-PE and IgG-APC were used as isotype controls. (D) Immunostaining of WT and  RelA −/−  VECs with VEC-specific markers, vWF and CD31. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (E) Immunostaining of WT and  RelA −/−  VSMCs with VSMC-specific markers, SM22 and Calponin. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (F) Western blot analysis of RelA protein in WT and  RelA −/−  VECs, VSMCs and MSCs, respectively. β-Actin was used as a loading control. (G) Immunostaining of RelA in WT and  RelA −/−  VECs, VSMCs and MSCs under basal condition. DNA was labeled by Hoechst 33342. Scale bar, 10 μm
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    Comparison of five automated DNA extraction systems. Illustration of relative DNA concentrations of samples 1–10 measured by the <t>NanoDrop</t> <t>2000c</t> spectrophotometer (A) and Qubit 2.0 fluorometer (B). The concentrations of the Maxwell 16 extracts were set to 100% for each sample.
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    Comparison of five automated DNA extraction systems. Illustration of relative DNA concentrations of samples 1–10 measured by the <t>NanoDrop</t> <t>2000c</t> spectrophotometer (A) and Qubit 2.0 fluorometer (B). The concentrations of the Maxwell 16 extracts were set to 100% for each sample.
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    Comparison of five automated DNA extraction systems. Illustration of relative DNA concentrations of samples 1–10 measured by the <t>NanoDrop</t> <t>2000c</t> spectrophotometer (A) and Qubit 2.0 fluorometer (B). The concentrations of the Maxwell 16 extracts were set to 100% for each sample.
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    Comparison of five automated DNA extraction systems. Illustration of relative DNA concentrations of samples 1–10 measured by the <t>NanoDrop</t> <t>2000c</t> spectrophotometer (A) and Qubit 2.0 fluorometer (B). The concentrations of the Maxwell 16 extracts were set to 100% for each sample.
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    Image Search Results


    RT progression assay determining G-quartet formation in the Sp1 binding region. Cation-dependent pausing of reverse transcription at the guanine-rich elements in the U3 region was analyzed with RNA and DNA templates. A fragment of the RNA/DNA sequence of the Sp1 binding region is shown (top) with G-rich elements (shaded). Strong pauses of the RT near G-rich elements were observed in the presence of 50 mM of KCl, but not LiCl, indicating that these elements are involved in the formation of structure, which is stabilized by potassium ions but destabilized by lithium ions. This is indicative of a G-quadruplex. The cation-independent RT pauses are likely caused by hairpin structures. DNA primer, P; DNA marker, M; KCl, K + ; LiCl, Li + .

    Journal: Biochemistry

    Article Title: U3 Region in the HIV-1 Genome Adopts a G-Quadruplex Structure in Its RNA and DNA Sequence

    doi: 10.1021/bi4016692

    Figure Lengend Snippet: RT progression assay determining G-quartet formation in the Sp1 binding region. Cation-dependent pausing of reverse transcription at the guanine-rich elements in the U3 region was analyzed with RNA and DNA templates. A fragment of the RNA/DNA sequence of the Sp1 binding region is shown (top) with G-rich elements (shaded). Strong pauses of the RT near G-rich elements were observed in the presence of 50 mM of KCl, but not LiCl, indicating that these elements are involved in the formation of structure, which is stabilized by potassium ions but destabilized by lithium ions. This is indicative of a G-quadruplex. The cation-independent RT pauses are likely caused by hairpin structures. DNA primer, P; DNA marker, M; KCl, K + ; LiCl, Li + .

    Article Snippet: Preparation of RNA Templates RNA molecules were transcribed in vitro (Ambion T7-MEGAshortscript kit; Applied Biosystems) from DNA templates amplified by PCR using Vent DNA polymerase (New England BioLabs, Inc.) and two overlapping oligomers with the sequence of the desired region.

    Techniques: Binding Assay, Sequencing, Marker

    Formation of the structure stabilized by potassium ions in the HIV-1 U3 region facilitates RT template switching during reverse transcription. (A) Reconstituted system to analyze the influence of G-rich elements on strand transfer during HIV-1 minus strand DNA synthesis in vitro . Donor and acceptor RNA templates represent two copies of the viral RNA genome; in which reverse transcription is initiated from a 32 P-labeled DNA primer annealed to the donor RNA. The acceptor RNA does not share a homology (circle) with two nucleotides at the 5′ end of the donor RNA. TP, transfer product; DE, donor extension product; and P, DNA primer. (B) A time course of strand transfer reactions performed in the presence of potassium and lithium ions. Samples were collected at 1, 5, 15, and 30 min after the reaction was initiated. Formation of a potassium-dependent structure, anticipated to be a G-quadruplex, in the RNA template paused the RT during minus strand DNA synthesis and influenced the yield of the final products. The transfer efficiency decreased about 37% in reactions with lithium ions, presumably because the templates could not form a G-quadruplex.

    Journal: Biochemistry

    Article Title: U3 Region in the HIV-1 Genome Adopts a G-Quadruplex Structure in Its RNA and DNA Sequence

    doi: 10.1021/bi4016692

    Figure Lengend Snippet: Formation of the structure stabilized by potassium ions in the HIV-1 U3 region facilitates RT template switching during reverse transcription. (A) Reconstituted system to analyze the influence of G-rich elements on strand transfer during HIV-1 minus strand DNA synthesis in vitro . Donor and acceptor RNA templates represent two copies of the viral RNA genome; in which reverse transcription is initiated from a 32 P-labeled DNA primer annealed to the donor RNA. The acceptor RNA does not share a homology (circle) with two nucleotides at the 5′ end of the donor RNA. TP, transfer product; DE, donor extension product; and P, DNA primer. (B) A time course of strand transfer reactions performed in the presence of potassium and lithium ions. Samples were collected at 1, 5, 15, and 30 min after the reaction was initiated. Formation of a potassium-dependent structure, anticipated to be a G-quadruplex, in the RNA template paused the RT during minus strand DNA synthesis and influenced the yield of the final products. The transfer efficiency decreased about 37% in reactions with lithium ions, presumably because the templates could not form a G-quadruplex.

    Article Snippet: Preparation of RNA Templates RNA molecules were transcribed in vitro (Ambion T7-MEGAshortscript kit; Applied Biosystems) from DNA templates amplified by PCR using Vent DNA polymerase (New England BioLabs, Inc.) and two overlapping oligomers with the sequence of the desired region.

    Techniques: DNA Synthesis, In Vitro, Labeling

    CD spectral analysis of the RNA and single stranded DNA with Sp1 binding sites in HIV-1. CD spectra indicate the formation of the parallel G-quadruplex for the RNA template and an antiparallel or hybrid G-quadruplex for single stranded DNA. For reference, a profile of the G-rich sequence (50% of Gs; GGGGGGAUUGUG UGGUACAGUGCAGAGA), which is unable to adopt G-quadruplex structure, is shown in gray.

    Journal: Biochemistry

    Article Title: U3 Region in the HIV-1 Genome Adopts a G-Quadruplex Structure in Its RNA and DNA Sequence

    doi: 10.1021/bi4016692

    Figure Lengend Snippet: CD spectral analysis of the RNA and single stranded DNA with Sp1 binding sites in HIV-1. CD spectra indicate the formation of the parallel G-quadruplex for the RNA template and an antiparallel or hybrid G-quadruplex for single stranded DNA. For reference, a profile of the G-rich sequence (50% of Gs; GGGGGGAUUGUG UGGUACAGUGCAGAGA), which is unable to adopt G-quadruplex structure, is shown in gray.

    Article Snippet: Preparation of RNA Templates RNA molecules were transcribed in vitro (Ambion T7-MEGAshortscript kit; Applied Biosystems) from DNA templates amplified by PCR using Vent DNA polymerase (New England BioLabs, Inc.) and two overlapping oligomers with the sequence of the desired region.

    Techniques: Binding Assay, Sequencing

    DNA sequences at the Dissociation ( Ds ) -NAT1 excision and reinsertion sites. (A) Ds excision footprints of 128 independent KMY103G1 ADE2 revertant colonies. The top row shows the ade2 :: Ds-NAT1 sequence on chromosome 3. Rows 1–28 show the recovered Ds excision footprints and their incidence ( F ) in independent ADE2 revertants from Ac TPase4xCa-expressing KMY103G1 cells grown in maltose-containing medium. Arrows above the sequences indicate inverted repeats centered around the complementary bases C and A (boldface red letters) of the nucleotides bordering the Ds-NAT1 transposon that result from the resolution of intermediate hairpin structures. Lower case letters indicate nucleotides that are not explained with the hairpin model. (B) Target site duplications at Ds-NAT1 reinsertion sites in transposants “1,” “2,” and “3”; see Table S2 in File S1 .

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Maize Transposable Elements Ac/Ds as Insertion Mutagenesis Tools in Candida albicans

    doi: 10.1534/g3.117.300388

    Figure Lengend Snippet: DNA sequences at the Dissociation ( Ds ) -NAT1 excision and reinsertion sites. (A) Ds excision footprints of 128 independent KMY103G1 ADE2 revertant colonies. The top row shows the ade2 :: Ds-NAT1 sequence on chromosome 3. Rows 1–28 show the recovered Ds excision footprints and their incidence ( F ) in independent ADE2 revertants from Ac TPase4xCa-expressing KMY103G1 cells grown in maltose-containing medium. Arrows above the sequences indicate inverted repeats centered around the complementary bases C and A (boldface red letters) of the nucleotides bordering the Ds-NAT1 transposon that result from the resolution of intermediate hairpin structures. Lower case letters indicate nucleotides that are not explained with the hairpin model. (B) Target site duplications at Ds-NAT1 reinsertion sites in transposants “1,” “2,” and “3”; see Table S2 in File S1 .

    Article Snippet: Construction of Ac/Ds components The codon-adapted Ac TPase4xCa open reading frame (Supplemental Material, Figure S1 in File S1 ) was constructed by fusing three de novo -synthesized DNA fragments (Thermo Fisher Scientific GENEART GmbH, Regensburg, Germany) and cloned into pJET1.2 via the CloneJET kit (Thermo Fisher Scientific).

    Techniques: Sequencing, Expressing

    Diabetes-induced increase in inflammatory proteins in photoreceptor cells. There was no detection of iNOS in the photoreceptor region in the nondiabetic retina ( A ), but in diabetes, there were increased levels of iNOS in the photoreceptor region ( B ). There was no detection of COX2 in the photoreceptor region of the nondiabetic retina ( C ), but in diabetes, there were increased levels of COX2 in the photoreceptor region in diabetes ( D ). The nuclei were stained with Hoechst DNA dye ( blue ). Micrographs are representative of three or more animals per group.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Photoreceptor Cells Produce Inflammatory Mediators That Contribute to Endothelial Cell Death in Diabetes

    doi: 10.1167/iovs.16-19859

    Figure Lengend Snippet: Diabetes-induced increase in inflammatory proteins in photoreceptor cells. There was no detection of iNOS in the photoreceptor region in the nondiabetic retina ( A ), but in diabetes, there were increased levels of iNOS in the photoreceptor region ( B ). There was no detection of COX2 in the photoreceptor region of the nondiabetic retina ( C ), but in diabetes, there were increased levels of COX2 in the photoreceptor region in diabetes ( D ). The nuclei were stained with Hoechst DNA dye ( blue ). Micrographs are representative of three or more animals per group.

    Article Snippet: Hoechst deoxyribonucleic acid dye (Thermo, Rockford, IL, USA) at 1:1000 was used to stain nuclei.

    Techniques: Staining

    Osteoclastogenesis in wild-type (B6) and Fas deficient (Fas −/−) mice 4 weeks after ovariectomy (OVX) Osteoclastogenesis in vivo was assessed by histomorphometric analysis of distal femoral sections from sham-operated (SH) and OVX B6 and Fas −/− mice stained histochemically for the activity of tartrate-resistant acid phosphatase (TRAP). Osteoclastogenesis in vitro was assessed in cultures prepared from bone marrow of SH and OVX B6 and Fas −/− mice. (A) Number of TRAP positive osteoclasts per millimeter bone perimeter in distal femoral metaphyseal sections (mean±SD, t-test, *p≤0.03 vs. SH mice). (B) Number of TRAP positive osteoclasts (mean±SD, t-test, *p≤0.02 vs. SH B6 mice) on day 6 of cell culture. (C) Gene expression pattern in osteoclastogenic cultures from SH and OVX B6 and Fas −/− mice. For each time point in each group, cells were cultured in quadruplicates and pooled for RNA isolation. Day 0 represents gene expression in freshly isolated bone marrow cells. Values were calculated according to the standard curve of gene expression in the calibrator sample (cDNA from osteoclastogenic cultures) and normalized to the expression of the gene for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time PCR reaction duplicates prepared from the same sample of the representative experiment. Csf1r, colony stimulating factor 1 receptor; Calcr, calcitonin receptor; Tnfrsf11a, tumor necrosis factor receptor superfamily member 11a, RANK.

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Fas receptor is required for estrogen deficiency-induced bone loss in mice

    doi: 10.1038/labinvest.2009.144

    Figure Lengend Snippet: Osteoclastogenesis in wild-type (B6) and Fas deficient (Fas −/−) mice 4 weeks after ovariectomy (OVX) Osteoclastogenesis in vivo was assessed by histomorphometric analysis of distal femoral sections from sham-operated (SH) and OVX B6 and Fas −/− mice stained histochemically for the activity of tartrate-resistant acid phosphatase (TRAP). Osteoclastogenesis in vitro was assessed in cultures prepared from bone marrow of SH and OVX B6 and Fas −/− mice. (A) Number of TRAP positive osteoclasts per millimeter bone perimeter in distal femoral metaphyseal sections (mean±SD, t-test, *p≤0.03 vs. SH mice). (B) Number of TRAP positive osteoclasts (mean±SD, t-test, *p≤0.02 vs. SH B6 mice) on day 6 of cell culture. (C) Gene expression pattern in osteoclastogenic cultures from SH and OVX B6 and Fas −/− mice. For each time point in each group, cells were cultured in quadruplicates and pooled for RNA isolation. Day 0 represents gene expression in freshly isolated bone marrow cells. Values were calculated according to the standard curve of gene expression in the calibrator sample (cDNA from osteoclastogenic cultures) and normalized to the expression of the gene for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time PCR reaction duplicates prepared from the same sample of the representative experiment. Csf1r, colony stimulating factor 1 receptor; Calcr, calcitonin receptor; Tnfrsf11a, tumor necrosis factor receptor superfamily member 11a, RANK.

    Article Snippet: For PCR amplification, 2 μg of total RNA was converted to complementary deoxyribonucleic acid (cDNA) by reverse transcriptase (Applied Biosystems, Foster City, CA, USA).

    Techniques: Mouse Assay, In Vivo, Staining, Activity Assay, In Vitro, Cell Culture, Expressing, Isolation, Real-time Polymerase Chain Reaction

    Osteoblast differentiation and activity in wild-type (B6) and Fas deficient (Fas −/−) mice 4 weeks after ovariectomy (OVX) Osteoblast activity in vivo was assessed by dynamic histomorphometry. Osteoblastogenic cultures were prepared from bone marrow of sham-operated (SH) and OVX B6 and Fas −/− mice. (A) Mineral apposition rate (MAR) in SH and OVX B6 and Fas −/− mice was assessed by histomorphometric analysis of distal femoral sections:* p≤ 0.001 (t-test) vs. SH mice, ** p≤ 0.001 (t-test) vs. B6 mice. (B) Osteoblastic colonies from SH and OVX B6 and Fas −/− mice on day 14 of cell culture, stained red for alkaline phosphatase (AP). Number of osteoblast colonies is presented as mean±SD of culture duplicates: t-test, * p≤ 0.05 vs. SH mice, ** p≤ 0.05 vs. B6 mice (C) Percent increase in the number of osteoblast colonies in OVX mice compared to SH mice (mean±SD increase in four repeated experiments; t-test, * p=0.002 vs. B6 mice). (D) Gene expression pattern during in vitro osteoblast differentiation. For each time point in each group, cells were cultured in triplicates pooled for RNA isolation. Day 0 represents gene expression in freshly isolated bone marrow cells. Values were calculated according to the standard curve of gene expression in the calibrator sample (cDNA from osteoblastogenic culture), and normalized to the expression of the gene for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time PCR reaction duplicates prepared from the same sample of the representative experiment. Akp, alkaline phosphatase gene; AP, alkaline phosphatase; Bglap2, osteocalcin; Runx2, runt-related transcription factor 2; Tnfsf11, tumor necrosis factor receptor superfamily member 11, RANKL; Tnfrsf11b, tumor necrosis factor receptor superfamily member 11b, OPG.

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Fas receptor is required for estrogen deficiency-induced bone loss in mice

    doi: 10.1038/labinvest.2009.144

    Figure Lengend Snippet: Osteoblast differentiation and activity in wild-type (B6) and Fas deficient (Fas −/−) mice 4 weeks after ovariectomy (OVX) Osteoblast activity in vivo was assessed by dynamic histomorphometry. Osteoblastogenic cultures were prepared from bone marrow of sham-operated (SH) and OVX B6 and Fas −/− mice. (A) Mineral apposition rate (MAR) in SH and OVX B6 and Fas −/− mice was assessed by histomorphometric analysis of distal femoral sections:* p≤ 0.001 (t-test) vs. SH mice, ** p≤ 0.001 (t-test) vs. B6 mice. (B) Osteoblastic colonies from SH and OVX B6 and Fas −/− mice on day 14 of cell culture, stained red for alkaline phosphatase (AP). Number of osteoblast colonies is presented as mean±SD of culture duplicates: t-test, * p≤ 0.05 vs. SH mice, ** p≤ 0.05 vs. B6 mice (C) Percent increase in the number of osteoblast colonies in OVX mice compared to SH mice (mean±SD increase in four repeated experiments; t-test, * p=0.002 vs. B6 mice). (D) Gene expression pattern during in vitro osteoblast differentiation. For each time point in each group, cells were cultured in triplicates pooled for RNA isolation. Day 0 represents gene expression in freshly isolated bone marrow cells. Values were calculated according to the standard curve of gene expression in the calibrator sample (cDNA from osteoblastogenic culture), and normalized to the expression of the gene for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time PCR reaction duplicates prepared from the same sample of the representative experiment. Akp, alkaline phosphatase gene; AP, alkaline phosphatase; Bglap2, osteocalcin; Runx2, runt-related transcription factor 2; Tnfsf11, tumor necrosis factor receptor superfamily member 11, RANKL; Tnfrsf11b, tumor necrosis factor receptor superfamily member 11b, OPG.

    Article Snippet: For PCR amplification, 2 μg of total RNA was converted to complementary deoxyribonucleic acid (cDNA) by reverse transcriptase (Applied Biosystems, Foster City, CA, USA).

    Techniques: Activity Assay, Mouse Assay, In Vivo, T-Test, Cell Culture, Staining, Expressing, In Vitro, Isolation, Real-time Polymerase Chain Reaction, ALP Assay

    Estradiol does not influence Fas mediated osteoclast apoptosis Osteoclastogenic cultures were prepared from bone marrow of wt mice and treated with 0.5 μg/mL of anti-Fas antibody (Jo-1) and 5 μg/mL protein G at day 1 and 4. Cells treated with 0.5 μg/mL normal hamster IgG, and 5 μg/mL protein G were used as negative controls. 10 nM estradiol was added to osteoclastogenic cultures with each medium exchange and a corresponding volume of ethanol (used as a solvent for estradiol) was used as a negative control. (A) Expression of Fas in osteoclastogenic cultures after treatment with estradiol. Values were calculated according to the standard curve of gene expression in the calibrator sample (cDNA from osteoclastogenic cultures) and normalized to the expression of the gene for β-actin (“endogenous” control). Cell treatment was performed in six wells and three wells were pooled for RNA isolation. Results are arithmetic mean±SD of relative Fas mRNA expression in two pooled samples. (B) Representative data of the Annexin V-FITC/PI labeling of apoptotic cells. Numbers represent percentages of cells in each quadrant. Apoptotic cells are in the lower right quadrant, and dead cells are in the upper right quadrant.

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Fas receptor is required for estrogen deficiency-induced bone loss in mice

    doi: 10.1038/labinvest.2009.144

    Figure Lengend Snippet: Estradiol does not influence Fas mediated osteoclast apoptosis Osteoclastogenic cultures were prepared from bone marrow of wt mice and treated with 0.5 μg/mL of anti-Fas antibody (Jo-1) and 5 μg/mL protein G at day 1 and 4. Cells treated with 0.5 μg/mL normal hamster IgG, and 5 μg/mL protein G were used as negative controls. 10 nM estradiol was added to osteoclastogenic cultures with each medium exchange and a corresponding volume of ethanol (used as a solvent for estradiol) was used as a negative control. (A) Expression of Fas in osteoclastogenic cultures after treatment with estradiol. Values were calculated according to the standard curve of gene expression in the calibrator sample (cDNA from osteoclastogenic cultures) and normalized to the expression of the gene for β-actin (“endogenous” control). Cell treatment was performed in six wells and three wells were pooled for RNA isolation. Results are arithmetic mean±SD of relative Fas mRNA expression in two pooled samples. (B) Representative data of the Annexin V-FITC/PI labeling of apoptotic cells. Numbers represent percentages of cells in each quadrant. Apoptotic cells are in the lower right quadrant, and dead cells are in the upper right quadrant.

    Article Snippet: For PCR amplification, 2 μg of total RNA was converted to complementary deoxyribonucleic acid (cDNA) by reverse transcriptase (Applied Biosystems, Foster City, CA, USA).

    Techniques: Mouse Assay, Negative Control, Expressing, Isolation, Labeling

    Gene expression of Fas and Fasl in bone, bone marrow, osteoblast, and osteoclast lineage cells in wild-type mice 4 weeks after ovariectomy (OVX) (A) Expression of Fas/Fasl mRNA in bone and bone marrow. (B) Expression of Fas/Fasl mRNA in osteoclastogenic cultures. (C) Expression of Fas/Fasl mRNA in osteoblastogenic cultures. Expression was calculated according to the standard curve for Fas/Fasl expression in the calibrator sample (cDNA from bone, bone marrow, osteoblastogenic or osteoblastogenic cultures), and normalized to the mRNA quantity for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time PCR reaction duplicates prepared from the same sample of the representative experiment.

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Fas receptor is required for estrogen deficiency-induced bone loss in mice

    doi: 10.1038/labinvest.2009.144

    Figure Lengend Snippet: Gene expression of Fas and Fasl in bone, bone marrow, osteoblast, and osteoclast lineage cells in wild-type mice 4 weeks after ovariectomy (OVX) (A) Expression of Fas/Fasl mRNA in bone and bone marrow. (B) Expression of Fas/Fasl mRNA in osteoclastogenic cultures. (C) Expression of Fas/Fasl mRNA in osteoblastogenic cultures. Expression was calculated according to the standard curve for Fas/Fasl expression in the calibrator sample (cDNA from bone, bone marrow, osteoblastogenic or osteoblastogenic cultures), and normalized to the mRNA quantity for β-actin (“endogenous” control). Results are arithmetic mean±SD of real-time PCR reaction duplicates prepared from the same sample of the representative experiment.

    Article Snippet: For PCR amplification, 2 μg of total RNA was converted to complementary deoxyribonucleic acid (cDNA) by reverse transcriptase (Applied Biosystems, Foster City, CA, USA).

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    TK and CD gene expression in HTF cells. (A) The plasmid map showing the expression vector, pAcGFP1-Hyg, carrying the TK and CD genes. (B) The 1,617 bp fragment of the TK-CD gene was confirmed by restriction enzyme digestion of the plasmid with  Xho I and  Kpn I followed by 1% DNA gel electrophoresis. Lane M, DNA marker; lane 1, vector pAcGFP1-Hyg; and lane 2, pAcGFP1-Hyg-TK-CD. (C) Reverse transcription-polymerase chain reaction analysis of HFT cells transfected with pAcGFP1-Hyg-TK-CD. Lane M, DL2000 marker; lane 1, fragment of TK-CD (403 bp) and β-actin (561 bp) in the transfected cells; lane 2, untransfected; and lane 3, negative control groups. (D) TK and CD gene expression mediated either by Lipo or G5-PAMAM-D was accessed by fluorescence microscopy (magnification, ×100). TK, thymidine kinase; CD, cytosine deaminase; HTFs, human Tenon's capsule fibroblasts; G5-PAMAM-D, 5-polyamidoamine dendrimers; bp, base pairs; Lipo, Lipofectamine 2000; ctrl, control.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Antiproliferative effect of double suicide gene delivery mediated by polyamidoamine dendrimers in human Tenon's capsule fibroblasts

    doi: 10.3892/etm.2017.5235

    Figure Lengend Snippet: TK and CD gene expression in HTF cells. (A) The plasmid map showing the expression vector, pAcGFP1-Hyg, carrying the TK and CD genes. (B) The 1,617 bp fragment of the TK-CD gene was confirmed by restriction enzyme digestion of the plasmid with Xho I and Kpn I followed by 1% DNA gel electrophoresis. Lane M, DNA marker; lane 1, vector pAcGFP1-Hyg; and lane 2, pAcGFP1-Hyg-TK-CD. (C) Reverse transcription-polymerase chain reaction analysis of HFT cells transfected with pAcGFP1-Hyg-TK-CD. Lane M, DL2000 marker; lane 1, fragment of TK-CD (403 bp) and β-actin (561 bp) in the transfected cells; lane 2, untransfected; and lane 3, negative control groups. (D) TK and CD gene expression mediated either by Lipo or G5-PAMAM-D was accessed by fluorescence microscopy (magnification, ×100). TK, thymidine kinase; CD, cytosine deaminase; HTFs, human Tenon's capsule fibroblasts; G5-PAMAM-D, 5-polyamidoamine dendrimers; bp, base pairs; Lipo, Lipofectamine 2000; ctrl, control.

    Article Snippet: Lipofectamine® 2000 was purchased from Invitrogen (Thermo Fisher Scientific, Inc.).

    Techniques: Expressing, Plasmid Preparation, DNA Gel Electrophoresis, Marker, Reverse Transcription Polymerase Chain Reaction, Transfection, Negative Control, Fluorescence, Microscopy

    Splicing to include exon 3a is a developmentally regulated event in skeletal muscle. ( A ) Microscopy of cell cultures before, during and after differentiation. C2C12 myoblasts were grown to 70% confluence and induced to differentiate in DMEM+10% horse serum. Partially differentiated cultures containing both myoblasts and myotubes were observed by 2 days post-differentiation. After 48 hours, medium was replaced with DMEM+2% horse serum and 10 µM Ara-C and cultured for an additional 4 days. ( B ) qPCR of RNA harvested in Trizol showed that concurrent with a decrease in normal Actg1 , splicing to generate alternative Actg1 increases during differentiation into myotubes. Expression of both the normal and alternative transcripts was normalized to Ppia and is presented as fold-difference compared to skeletal muscle. A two-tailed type 2 Student's T-test was used to compare expression differences between time points. For all time points compared, p

    Journal: PLoS Genetics

    Article Title: A Novel Actin mRNA Splice Variant Regulates ACTG1 Expression

    doi: 10.1371/journal.pgen.1003743

    Figure Lengend Snippet: Splicing to include exon 3a is a developmentally regulated event in skeletal muscle. ( A ) Microscopy of cell cultures before, during and after differentiation. C2C12 myoblasts were grown to 70% confluence and induced to differentiate in DMEM+10% horse serum. Partially differentiated cultures containing both myoblasts and myotubes were observed by 2 days post-differentiation. After 48 hours, medium was replaced with DMEM+2% horse serum and 10 µM Ara-C and cultured for an additional 4 days. ( B ) qPCR of RNA harvested in Trizol showed that concurrent with a decrease in normal Actg1 , splicing to generate alternative Actg1 increases during differentiation into myotubes. Expression of both the normal and alternative transcripts was normalized to Ppia and is presented as fold-difference compared to skeletal muscle. A two-tailed type 2 Student's T-test was used to compare expression differences between time points. For all time points compared, p

    Article Snippet: RNA isolation and cDNA synthesis Total RNA isolation from tissue and cell culture samples was achieved using TRIzol (Invitrogen, Carlsbad, CA) purification followed by a DNaseI treatment using RNeasy mini-columns (Qiagen, Hilden, Germany).

    Techniques: Microscopy, Acetylene Reduction Assay, Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test

    SMC4 coregulates loci silenced by maintenance DNA methylation and histone modifications associated with heterochromatin. ( A ) SMC4 silences a significant subset of MET1 and DDM1 targets. Venn diagram describing the relationship between TEs derepressed in the smc4-1 , met1-3 , and ddm1-2 mutants. Asterisks denote statistically significant overlaps. ( B ) Venn diagram showing the overlap between TEs derepressed in the smc4-1 , met1-3 , and cmt3-11t mutants. Asterisks denote statistically significant overlaps. ( C ) Venn diagram describing the relationships between TEs derepressed in the smc4-1 , ddm1-2 , and cmt3-11t mutants. Asterisks denote statistically significant overlaps. ( D ) Overlap between TEs derepressed in smc4-1 and suvh4 suvh5 suvh6 triple mutants. Asterisks denote statistically significant overlaps. ( E ) ATXR5 and ATXR6 help silence a subset of TEs that also require MET1, DDM1, and SMC4. The Venn diagram compares the 100 TEs derepressed in an atxr5 atxr6 double mutant with the 169 TEs that represent the overlap between the set of TEs derepressed in met1 , ddm1 , and smc4-1 . ( F ) Genome browser snapshot of H3K9me2 enrichment, H3K27me1 enrichment, localization of SMC4-dependent TEs, and the centromeric position on chromosome 1 in wild-type Col-0. Raw counts of fragment pileup for the ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data are shown in the top two tracks. Vertical bars in the third and fourth tracks represent SMC4-dependent TEs or centromeric repeats, respectively. ( G ) RT–PCR verification of the derepression of four TEs ( AT4TE15030 , AT2TE15880 , AT2TE19625 , and 106B ) predicted from RNA-seq data to be silenced via the partnership of MET1, DDM1, ATXR5/6, and SMC4. Genotypes tested are indicated at the top of the figure. UBQ reactions served as loading controls. Reactions omitting reverse transcriptase (no RT) control for DNA contamination. ( H ) Hierarchical clustering of the 169 TEs coregulated by SMC4, MET1, and DDM1, with TE expression levels displayed as a heat map. Expression levels were determined as RNA-seq reads corresponding to the TEs, normalized to the total number of mapped reads per genotype.

    Journal: Genes & Development

    Article Title: Mutation of Arabidopsis SMC4 identifies condensin as a corepressor of pericentromeric transposons and conditionally expressed genes

    doi: 10.1101/gad.301499.117

    Figure Lengend Snippet: SMC4 coregulates loci silenced by maintenance DNA methylation and histone modifications associated with heterochromatin. ( A ) SMC4 silences a significant subset of MET1 and DDM1 targets. Venn diagram describing the relationship between TEs derepressed in the smc4-1 , met1-3 , and ddm1-2 mutants. Asterisks denote statistically significant overlaps. ( B ) Venn diagram showing the overlap between TEs derepressed in the smc4-1 , met1-3 , and cmt3-11t mutants. Asterisks denote statistically significant overlaps. ( C ) Venn diagram describing the relationships between TEs derepressed in the smc4-1 , ddm1-2 , and cmt3-11t mutants. Asterisks denote statistically significant overlaps. ( D ) Overlap between TEs derepressed in smc4-1 and suvh4 suvh5 suvh6 triple mutants. Asterisks denote statistically significant overlaps. ( E ) ATXR5 and ATXR6 help silence a subset of TEs that also require MET1, DDM1, and SMC4. The Venn diagram compares the 100 TEs derepressed in an atxr5 atxr6 double mutant with the 169 TEs that represent the overlap between the set of TEs derepressed in met1 , ddm1 , and smc4-1 . ( F ) Genome browser snapshot of H3K9me2 enrichment, H3K27me1 enrichment, localization of SMC4-dependent TEs, and the centromeric position on chromosome 1 in wild-type Col-0. Raw counts of fragment pileup for the ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data are shown in the top two tracks. Vertical bars in the third and fourth tracks represent SMC4-dependent TEs or centromeric repeats, respectively. ( G ) RT–PCR verification of the derepression of four TEs ( AT4TE15030 , AT2TE15880 , AT2TE19625 , and 106B ) predicted from RNA-seq data to be silenced via the partnership of MET1, DDM1, ATXR5/6, and SMC4. Genotypes tested are indicated at the top of the figure. UBQ reactions served as loading controls. Reactions omitting reverse transcriptase (no RT) control for DNA contamination. ( H ) Hierarchical clustering of the 169 TEs coregulated by SMC4, MET1, and DDM1, with TE expression levels displayed as a heat map. Expression levels were determined as RNA-seq reads corresponding to the TEs, normalized to the total number of mapped reads per genotype.

    Article Snippet: RNA (1.5 µg) was then treated using a Turbo DNA-free kit (Thermo Fisher Scientific) and used for random-primed cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen).

    Techniques: DNA Methylation Assay, Mutagenesis, Chromatin Immunoprecipitation, Next-Generation Sequencing, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Expressing

    SMC4 represses protein-coding genes in addition to TEs. ( A ) Chromosomal positions and expression levels for protein-coding genes up-regulated in smc4-1 . Estimated positions of centromeres are shown as boxes numbered accordingly for the five chromosomes. ( B ) Hierarchical clustering of the 533 SMC4-dependent protein-coding genes, displaying their relative DNA methylation levels on a scale of 0.0 (white) to 1.0 (red) in wild type and smc4-1 mutants. Columns represent data for each indicated genotype, and rows represent 200-bp windows covering the 533 genes. The rows were clustered by the complete agglomeration hierarchical clustering method, with Euclidean distance as a distance measure. ( C ) Functional annotation of genes up-regulated in smc4-1 . The color gradient reflects the degree of up-regulation in smc4-1 relative to wild type on a log 2 scale. ( D ) RT–PCR analysis of four DNA repair genes ( GMI1 , BRCA1 , XRI1 , and RAD51 ) and the flowering gene Flowering Locus T ( FT ) predicted by RNA-seq to be up-regulated in smc4-1 . UBQ and no reverse transcriptase (no RT) reactions served as controls. ( E ) smc4-1 nuclei display RAD51 enrichment foci indicative of DNA damage, consistent with the up-regulation of DNA repair genes. Images show immunolocalization of RAD51 in leaf nuclei counterstained with DAPI. The atxr5 atxr6 ), served as a positive control for enhanced RAD51 foci.

    Journal: Genes & Development

    Article Title: Mutation of Arabidopsis SMC4 identifies condensin as a corepressor of pericentromeric transposons and conditionally expressed genes

    doi: 10.1101/gad.301499.117

    Figure Lengend Snippet: SMC4 represses protein-coding genes in addition to TEs. ( A ) Chromosomal positions and expression levels for protein-coding genes up-regulated in smc4-1 . Estimated positions of centromeres are shown as boxes numbered accordingly for the five chromosomes. ( B ) Hierarchical clustering of the 533 SMC4-dependent protein-coding genes, displaying their relative DNA methylation levels on a scale of 0.0 (white) to 1.0 (red) in wild type and smc4-1 mutants. Columns represent data for each indicated genotype, and rows represent 200-bp windows covering the 533 genes. The rows were clustered by the complete agglomeration hierarchical clustering method, with Euclidean distance as a distance measure. ( C ) Functional annotation of genes up-regulated in smc4-1 . The color gradient reflects the degree of up-regulation in smc4-1 relative to wild type on a log 2 scale. ( D ) RT–PCR analysis of four DNA repair genes ( GMI1 , BRCA1 , XRI1 , and RAD51 ) and the flowering gene Flowering Locus T ( FT ) predicted by RNA-seq to be up-regulated in smc4-1 . UBQ and no reverse transcriptase (no RT) reactions served as controls. ( E ) smc4-1 nuclei display RAD51 enrichment foci indicative of DNA damage, consistent with the up-regulation of DNA repair genes. Images show immunolocalization of RAD51 in leaf nuclei counterstained with DAPI. The atxr5 atxr6 ), served as a positive control for enhanced RAD51 foci.

    Article Snippet: RNA (1.5 µg) was then treated using a Turbo DNA-free kit (Thermo Fisher Scientific) and used for random-primed cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Expressing, DNA Methylation Assay, Functional Assay, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Positive Control

    SMC4 does not affect cytosine methylation or siRNA levels and acts in the context of both condensins I and II. ( A ) Hierarchical clustering of the 286 SMC4-dependent TEs displaying DNA methylation levels on a scale of 0.0 (white) to 1.0 (black) in smc4-1 , atxr5/6 , nrpe1 ( pol V ), drm1 drm2 , cmt2 , cmt3 , met1 , and ddm1 . Columns represent data for each indicated genotype, and rows represent 200-base-pair (bp) windows covering the 286 TEs. The rows were clustered by complete agglomeration hierarchical clustering method, with Euclidean distance as a distance measure. ( B ) RNA-seq and methylation profiles for three TEs ( AT2TE19625 , AT4TE15030 , and AT2TE15880 ) cooperatively regulated by SMC4, MET1, DDM1, and ATXR5/6. The first two data tracks show mapped RNA-seq reads (black vertical bars) in the wild-type Col-0 and smc4-1 mutant. The remaining six data tracks show cytosine methylation levels in each of the three sequence contexts (CG, CHG, and CHH). Methylation on the plus strand is plotted with blue vertical bars, and methylation on the minus strand is plotted with red vertical bars. TEs (black bars) are shown above the data tracks. ( C ) Box plot analyses comparing wild-type Col-0 and smc4-1 with respect to 21- and 24-nt small RNA abundance genome-wide. All read counts were normalized to total mapped reads. ( D ) Involvement of condensins I and II in SMC4-dependent silencing. The cartoons at the left show the subunit compositions of A. thaliana condensins I and II. The gel images show RT–PCR results for SMC4-dependent loci, tested in the indicated condensin subunit mutants.

    Journal: Genes & Development

    Article Title: Mutation of Arabidopsis SMC4 identifies condensin as a corepressor of pericentromeric transposons and conditionally expressed genes

    doi: 10.1101/gad.301499.117

    Figure Lengend Snippet: SMC4 does not affect cytosine methylation or siRNA levels and acts in the context of both condensins I and II. ( A ) Hierarchical clustering of the 286 SMC4-dependent TEs displaying DNA methylation levels on a scale of 0.0 (white) to 1.0 (black) in smc4-1 , atxr5/6 , nrpe1 ( pol V ), drm1 drm2 , cmt2 , cmt3 , met1 , and ddm1 . Columns represent data for each indicated genotype, and rows represent 200-base-pair (bp) windows covering the 286 TEs. The rows were clustered by complete agglomeration hierarchical clustering method, with Euclidean distance as a distance measure. ( B ) RNA-seq and methylation profiles for three TEs ( AT2TE19625 , AT4TE15030 , and AT2TE15880 ) cooperatively regulated by SMC4, MET1, DDM1, and ATXR5/6. The first two data tracks show mapped RNA-seq reads (black vertical bars) in the wild-type Col-0 and smc4-1 mutant. The remaining six data tracks show cytosine methylation levels in each of the three sequence contexts (CG, CHG, and CHH). Methylation on the plus strand is plotted with blue vertical bars, and methylation on the minus strand is plotted with red vertical bars. TEs (black bars) are shown above the data tracks. ( C ) Box plot analyses comparing wild-type Col-0 and smc4-1 with respect to 21- and 24-nt small RNA abundance genome-wide. All read counts were normalized to total mapped reads. ( D ) Involvement of condensins I and II in SMC4-dependent silencing. The cartoons at the left show the subunit compositions of A. thaliana condensins I and II. The gel images show RT–PCR results for SMC4-dependent loci, tested in the indicated condensin subunit mutants.

    Article Snippet: RNA (1.5 µg) was then treated using a Turbo DNA-free kit (Thermo Fisher Scientific) and used for random-primed cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Methylation, DNA Methylation Assay, RNA Sequencing Assay, Mutagenesis, Sequencing, Genome Wide, Reverse Transcription Polymerase Chain Reaction

    Overexpression of the Pol V largest subunit CTD induces a dominant-negative RdDM phenotype suppressed in EMS-induced mutants. ( A ) RNA blot analysis of small RNA of wild-type (Col-0), pol IV mutant ( nrpd1 ), pol V mutant ( nrpe1-11 ), or CTD-OX plants. The blot was sequentially probed for small RNAs matching the 45S rRNA gene promoter, 5S rRNA gene intergenic spacer ( siR1003 ), or microRNA miR160 . An image of the ethidium bromide (EtBr)-stained gel is shown at the bottom . ( B ) Analysis of AtSN1 and SoloLTR transposon DNA methylation levels using Chop-PCR. Genomic DNA of wild-type Col-0, Pol V mutant ( nrpe1 - 11 ), or CTD-OX plants was digested (chopped) with the indicated methylation-sensitive endonucleases (the sequence context of queried cytosines are shown in parentheses) or left uncut as a control and then amplified using PCR primers specific for AtSN1 or soloLTR retrotransposons. PCR products were resolved by agarose gel electrophoresis and visualized with EtBr staining. ( C ) RT–PCR analyses of SDC , CTD , AtSN1 , and soloLTR expression levels relative to a ubiquitin ( UBQ ) control. The genotypes of plants tested are indicated at the top of each lane. Reactions in which reverse transcriptase was omitted (no RT) control for DNA contamination. The drm1 drm2 cmt3 genotype is known to induce SDC overexpression, serving as a positive control for the cmt3 CTD-OX genotype. The CTD reactions control for transgene expression. ( D ) CHG and CHH methylation-deficient cmt3 CTD-OX plants display the SDC overexpression phenotype. ( E ) RT–PCR analysis of CTD and native NRPE1 expression. The genotypes of plants tested are indicated at the top of each lane. Reactions lacking reverse transcriptase (no RT) control for DNA contamination. UBQ reactions control for the amount of RNA tested. ( F ) RT–PCR analysis of SDC , AtSN1 , soloLTR , and CTD expression in the cmt3 CTD-OX parental line and in the suppressor mutants m17, m65, m71, and m73. The nrpe1 - 11 ( pol V ) mutant served as control for derepression of AtSN1 and SoloLTR elements silenced by RdDM in wild type (Col-0). UBQ served as a loading control. Reactions without reverse transcriptase (no RT) served as controls for DNA contamination. ( G ) Analysis of AtSN1 and SoloLTR DNA methylation levels using Chop-PCR. Assays were conducted as in B , comparing wild-type Col-0 with the indicated mutants.

    Journal: Genes & Development

    Article Title: Mutation of Arabidopsis SMC4 identifies condensin as a corepressor of pericentromeric transposons and conditionally expressed genes

    doi: 10.1101/gad.301499.117

    Figure Lengend Snippet: Overexpression of the Pol V largest subunit CTD induces a dominant-negative RdDM phenotype suppressed in EMS-induced mutants. ( A ) RNA blot analysis of small RNA of wild-type (Col-0), pol IV mutant ( nrpd1 ), pol V mutant ( nrpe1-11 ), or CTD-OX plants. The blot was sequentially probed for small RNAs matching the 45S rRNA gene promoter, 5S rRNA gene intergenic spacer ( siR1003 ), or microRNA miR160 . An image of the ethidium bromide (EtBr)-stained gel is shown at the bottom . ( B ) Analysis of AtSN1 and SoloLTR transposon DNA methylation levels using Chop-PCR. Genomic DNA of wild-type Col-0, Pol V mutant ( nrpe1 - 11 ), or CTD-OX plants was digested (chopped) with the indicated methylation-sensitive endonucleases (the sequence context of queried cytosines are shown in parentheses) or left uncut as a control and then amplified using PCR primers specific for AtSN1 or soloLTR retrotransposons. PCR products were resolved by agarose gel electrophoresis and visualized with EtBr staining. ( C ) RT–PCR analyses of SDC , CTD , AtSN1 , and soloLTR expression levels relative to a ubiquitin ( UBQ ) control. The genotypes of plants tested are indicated at the top of each lane. Reactions in which reverse transcriptase was omitted (no RT) control for DNA contamination. The drm1 drm2 cmt3 genotype is known to induce SDC overexpression, serving as a positive control for the cmt3 CTD-OX genotype. The CTD reactions control for transgene expression. ( D ) CHG and CHH methylation-deficient cmt3 CTD-OX plants display the SDC overexpression phenotype. ( E ) RT–PCR analysis of CTD and native NRPE1 expression. The genotypes of plants tested are indicated at the top of each lane. Reactions lacking reverse transcriptase (no RT) control for DNA contamination. UBQ reactions control for the amount of RNA tested. ( F ) RT–PCR analysis of SDC , AtSN1 , soloLTR , and CTD expression in the cmt3 CTD-OX parental line and in the suppressor mutants m17, m65, m71, and m73. The nrpe1 - 11 ( pol V ) mutant served as control for derepression of AtSN1 and SoloLTR elements silenced by RdDM in wild type (Col-0). UBQ served as a loading control. Reactions without reverse transcriptase (no RT) served as controls for DNA contamination. ( G ) Analysis of AtSN1 and SoloLTR DNA methylation levels using Chop-PCR. Assays were conducted as in B , comparing wild-type Col-0 with the indicated mutants.

    Article Snippet: RNA (1.5 µg) was then treated using a Turbo DNA-free kit (Thermo Fisher Scientific) and used for random-primed cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Over Expression, Dominant Negative Mutation, Northern blot, Mutagenesis, Staining, DNA Methylation Assay, Polymerase Chain Reaction, Methylation, Sequencing, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control

    ( a ) TLC identification of 5hmC in A. mellifera. Single radiolabelled nucleotides derived from a variety of samples were separated by TLC on PEI cellulose. Samples obtained from single dNTPs were used as standards. Three genomic DNA preparations were digested to single nucleotides and immunoprecipitated with anti-5hmC antibodies. Purified nucleotides were radiolabelled and resolved by TLC. A spot representing 5hmC is present in drone testes DNA, whereas a much stronger spot can be seen for mouse brain known to be enriched in 5hmC (white arrows). No 5hmC spot can be found in λ phage (Dam − Dcm − ). All lanes are from the same TLC plate. ( b ) An image of a DNA dot-blot hybridized with an anti-5hmC antibody. For each sample, 1 and 2 µg of DNA were spotted on the membrane. A PCR product with dCTP substituted for d5hmCTP was used as control. ( c ) 5hmC quantification in various honeybee DNA samples. The data points were obtained using two methods; a densitometry scan of the dot-blots shown in ( b ), and a β-glucosyltransferase assay (see Material and methods). The resulting values were plotted as either fractions of total cytosines (top) or as a number of 5hmCs in a haploid genome (bottom). The overall correlation between two methods is 0.712. Q, W, D and E refer to queen, worker, drone and embryos, respectively.

    Journal: Open Biology

    Article Title: Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase

    doi: 10.1098/rsob.140110

    Figure Lengend Snippet: ( a ) TLC identification of 5hmC in A. mellifera. Single radiolabelled nucleotides derived from a variety of samples were separated by TLC on PEI cellulose. Samples obtained from single dNTPs were used as standards. Three genomic DNA preparations were digested to single nucleotides and immunoprecipitated with anti-5hmC antibodies. Purified nucleotides were radiolabelled and resolved by TLC. A spot representing 5hmC is present in drone testes DNA, whereas a much stronger spot can be seen for mouse brain known to be enriched in 5hmC (white arrows). No 5hmC spot can be found in λ phage (Dam − Dcm − ). All lanes are from the same TLC plate. ( b ) An image of a DNA dot-blot hybridized with an anti-5hmC antibody. For each sample, 1 and 2 µg of DNA were spotted on the membrane. A PCR product with dCTP substituted for d5hmCTP was used as control. ( c ) 5hmC quantification in various honeybee DNA samples. The data points were obtained using two methods; a densitometry scan of the dot-blots shown in ( b ), and a β-glucosyltransferase assay (see Material and methods). The resulting values were plotted as either fractions of total cytosines (top) or as a number of 5hmCs in a haploid genome (bottom). The overall correlation between two methods is 0.712. Q, W, D and E refer to queen, worker, drone and embryos, respectively.

    Article Snippet: Thin layer chromatography 5hmC pulldown Honeybee genomic DNA (20 µg), 20 µg of phage lambda dam− dcm− DNA (Thermo Scientific), 4 µg of mouse brain DNA and 80 ng of 5hmC PCR product (with all cytosines replaced with 5hmC) were digested overnight with DNase I at 37°C.

    Techniques: Thin Layer Chromatography, Derivative Assay, Immunoprecipitation, Purification, Dot Blot, Polymerase Chain Reaction

    Predicted domain organization and transglutaminase activity of TgpA protein. (A) Map of the predicted domains DUF3488 (PF11992) and TG (PF01841) along the primary sequence of the PA2873 gene product, called TgpA. The sequence of TgpA spanning aa 396 to 467 of the TG domain is highlighted and aligned to homologous functional TG domains of human coagulation Factor XIII, fish-derived transglutaminase (FTG) and WbmE protein from B. bronchiseptica . Conserved aminoacids of catalytic triad are indicated by an asterisk. (B) Colorimetric assay of transglutaminase activity of purified TgpA TG 180–544 domain by Transglutaminase Colorimetric Microassay Kit (TCM kit; Covalab). TCM kit uses immobilized N-carbobenzoxy(CBZ)-Gln-Gly as the amine acceptor and biotin-conjugated cadaverine as the amine donor. The indicated amounts of purified TgpA TG 180–544 (stock: 2.7 mg/ml, 95% purity) were incubated in 96-well microtiter plate coated with CBZ-Gln-Gly at 37°C for 15 min with calcium, DTT and biotinylated cadaverine, both in the presence and the absence of EDTA supplied in the kit. As a reference for TGase activity, the indicated amounts of kit-included purified guinea pig TGase with specific activity of 0.1 U/mg were incubated under the same conditions. The wells were washed extensively and filled with streptavidin-labelled horseradish peroxidase (HRP) to assay the formation of immobilized γ-glutamyl-cadaverine-biotin by OD 450 measurement of HRP activity using H 2 O 2 as substrate and tetramethyl benzidine as electron acceptor (chromogen).

    Journal: PLoS ONE

    Article Title: TgpA, a Protein with a Eukaryotic-Like Transglutaminase Domain, Plays a Critical Role in the Viability of Pseudomonas aeruginosa

    doi: 10.1371/journal.pone.0050323

    Figure Lengend Snippet: Predicted domain organization and transglutaminase activity of TgpA protein. (A) Map of the predicted domains DUF3488 (PF11992) and TG (PF01841) along the primary sequence of the PA2873 gene product, called TgpA. The sequence of TgpA spanning aa 396 to 467 of the TG domain is highlighted and aligned to homologous functional TG domains of human coagulation Factor XIII, fish-derived transglutaminase (FTG) and WbmE protein from B. bronchiseptica . Conserved aminoacids of catalytic triad are indicated by an asterisk. (B) Colorimetric assay of transglutaminase activity of purified TgpA TG 180–544 domain by Transglutaminase Colorimetric Microassay Kit (TCM kit; Covalab). TCM kit uses immobilized N-carbobenzoxy(CBZ)-Gln-Gly as the amine acceptor and biotin-conjugated cadaverine as the amine donor. The indicated amounts of purified TgpA TG 180–544 (stock: 2.7 mg/ml, 95% purity) were incubated in 96-well microtiter plate coated with CBZ-Gln-Gly at 37°C for 15 min with calcium, DTT and biotinylated cadaverine, both in the presence and the absence of EDTA supplied in the kit. As a reference for TGase activity, the indicated amounts of kit-included purified guinea pig TGase with specific activity of 0.1 U/mg were incubated under the same conditions. The wells were washed extensively and filled with streptavidin-labelled horseradish peroxidase (HRP) to assay the formation of immobilized γ-glutamyl-cadaverine-biotin by OD 450 measurement of HRP activity using H 2 O 2 as substrate and tetramethyl benzidine as electron acceptor (chromogen).

    Article Snippet: Residual DNA was removed from purified RNA by further treatment with RNA-free DNase I (Ambion) at 37°C for 15 min, followed by DNase I inactivation with 2.5 mM EDTA at 65°C for 10 min. cDNA was generated by incubating 1 µg of RNA with Superscript II Reverse Transcriptase (RT) (Invitrogen), 100 pg of random primers (Invitrogen) and buffer supplied by the manufacturer for 50 min at 42°C.

    Techniques: Activity Assay, Sequencing, Functional Assay, Coagulation, Fluorescence In Situ Hybridization, Derivative Assay, Colorimetric Assay, Purification, Incubation

    Recombinant CMV mutant lacking expression of pUL31-YFP exhibits an MOI-dependent replication defect. (A) UL31 was disrupted in CMV TB40/E (TB wt ) by replacing 1,200 bp of the UL31 ORF with the galK gene (UL31 del ) or insertion of galK into the first ATG of UL31 (UL31 in ). The locations of the first two ATG sequences are indicated. (B) MRC-5 fibroblasts were infected using TB GFP , UL31 del , or UL31 in virus at an MOI of 3. Viral titers from culture supernatants at 72 and 96 hpi were quantified using a TCID 50 assay. Data are from three biological replicates and two technical replicates, with error bars representing standard deviations from the means. (C) Whole-cell lysates were collected at 96 hpi and analyzed using Western blotting and the indicated antibodies. (D) A stop codon, TAG, was introduced into the second ATG of AD169 UL31 YFP , resulting in UL31 stop virus. (E) Fibroblasts were infected with UL31 YFP or UL31 stop virus at an MOI of 3. Total RNA was collected at 96 hpi and analyzed using qRT-PCR with primers to UL32 (pp150) and GAPDH. Data include three biological replicates and two technical replicates, with error bars representing standard deviations from the means. Whole-cell lysates were analyzed by Western blotting. (F) Fibroblasts were infected with UL31 YFP or UL31 stop virus at an MOI of 0.05 IU per cell. Whole-cell DNA was isolated at the indicated times, and relative viral genomes were determined by qPCR using primers for UL123 and GAPDH. Viral titers were determined from cell-free virus at 120 hpi. (G) Titers were also determined following infection at an MOI of 3. Data include three biological replicates and two technical replicates, with error bars representing standard deviations from the means.

    Journal: Journal of Virology

    Article Title: Cytomegalovirus Late Protein pUL31 Alters Pre-rRNA Expression and Nuclear Organization during Infection

    doi: 10.1128/JVI.00593-17

    Figure Lengend Snippet: Recombinant CMV mutant lacking expression of pUL31-YFP exhibits an MOI-dependent replication defect. (A) UL31 was disrupted in CMV TB40/E (TB wt ) by replacing 1,200 bp of the UL31 ORF with the galK gene (UL31 del ) or insertion of galK into the first ATG of UL31 (UL31 in ). The locations of the first two ATG sequences are indicated. (B) MRC-5 fibroblasts were infected using TB GFP , UL31 del , or UL31 in virus at an MOI of 3. Viral titers from culture supernatants at 72 and 96 hpi were quantified using a TCID 50 assay. Data are from three biological replicates and two technical replicates, with error bars representing standard deviations from the means. (C) Whole-cell lysates were collected at 96 hpi and analyzed using Western blotting and the indicated antibodies. (D) A stop codon, TAG, was introduced into the second ATG of AD169 UL31 YFP , resulting in UL31 stop virus. (E) Fibroblasts were infected with UL31 YFP or UL31 stop virus at an MOI of 3. Total RNA was collected at 96 hpi and analyzed using qRT-PCR with primers to UL32 (pp150) and GAPDH. Data include three biological replicates and two technical replicates, with error bars representing standard deviations from the means. Whole-cell lysates were analyzed by Western blotting. (F) Fibroblasts were infected with UL31 YFP or UL31 stop virus at an MOI of 0.05 IU per cell. Whole-cell DNA was isolated at the indicated times, and relative viral genomes were determined by qPCR using primers for UL123 and GAPDH. Viral titers were determined from cell-free virus at 120 hpi. (G) Titers were also determined following infection at an MOI of 3. Data include three biological replicates and two technical replicates, with error bars representing standard deviations from the means.

    Article Snippet: Approximately 2 μg of RNA was treated with DNA-free DNA removal kit (ThermoFisher Scientific) and used to synthesize cDNA with random hexamers and Superscript III reverse transcriptase (ThermoFisher Scientific).

    Techniques: Recombinant, Mutagenesis, Expressing, Infection, Western Blot, Quantitative RT-PCR, Isolation, Real-time Polymerase Chain Reaction

    Real-time one-step RT-PCR evaluation of cDNA priming strategies and the effect of unbalanced target concentrations . A) Real-time one-step RT-PCR evaluation of the effect of different RT primers on quantification of RNA expression in singleplex and in triplex. Reactions employed either an oligo(dT) 18 RT primer, a random decamer RT primer, or a combination of oligo(dT) 18 and random decamer RT primers for cDNA synthesis. In addition, each one-step RT-PCR protocol employed Taq DNA polymerase, M-MLV RT, 0.8 μg of human thymus total RNA, and CleanAmp™ Precision PCR primers. Reactions were performed in triplicate. B) Real-time one-step RT-PCR evaluation of triplex one-step RT-PCR amplifications using different custom prepared mixes containing three RNA standards in different ratios. The relative abundance for each mixture A through H is represented in the following format: (X:Y:Z), where the copies of the ABCA5 RNA standard is present at 10^X copies, the ABCA6 RNA standard is present at 10^Y copies, and the ABCA7 RNA standard is present at 10^Z copies. The observed copy number for each reaction, which was performed in triplicate, was obtained by extrapolation of the Cq to a standard curve for the ABCA5, ABCA6, and ABCA7 RNA standards. The resultant data for each RNA sample was normalized to ABCA7 and was plotted graphically.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Real-time one-step RT-PCR evaluation of cDNA priming strategies and the effect of unbalanced target concentrations . A) Real-time one-step RT-PCR evaluation of the effect of different RT primers on quantification of RNA expression in singleplex and in triplex. Reactions employed either an oligo(dT) 18 RT primer, a random decamer RT primer, or a combination of oligo(dT) 18 and random decamer RT primers for cDNA synthesis. In addition, each one-step RT-PCR protocol employed Taq DNA polymerase, M-MLV RT, 0.8 μg of human thymus total RNA, and CleanAmp™ Precision PCR primers. Reactions were performed in triplicate. B) Real-time one-step RT-PCR evaluation of triplex one-step RT-PCR amplifications using different custom prepared mixes containing three RNA standards in different ratios. The relative abundance for each mixture A through H is represented in the following format: (X:Y:Z), where the copies of the ABCA5 RNA standard is present at 10^X copies, the ABCA6 RNA standard is present at 10^Y copies, and the ABCA7 RNA standard is present at 10^Z copies. The observed copy number for each reaction, which was performed in triplicate, was obtained by extrapolation of the Cq to a standard curve for the ABCA5, ABCA6, and ABCA7 RNA standards. The resultant data for each RNA sample was normalized to ABCA7 and was plotted graphically.

    Article Snippet: M-MLV reverse transcriptase (25 U) (Invitrogen) and Taq DNA polymerase (2.5 U) (Invitrogen) were used in all quantitative real-time experiments.

    Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Expression, Polymerase Chain Reaction

    One-step RT-PCR evaluation of unmodified and thermolabile CleanAmp™ Precision primers to amplify three different targets of ABCA transporters in singleplex, duplex, and triplex amplifications . For each gene of interest (ABCA5, ABCA6, and ABCA7), the PCR primers were unmodified or contained CleanAmp™ Precision modifications. Reverse transcription utilized an oligo(dT) 18 primer. Reactions contained Taq DNA polymerase, the appropriate reverse transcriptase, and 0.82 μg of human trachea total RNA. A) Reactions employed M-MLV reverse transcriptase and utilized an RT extension temperature of 42°C. B) Reactions employed SuperScript ® III reverse transcriptase (SSIII RT) and utilized an RT extension temperature of 55°C.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: One-step RT-PCR evaluation of unmodified and thermolabile CleanAmp™ Precision primers to amplify three different targets of ABCA transporters in singleplex, duplex, and triplex amplifications . For each gene of interest (ABCA5, ABCA6, and ABCA7), the PCR primers were unmodified or contained CleanAmp™ Precision modifications. Reverse transcription utilized an oligo(dT) 18 primer. Reactions contained Taq DNA polymerase, the appropriate reverse transcriptase, and 0.82 μg of human trachea total RNA. A) Reactions employed M-MLV reverse transcriptase and utilized an RT extension temperature of 42°C. B) Reactions employed SuperScript ® III reverse transcriptase (SSIII RT) and utilized an RT extension temperature of 55°C.

    Article Snippet: M-MLV reverse transcriptase (25 U) (Invitrogen) and Taq DNA polymerase (2.5 U) (Invitrogen) were used in all quantitative real-time experiments.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Hot Start DNA polymerase evaluation in triplex one-step RT-PCR amplification of ABCA5, ABCA6 and ABCA7 targets . Reactions contained 0.82 μg of human trachea total RNA and an unmodified oligo(dT) 18 RT primer. The PCR primers were either unmodified, or contained CleanAmp™ Precision modifications. These reactions contained one of the following DNA polymerases: Taq , Platinum ® Taq , or AmpliTaq Gold ® and one of the following reverse transcriptases: M-MLV or SSIII. Reactions with M-MLV were incubated at 42°C, while reactions with SSIII were incubated at 55°C.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Hot Start DNA polymerase evaluation in triplex one-step RT-PCR amplification of ABCA5, ABCA6 and ABCA7 targets . Reactions contained 0.82 μg of human trachea total RNA and an unmodified oligo(dT) 18 RT primer. The PCR primers were either unmodified, or contained CleanAmp™ Precision modifications. These reactions contained one of the following DNA polymerases: Taq , Platinum ® Taq , or AmpliTaq Gold ® and one of the following reverse transcriptases: M-MLV or SSIII. Reactions with M-MLV were incubated at 42°C, while reactions with SSIII were incubated at 55°C.

    Article Snippet: M-MLV reverse transcriptase (25 U) (Invitrogen) and Taq DNA polymerase (2.5 U) (Invitrogen) were used in all quantitative real-time experiments.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Incubation

    Singleplex and triplex real-time one-step RT-PCR detection of ABCA5, ABCA6, and ABCA7 in three different tissues . Reactions, which were performed in triplicate, contained M-MLV reverse transcriptase, an unmodified oligo(dT) 18 primer, Taq DNA polymerase and CleanAmp™ Precision PCR primers for the ABCA5, ABCA6 and ABCA7 genes. A standard curve for ABCA5, ABCA6, and ABCA7 was determined by employing ~10 1 to ~10 8 copies of the appropriate RNA standard. Each of the three human total RNA tissue samples (brain (0.78 μg), thymus (0.8 μg), and trachea (0.82 μg)) was amplified in singleplex and triplex format for detection of ABCA5, ABCA6, and ABCA7. The number of copies of each target in a given tissue was determined by extrapolating the resultant Cq values to the standard curve and normalizing the resultant values to the micrograms of input total RNA. A) The relative number of copies per microgram and standard deviation for each target in brain, thymus, and trachea total RNA is represented in a bar graph, which displays the results for singleplex and triplex amplifications. B) The corresponding agarose gel analysis of the three tissue samples amplified in singleplex and in triplex.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Singleplex and triplex real-time one-step RT-PCR detection of ABCA5, ABCA6, and ABCA7 in three different tissues . Reactions, which were performed in triplicate, contained M-MLV reverse transcriptase, an unmodified oligo(dT) 18 primer, Taq DNA polymerase and CleanAmp™ Precision PCR primers for the ABCA5, ABCA6 and ABCA7 genes. A standard curve for ABCA5, ABCA6, and ABCA7 was determined by employing ~10 1 to ~10 8 copies of the appropriate RNA standard. Each of the three human total RNA tissue samples (brain (0.78 μg), thymus (0.8 μg), and trachea (0.82 μg)) was amplified in singleplex and triplex format for detection of ABCA5, ABCA6, and ABCA7. The number of copies of each target in a given tissue was determined by extrapolating the resultant Cq values to the standard curve and normalizing the resultant values to the micrograms of input total RNA. A) The relative number of copies per microgram and standard deviation for each target in brain, thymus, and trachea total RNA is represented in a bar graph, which displays the results for singleplex and triplex amplifications. B) The corresponding agarose gel analysis of the three tissue samples amplified in singleplex and in triplex.

    Article Snippet: M-MLV reverse transcriptase (25 U) (Invitrogen) and Taq DNA polymerase (2.5 U) (Invitrogen) were used in all quantitative real-time experiments.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Standard Deviation, Agarose Gel Electrophoresis

    Evaluation of M-MLV and SSIII reverse transcriptases in multiplex one-step RT-PCR amplification of up to five targets . The amplification of increasing number of targets was evaluated by using either M-MLV RT (42°C) or SSIII RT (55°C). Reactions contained an oligo(dT) 18 primer, 0.82 μg of human trachea total RNA, CleanAmp™ Precision primers, and Taq DNA polymerase.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Evaluation of M-MLV and SSIII reverse transcriptases in multiplex one-step RT-PCR amplification of up to five targets . The amplification of increasing number of targets was evaluated by using either M-MLV RT (42°C) or SSIII RT (55°C). Reactions contained an oligo(dT) 18 primer, 0.82 μg of human trachea total RNA, CleanAmp™ Precision primers, and Taq DNA polymerase.

    Article Snippet: M-MLV reverse transcriptase (25 U) (Invitrogen) and Taq DNA polymerase (2.5 U) (Invitrogen) were used in all quantitative real-time experiments.

    Techniques: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

    Derivation of RelA −/− VECs, VSMCs, and MSCs from RelA −/− ESCs . (A) Flow cytometric analysis of WT and  RelA −/−  VECs with VEC-specific markers CD34 and CD201. IgG-FITC and IgG-PE were used as isotype controls. (B) Flow cytometric analysis of WT and  RelA −/−  VSMCs with VSMC-specific marker, CD140b. IgG-APC was used as an isotype control. (C) Flow cytometric analysis of WT and  RelA −/−  MSCs with MSC-specific markers, CD73, CD90 and CD105. IgG-FITC, IgG-PE and IgG-APC were used as isotype controls. (D) Immunostaining of WT and  RelA −/−  VECs with VEC-specific markers, vWF and CD31. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (E) Immunostaining of WT and  RelA −/−  VSMCs with VSMC-specific markers, SM22 and Calponin. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (F) Western blot analysis of RelA protein in WT and  RelA −/−  VECs, VSMCs and MSCs, respectively. β-Actin was used as a loading control. (G) Immunostaining of RelA in WT and  RelA −/−  VECs, VSMCs and MSCs under basal condition. DNA was labeled by Hoechst 33342. Scale bar, 10 μm

    Journal: Protein & Cell

    Article Title: CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells

    doi: 10.1007/s13238-018-0560-5

    Figure Lengend Snippet: Derivation of RelA −/− VECs, VSMCs, and MSCs from RelA −/− ESCs . (A) Flow cytometric analysis of WT and RelA −/− VECs with VEC-specific markers CD34 and CD201. IgG-FITC and IgG-PE were used as isotype controls. (B) Flow cytometric analysis of WT and RelA −/− VSMCs with VSMC-specific marker, CD140b. IgG-APC was used as an isotype control. (C) Flow cytometric analysis of WT and RelA −/− MSCs with MSC-specific markers, CD73, CD90 and CD105. IgG-FITC, IgG-PE and IgG-APC were used as isotype controls. (D) Immunostaining of WT and RelA −/− VECs with VEC-specific markers, vWF and CD31. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (E) Immunostaining of WT and RelA −/− VSMCs with VSMC-specific markers, SM22 and Calponin. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (F) Western blot analysis of RelA protein in WT and RelA −/− VECs, VSMCs and MSCs, respectively. β-Actin was used as a loading control. (G) Immunostaining of RelA in WT and RelA −/− VECs, VSMCs and MSCs under basal condition. DNA was labeled by Hoechst 33342. Scale bar, 10 μm

    Article Snippet: Nuclear DNA was stained by Hoechst 33342 (Invitrogen, H3570).

    Techniques: Flow Cytometry, Marker, Immunostaining, Labeling, Western Blot

    Transcriptomic analysis revealed the effect of IκBα deficiency on RelA signaling . (A) Schemic diagram of  IκB α knockout strategy via CRISPR/Cas9 in human ESCs. A neomycin-resistant cassette (Neo) was included for positive selection. (B) Genomic PCR verification of the deletion of  IκB α exon 1 in  IκB α −/−  ESCs. Water was used as a negative control (NC). (C) Western blot analysis showing IκBα protein levels in WT and  IκB α −/−  ESCs. β-Actin was used as a loading control. (D) Transcriptional signal of IκBα in WT and  IκB α −/−  in VECs, VSMCs and MSCs. Transcriptional signals were normalized by RPKM at bin size 10 bp. (E) Venn diagrams showing the overlap between upregulated genes in  IκB α −/−  vascular cells and downregulated genes in  RelA −/−  vascular cells compared to WT vascular cells under basal condition. (F) Heatmaps revealing the transcriptional patterns of genes upregulated only in  IκB α −/−  vascular cells (pink), downregulated only in  RelA −/−  vascular cells (green), and genes overlapped (blue) under basal condition. (G) Venn diagrams showing the overalp between upregulated genes in  IκB α −/−  vascular cells and downregulated genes in  RelA −/−  vascular cells compared to WT vascular cells upon TNFα treatment. (H) Heatmaps revealing the transcriptional patterns of genes upregulated only in  IκB α −/−  vascular cells (pink), downregulated only in  RelA −/−  vascular cells (green) and genes overlapped (blue) upon TNFα treatment. (I) Immunostaining of RelA in WT and  IκB α −/−  MSCs under basal and TNFα-treated conditions. DNA was labeled by Hoechst 33342. Scale bar, 10 μm

    Journal: Protein & Cell

    Article Title: CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells

    doi: 10.1007/s13238-018-0560-5

    Figure Lengend Snippet: Transcriptomic analysis revealed the effect of IκBα deficiency on RelA signaling . (A) Schemic diagram of IκB α knockout strategy via CRISPR/Cas9 in human ESCs. A neomycin-resistant cassette (Neo) was included for positive selection. (B) Genomic PCR verification of the deletion of IκB α exon 1 in IκB α −/− ESCs. Water was used as a negative control (NC). (C) Western blot analysis showing IκBα protein levels in WT and IκB α −/− ESCs. β-Actin was used as a loading control. (D) Transcriptional signal of IκBα in WT and IκB α −/− in VECs, VSMCs and MSCs. Transcriptional signals were normalized by RPKM at bin size 10 bp. (E) Venn diagrams showing the overlap between upregulated genes in IκB α −/− vascular cells and downregulated genes in RelA −/− vascular cells compared to WT vascular cells under basal condition. (F) Heatmaps revealing the transcriptional patterns of genes upregulated only in IκB α −/− vascular cells (pink), downregulated only in RelA −/− vascular cells (green), and genes overlapped (blue) under basal condition. (G) Venn diagrams showing the overalp between upregulated genes in IκB α −/− vascular cells and downregulated genes in RelA −/− vascular cells compared to WT vascular cells upon TNFα treatment. (H) Heatmaps revealing the transcriptional patterns of genes upregulated only in IκB α −/− vascular cells (pink), downregulated only in RelA −/− vascular cells (green) and genes overlapped (blue) upon TNFα treatment. (I) Immunostaining of RelA in WT and IκB α −/− MSCs under basal and TNFα-treated conditions. DNA was labeled by Hoechst 33342. Scale bar, 10 μm

    Article Snippet: Nuclear DNA was stained by Hoechst 33342 (Invitrogen, H3570).

    Techniques: Knock-Out, CRISPR, Selection, Polymerase Chain Reaction, Negative Control, Western Blot, Immunostaining, Labeling

    RelA deficiency affected vascular cell homeostasis . (A) Immunostaining and flow cytometry analysis of the Dil-Ac-LDL uptake capacity in WT and  RelA −/−  VECs. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (B) Representative micrographs of matrigel tubes formed by WT and  RelA −/−  VECs  in vitro  ( n  = 3). Scale bar, 3 mm. (C) Oil red O staining of WT and  RelA −/−  adipocytes derived from MSCs, respectively. The quantification of adipocytes was measured by absorbance at 510 nm ( n  = 4). ***  P

    Journal: Protein & Cell

    Article Title: CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells

    doi: 10.1007/s13238-018-0560-5

    Figure Lengend Snippet: RelA deficiency affected vascular cell homeostasis . (A) Immunostaining and flow cytometry analysis of the Dil-Ac-LDL uptake capacity in WT and RelA −/− VECs. DNA was labeled by Hoechst 33342. Scale bar, 30 μm. (B) Representative micrographs of matrigel tubes formed by WT and RelA −/− VECs in vitro ( n  = 3). Scale bar, 3 mm. (C) Oil red O staining of WT and RelA −/− adipocytes derived from MSCs, respectively. The quantification of adipocytes was measured by absorbance at 510 nm ( n  = 4). *** P

    Article Snippet: Nuclear DNA was stained by Hoechst 33342 (Invitrogen, H3570).

    Techniques: Immunostaining, Flow Cytometry, Cytometry, Labeling, In Vitro, Staining, Derivative Assay

    Generation and characterization of RelA −/− human ESCs . (A) Schemic diagram of  RelA  knockout strategy via CRISPR/Cas9 in human ESCs. A neomycin-resistant cassette (Neo) was included for positive selection. (B) Genomic PCR verification of  RelA  exon 1 knockout in  RelA −/−  ESCs. Water was used as a negative control (NC). (C) Western blot analysis of RelA protein levels in WT and  RelA −/−  ESCs. β-Actin was used as a loading control. (D) Representative colony morphology and immunostaining of pluripotency markers in WT and  RelA −/−  ESCs. Scale bar, 30 μm. (E) Measurement of the mRNA expression levels of pluripotency markers by semi-quantitative PCR in WT and  RelA −/−  ESCs.  18S  was used as a loading control. (F) Teratoma analysis of WT and  RelA −/−  ESCs with three germ layer markers. Markers were stained in red; DNA was labeled in blue by Hoechst 33342. Scale bar, 100 μm. (G) Karyotype analysis of WT and  RelA −/−  ESCs. (H) Ki67 immunostaining in WT and  RelA −/−  ESCs. Ki67 was stained in red; DNA was labeled by Hoechst 33342. Scale bar, 30 μm

    Journal: Protein & Cell

    Article Title: CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells

    doi: 10.1007/s13238-018-0560-5

    Figure Lengend Snippet: Generation and characterization of RelA −/− human ESCs . (A) Schemic diagram of RelA knockout strategy via CRISPR/Cas9 in human ESCs. A neomycin-resistant cassette (Neo) was included for positive selection. (B) Genomic PCR verification of RelA exon 1 knockout in RelA −/− ESCs. Water was used as a negative control (NC). (C) Western blot analysis of RelA protein levels in WT and RelA −/− ESCs. β-Actin was used as a loading control. (D) Representative colony morphology and immunostaining of pluripotency markers in WT and RelA −/− ESCs. Scale bar, 30 μm. (E) Measurement of the mRNA expression levels of pluripotency markers by semi-quantitative PCR in WT and RelA −/− ESCs. 18S was used as a loading control. (F) Teratoma analysis of WT and RelA −/− ESCs with three germ layer markers. Markers were stained in red; DNA was labeled in blue by Hoechst 33342. Scale bar, 100 μm. (G) Karyotype analysis of WT and RelA −/− ESCs. (H) Ki67 immunostaining in WT and RelA −/− ESCs. Ki67 was stained in red; DNA was labeled by Hoechst 33342. Scale bar, 30 μm

    Article Snippet: Nuclear DNA was stained by Hoechst 33342 (Invitrogen, H3570).

    Techniques: Knock-Out, CRISPR, Selection, Polymerase Chain Reaction, Negative Control, Western Blot, Immunostaining, Expressing, Real-time Polymerase Chain Reaction, Staining, Labeling

    Comparison of five automated DNA extraction systems. Illustration of relative DNA concentrations of samples 1–10 measured by the NanoDrop 2000c spectrophotometer (A) and Qubit 2.0 fluorometer (B). The concentrations of the Maxwell 16 extracts were set to 100% for each sample.

    Journal: PLoS ONE

    Article Title: Comparison of Pre-Analytical FFPE Sample Preparation Methods and Their Impact on Massively Parallel Sequencing in Routine Diagnostics

    doi: 10.1371/journal.pone.0104566

    Figure Lengend Snippet: Comparison of five automated DNA extraction systems. Illustration of relative DNA concentrations of samples 1–10 measured by the NanoDrop 2000c spectrophotometer (A) and Qubit 2.0 fluorometer (B). The concentrations of the Maxwell 16 extracts were set to 100% for each sample.

    Article Snippet: Quantification of DNA purity and concentration The genomic DNA extracted with each system was quantified in duplicates with the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: DNA Extraction, Spectrophotometry