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  • 99
    Thermo Fisher high capacity cdna rt kit
    Increased expression of GFAT in lung cancer cell lines and tissues. ( A ) Expression of GFAT mRNA in HBECs and lung cancer cell lines. Total <t>RNA</t> was extracted from cell lines; <t>cDNA</t> was synthesized by reverse transcription and used for PCR with specific primers for GFAT1, GFAT2, and β-actin as loading control. Products were run in agarose gel with EB. ( B ) GFAT protein and O-GlcNAcylation levels in HBECs and lung cancer cell lines as examined with Western blot in total cell lysates. β-Actin was probed as a loading control. ( C ) GFAT mRNA expression in human lung cancer tissues examined with TaqMan assay. GFAT expression in 12 adenocarcinomas, 12 squamous cell carcinomas, and their corresponding distant normal tissues was normalized to respective β-actin, and then cancer over normal expression was calculated. * P
    High Capacity Cdna Rt Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14000 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher revertaid rt reverse transcription kit
    Increased expression of GFAT in lung cancer cell lines and tissues. ( A ) Expression of GFAT mRNA in HBECs and lung cancer cell lines. Total <t>RNA</t> was extracted from cell lines; <t>cDNA</t> was synthesized by reverse transcription and used for PCR with specific primers for GFAT1, GFAT2, and β-actin as loading control. Products were run in agarose gel with EB. ( B ) GFAT protein and O-GlcNAcylation levels in HBECs and lung cancer cell lines as examined with Western blot in total cell lysates. β-Actin was probed as a loading control. ( C ) GFAT mRNA expression in human lung cancer tissues examined with TaqMan assay. GFAT expression in 12 adenocarcinomas, 12 squamous cell carcinomas, and their corresponding distant normal tissues was normalized to respective β-actin, and then cancer over normal expression was calculated. * P
    Revertaid Rt Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega reverse transcription pcr rt pcr
    Alteration of expression levels of several hematopoietic transcription factors in THAP11-overexpressing K562 cells. K562 cells infected with THAP11-lentiviruses (THAP11-LV) and control cells were treated with 10 nM PMA for 72 hours. Then total <t>RNA</t> was extracted for real-time <t>PCR</t> analysis. Real-time PCR results were expressed as fold induction relative to cells at day 0 and normalized to GAPDH mRNA. Each bar represented the mean ± SD for three independent experiments. The statistical difference between the samples was demonstrated as * P ≤0.05 or ** P ≤0.001.
    Reverse Transcription Pcr Rt Pcr, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 629 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa reverse transcription kit
    Alteration of expression levels of several hematopoietic transcription factors in THAP11-overexpressing K562 cells. K562 cells infected with THAP11-lentiviruses (THAP11-LV) and control cells were treated with 10 nM PMA for 72 hours. Then total <t>RNA</t> was extracted for real-time <t>PCR</t> analysis. Real-time PCR results were expressed as fold induction relative to cells at day 0 and normalized to GAPDH mRNA. Each bar represented the mean ± SD for three independent experiments. The statistical difference between the samples was demonstrated as * P ≤0.05 or ** P ≤0.001.
    Reverse Transcription Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7319 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen quantitect reverse transcription rt kit
    Alteration of expression levels of several hematopoietic transcription factors in THAP11-overexpressing K562 cells. K562 cells infected with THAP11-lentiviruses (THAP11-LV) and control cells were treated with 10 nM PMA for 72 hours. Then total <t>RNA</t> was extracted for real-time <t>PCR</t> analysis. Real-time PCR results were expressed as fold induction relative to cells at day 0 and normalized to GAPDH mRNA. Each bar represented the mean ± SD for three independent experiments. The statistical difference between the samples was demonstrated as * P ≤0.05 or ** P ≤0.001.
    Quantitect Reverse Transcription Rt Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 427 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman microrna reverse transcription kit
    Alteration of expression levels of several hematopoietic transcription factors in THAP11-overexpressing K562 cells. K562 cells infected with THAP11-lentiviruses (THAP11-LV) and control cells were treated with 10 nM PMA for 72 hours. Then total <t>RNA</t> was extracted for real-time <t>PCR</t> analysis. Real-time PCR results were expressed as fold induction relative to cells at day 0 and normalized to GAPDH mRNA. Each bar represented the mean ± SD for three independent experiments. The statistical difference between the samples was demonstrated as * P ≤0.05 or ** P ≤0.001.
    Taqman Microrna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 26114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen omniscript reverse transcription rt kit
    Alteration of expression levels of several hematopoietic transcription factors in THAP11-overexpressing K562 cells. K562 cells infected with THAP11-lentiviruses (THAP11-LV) and control cells were treated with 10 nM PMA for 72 hours. Then total <t>RNA</t> was extracted for real-time <t>PCR</t> analysis. Real-time PCR results were expressed as fold induction relative to cells at day 0 and normalized to GAPDH mRNA. Each bar represented the mean ± SD for three independent experiments. The statistical difference between the samples was demonstrated as * P ≤0.05 or ** P ≤0.001.
    Omniscript Reverse Transcription Rt Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad real time reverse transcription pcr rt pcr
    Downregulation of endogenous p52 reduces AR activity in C4-2 cells. A, C4-2 cells were cotransfected with p52 or control shRNAs and pGL3-PSA-Luc in FBS or CS-FBS, and luciferase activity was measured after 48 h. Columns, mean; bars, SD. Downregulation of endogenous p52 expression in C4-2 cells reduced the transactivation of PSA promoter. C4-2 cells were transfected with p52 or control shRNAs. B, PSA <t>qRT-PCR:</t> total RNAs were analyzed by qRT-PCR for PSA mRNA. Results are expressed as fold change in expression. PSA ChIP: extracts were analyzed by ChIP assay using PSA-AREIII primers. Recruitment of AR to AREIII was abolished when endogenous p52 expression was downregulated. C, NKX3.1 qRT-PCR: <t>cDNAs</t> were also analyzed for the expression of NKX3.1 mRNA by qRT-PCR. Results are expressed as fold change in expression. NKX3.1 ChIP: extracts were analyzed by ChIP assay using primers against NKX3.1-ARE site. Constitutive recruitment of AR to the ARE was dependent on endogenous p52 expression in C4-2 cells. D, immunoblotting of above cell lysates with p52 and AR antibodies.
    Real Time Reverse Transcription Pcr Rt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 340 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman microrna reverse transcription rt kit
    Heatmap showing the 28 microRNAs (miRNAs) found to be differentially expressed in skeletal muscle of non‐small cell lung cancer (NSCLC) patients with cachexia (P, n = 8) in comparison with age‐matched healthy controls (C, n = 8). The expression of miRNAs was measured by <t>TaqMan®</t> Array Human <t>MicroRNA</t> in vastus lateralis muscle biopsies as described in the Methods section. Red: up‐regulated miRNAs; blue: down‐regulated miRNAs. Only those miRNAs with a P
    Taqman Microrna Reverse Transcription Rt Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher reverse transcription quantitative polymerase chain reaction rt qpcr trizol reagent
    Heatmap showing the 28 microRNAs (miRNAs) found to be differentially expressed in skeletal muscle of non‐small cell lung cancer (NSCLC) patients with cachexia (P, n = 8) in comparison with age‐matched healthy controls (C, n = 8). The expression of miRNAs was measured by <t>TaqMan®</t> Array Human <t>MicroRNA</t> in vastus lateralis muscle biopsies as described in the Methods section. Red: up‐regulated miRNAs; blue: down‐regulated miRNAs. Only those miRNAs with a P
    Reverse Transcription Quantitative Polymerase Chain Reaction Rt Qpcr Trizol Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman reverse transcription reagents
    Heatmap showing the 28 microRNAs (miRNAs) found to be differentially expressed in skeletal muscle of non‐small cell lung cancer (NSCLC) patients with cachexia (P, n = 8) in comparison with age‐matched healthy controls (C, n = 8). The expression of miRNAs was measured by <t>TaqMan®</t> Array Human <t>MicroRNA</t> in vastus lateralis muscle biopsies as described in the Methods section. Red: up‐regulated miRNAs; blue: down‐regulated miRNAs. Only those miRNAs with a P
    Taqman Reverse Transcription Reagents, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10825 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad reverse transcription pcr rt pcr
    Fragmented Golgi structures are involved in secretory trafficking and cholesterol homeostasis. (A) DLD1 and DLD1/shAPC cells were stably transfected with pSELECT-zeo-SEAP plasmid, and the secretion of secreted embryonic alkaline phosphatase (SEAP) was monitored over time. SEAP secretion in DLD1/shAPC cells were also monitored in the presence of 1 μg/ml nocodazole or 5 μg/ml brefeldin A (BFA). (B) HCT116 and HCT116/A1309 cells were coimmunostained with Sec31 (red) and GM130 (green). RGB plot profiles illustrate the fluorescence intensity along the white line (see Fig. S7 in the supplemental material). (C) SRE promoter assay in DLD1/shAPC and its isogenic cells expressing A400, A900, and A1309 in response to 2.5 μM TASIN-1 or 5 μM simvastatin treatment for 24 h. (D to H) Quantitative <t>RT-PCR</t> analysis for SREBP2 target genes in DLD1 isogenic cell lines treated with or without 2.5 μM TASIN-1 or 5 μM simvastatin for 24 h. (I to K) Quantitative RT-PCR analysis for SREBP2 target genes in DLD1 (I), DLD1/shAPC (J), and DLD1/APC-2,3 (K) cell lines after incubation in low-serum media (−Serum) for 18 h. <t>mRNA</t> expression levels in low-serum media were normalized with the levels in high-serum media (+Serum) in each cell type. (L) Biosynthesis of cholesterol was measured using 14 ) in DLD1, DLD1/shAPC, and DLD1/shAPC+A1309 cells under low-serum conditions (0.2% serum).
    Reverse Transcription Pcr Rt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 164 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad real time reverse transcription polymerase chain reaction rt pcr
    Schematic representation of the experimental approach. (A) Mosquitoes were exposed to an infectious blood meal containing 10 8 TCID 50 /mL of either one of two DENV-1 isolates (Gen. IV and Gen. I). (B) Fully blood fed mosquitoes were sorted and incubated individually with permanent access to strips of filter paper soaked in 10 per cent sucrose solution to collect saliva samples non-sacrificially. Strips of filter paper were collected and replaced at 7, 10, 13, and 14 days after exposure. (C) Strips of filter paper were pooled by virus isolate and time point. Total <t>RNA</t> was extracted and purified from the pooled saliva samples and from the blood-meal samples, and the amount of viral RNA was estimated by real-time <t>RT-PCR.</t> The remainder of RNA was subjected to library preparation and high-throughput sequencing. (D) Sequencing reads from the blood-meal samples were trimmed according to their quality and used to assemble full-length viral genomes de novo . (E) After quality control, sequencing reads from the pooled saliva samples were aligned to the previously assembled reference genome. (F) For each nucleotide position of the reference viral genome, variants were called based on sequencing quality and depth. For each sample, variants with a frequency > 50 per cent would become the consensus sequence.
    Real Time Reverse Transcription Polymerase Chain Reaction Rt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad reverse transcription quantitative pcr rt qpcr
    Epigenetic modification of the STM locus. (A) <t>RT-PCR</t> with primers amplifying the REV , STM , or ACT2 coding regions on cDNAs generated from mRNA isolated from Col-0 wild-type inflorescence, stem, mature leaf (without the leaf axil region), root, and petal, respectively. ACT2 was used as a loading control. (B and C) Results of <t>ChIP-qPCR</t> performed on IP with antibodies against H3K27me3 (B) and H3K4me2/3 (C) on chromatin samples extracted from Col-0 wild-type inflorescences and mature leaves. The a-e regions (indicated as in Fig 5B ) were assayed. Error bars indicate SD. More controls are shown in (D). (D) ChIP enrichment test by PCR with an anti-H3K27me3 antibody and an anti-H3K4me2/3 antibody using Col-0 wild-type inflorescences and mature leaves, together with total DNA input (input) and no-antibody (mock) controls. An ACT2 promoter region was used as a negative control. (E) Up-regulation of STM expression in mutants affecting PRC1 and PRC2 requires REV . RT-qPCR analysis of STM in whole seedlings of Col-0 wild type and mutants affecting PRC1 and PRC2 w/ or w/o rev-6 . The vertical axis indicates relative mRNA amount compared with the amount in wild-type plants. Error bars indicate SD.
    Reverse Transcription Quantitative Pcr Rt Qpcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega goscript reverse transcription rt system
    Epigenetic modification of the STM locus. (A) <t>RT-PCR</t> with primers amplifying the REV , STM , or ACT2 coding regions on cDNAs generated from mRNA isolated from Col-0 wild-type inflorescence, stem, mature leaf (without the leaf axil region), root, and petal, respectively. ACT2 was used as a loading control. (B and C) Results of <t>ChIP-qPCR</t> performed on IP with antibodies against H3K27me3 (B) and H3K4me2/3 (C) on chromatin samples extracted from Col-0 wild-type inflorescences and mature leaves. The a-e regions (indicated as in Fig 5B ) were assayed. Error bars indicate SD. More controls are shown in (D). (D) ChIP enrichment test by PCR with an anti-H3K27me3 antibody and an anti-H3K4me2/3 antibody using Col-0 wild-type inflorescences and mature leaves, together with total DNA input (input) and no-antibody (mock) controls. An ACT2 promoter region was used as a negative control. (E) Up-regulation of STM expression in mutants affecting PRC1 and PRC2 requires REV . RT-qPCR analysis of STM in whole seedlings of Col-0 wild type and mutants affecting PRC1 and PRC2 w/ or w/o rev-6 . The vertical axis indicates relative mRNA amount compared with the amount in wild-type plants. Error bars indicate SD.
    Goscript Reverse Transcription Rt System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Increased expression of GFAT in lung cancer cell lines and tissues. ( A ) Expression of GFAT mRNA in HBECs and lung cancer cell lines. Total RNA was extracted from cell lines; cDNA was synthesized by reverse transcription and used for PCR with specific primers for GFAT1, GFAT2, and β-actin as loading control. Products were run in agarose gel with EB. ( B ) GFAT protein and O-GlcNAcylation levels in HBECs and lung cancer cell lines as examined with Western blot in total cell lysates. β-Actin was probed as a loading control. ( C ) GFAT mRNA expression in human lung cancer tissues examined with TaqMan assay. GFAT expression in 12 adenocarcinomas, 12 squamous cell carcinomas, and their corresponding distant normal tissues was normalized to respective β-actin, and then cancer over normal expression was calculated. * P

    Journal: Molecular carcinogenesis

    Article Title: Inhibition of the hexosamine biosynthesis pathway potentiates cisplatin cytotoxicity by decreasing BiP expression in non-small cell lung cancer cells

    doi: 10.1002/mc.22992

    Figure Lengend Snippet: Increased expression of GFAT in lung cancer cell lines and tissues. ( A ) Expression of GFAT mRNA in HBECs and lung cancer cell lines. Total RNA was extracted from cell lines; cDNA was synthesized by reverse transcription and used for PCR with specific primers for GFAT1, GFAT2, and β-actin as loading control. Products were run in agarose gel with EB. ( B ) GFAT protein and O-GlcNAcylation levels in HBECs and lung cancer cell lines as examined with Western blot in total cell lysates. β-Actin was probed as a loading control. ( C ) GFAT mRNA expression in human lung cancer tissues examined with TaqMan assay. GFAT expression in 12 adenocarcinomas, 12 squamous cell carcinomas, and their corresponding distant normal tissues was normalized to respective β-actin, and then cancer over normal expression was calculated. * P

    Article Snippet: Briefly, total RNA extracted with Trizol (Invitrogen) was used for reverse transcription with High Capacity cDNA RT kit (Applied Biosystems, Foster City, CA).

    Techniques: Expressing, Synthesized, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, TaqMan Assay

    2.3. Total RNA extraction, cDNA synthesis and quantitative polymerase chain reaction (qPCR)

    Journal: Food & function

    Article Title: Dietary resistant starch type 4-derived butyrate attenuates nuclear factor-kappa-B1 through modulation of lysine 27 trimethylation of histone H3

    doi: 10.1039/c6fo00856a

    Figure Lengend Snippet: 2.3. Total RNA extraction, cDNA synthesis and quantitative polymerase chain reaction (qPCR)

    Article Snippet: The cDNAs were synthesized using 3 µg of RNA for each sample using the High-Capacity cDNA Reverse Transcription (RT) Kit (Invitrogen, Grand Island, NY), following the manufacturers’ protocol.

    Techniques: RNA Extraction, Real-time Polymerase Chain Reaction

    Alteration of expression levels of several hematopoietic transcription factors in THAP11-overexpressing K562 cells. K562 cells infected with THAP11-lentiviruses (THAP11-LV) and control cells were treated with 10 nM PMA for 72 hours. Then total RNA was extracted for real-time PCR analysis. Real-time PCR results were expressed as fold induction relative to cells at day 0 and normalized to GAPDH mRNA. Each bar represented the mean ± SD for three independent experiments. The statistical difference between the samples was demonstrated as * P ≤0.05 or ** P ≤0.001.

    Journal: PLoS ONE

    Article Title: Effects of THAP11 on Erythroid Differentiation and Megakaryocytic Differentiation of K562 Cells

    doi: 10.1371/journal.pone.0091557

    Figure Lengend Snippet: Alteration of expression levels of several hematopoietic transcription factors in THAP11-overexpressing K562 cells. K562 cells infected with THAP11-lentiviruses (THAP11-LV) and control cells were treated with 10 nM PMA for 72 hours. Then total RNA was extracted for real-time PCR analysis. Real-time PCR results were expressed as fold induction relative to cells at day 0 and normalized to GAPDH mRNA. Each bar represented the mean ± SD for three independent experiments. The statistical difference between the samples was demonstrated as * P ≤0.05 or ** P ≤0.001.

    Article Snippet: Reverse transcription-PCR (RT-PCR) and quantitative real-time RT-PCR Total RNA was reverse-transcribed and amplified using reverse transcription and PCR kits, respectively (Promega Corp., Madison, WI, U.S.).

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

    Construction and phenotypic analysis of aspf2 Δ strains. (A) A 1.2-kb BstZ17I-XhoI DNA genomic fragment containing the complete coding sequence of aspf2 in CEA17 (delimited with triangles) was replaced by the lacI-pyrG-lacI cassette (gray arrow flanked by dotted arrows) in the aspf2 Δ (AF811) strain by the use of a 6.65-kb DNA fragment obtained from plasmid pASPF25 as transforming DNA. All strains harbored the correct integration event at the aspf2 locus, as verified by Southern blotting using as a probe a mixture of a DNA fragment obtained by PCR with the oligonucleotide pair JA187 and JA26 and plasmid pASPF25 as the template and a SmaI-BglII fragment obtained from plasmid pZRF39. Only relevant restriction sites are indicated. The source of the genomic DNA, the restriction enzymes used in the digestions, and the sizes of the fragments detected that match the expected sizes are specifically indicated in each panel. (B) Growth of A. fumigatus strains AF811 ( aspf2 Δ), AF881 ( aspf2 + ), and AF431 ( zrfC Δ) on both acid (SDAE; pH 4.5) and alkaline (SDNE; pH 7.5) zinc-limiting agar media not supplemented with Zn 2+ or supplemented with 1 to 100 μM Zn 2+ , as indicated at the top of each panel.

    Journal: Eukaryotic Cell

    Article Title: Aspergillus fumigatus Survival in Alkaline and Extreme Zinc-Limiting Environments Relies on the Induction of a Zinc Homeostasis System Encoded by the zrfC and aspf2 Genes ▿ Genes ▿ †

    doi: 10.1128/EC.00348-09

    Figure Lengend Snippet: Construction and phenotypic analysis of aspf2 Δ strains. (A) A 1.2-kb BstZ17I-XhoI DNA genomic fragment containing the complete coding sequence of aspf2 in CEA17 (delimited with triangles) was replaced by the lacI-pyrG-lacI cassette (gray arrow flanked by dotted arrows) in the aspf2 Δ (AF811) strain by the use of a 6.65-kb DNA fragment obtained from plasmid pASPF25 as transforming DNA. All strains harbored the correct integration event at the aspf2 locus, as verified by Southern blotting using as a probe a mixture of a DNA fragment obtained by PCR with the oligonucleotide pair JA187 and JA26 and plasmid pASPF25 as the template and a SmaI-BglII fragment obtained from plasmid pZRF39. Only relevant restriction sites are indicated. The source of the genomic DNA, the restriction enzymes used in the digestions, and the sizes of the fragments detected that match the expected sizes are specifically indicated in each panel. (B) Growth of A. fumigatus strains AF811 ( aspf2 Δ), AF881 ( aspf2 + ), and AF431 ( zrfC Δ) on both acid (SDAE; pH 4.5) and alkaline (SDNE; pH 7.5) zinc-limiting agar media not supplemented with Zn 2+ or supplemented with 1 to 100 μM Zn 2+ , as indicated at the top of each panel.

    Article Snippet: The cDNA of zrfC was obtained by reverse transcription (RT)-PCR using oligonucleotides JA299 (for retrotranscription) and JA60 and JA54 (both for PCR), and a 1.6-kb cDNA fragment was subcloned into pGEM-T Easy (Promega) to generate plasmid pZRF30 and sequenced.

    Techniques: Sequencing, Plasmid Preparation, Southern Blot, Polymerase Chain Reaction

    Construction and phenotypic analysis of zrfC Δ strains. (A) A 1.86-kb NheI-XmnI DNA genomic fragment containing the complete coding sequence of zrfC in the CEA17 and AF15 strains (delimited with triangles) was replaced by the lacI-pyrG-lacI cassette (gray arrow flanked by dotted arrows) in the zrfC Δ (AF431) and zrfA Δ zrfB Δ zrfC Δ (AF251) strains by the use of a 7.22-kb DNA fragment (delimited with closed squares) obtained from plasmid pZRF35 as transforming DNA. The thinner arrows indicate putative open reading frames surrounding the zrfC gene. The pyrG gene of AF251 was removed by spontaneous intrachromosomal recombination to generate the uridine-uracil-auxotrophic strain AF2511, in which the zrfC coding sequence had been replaced by only one lacI fragment (dotted arrow delimited with triangles). All strains harbored the correct integration event at the zrfC locus, as verified by Southern blotting analyses, using as a probe a DNA fragment obtained by PCR with the oligonucleotide pair JA8 and JA26 and plasmid pZRF35 as the template. Only relevant restriction sites are indicated. The source of the genomic DNA, the restriction enzymes used in the digestions, and the sizes of the fragments detected that match the expected sizes are specifically indicated in each panel. (B) Growth of A. fumigatus strains AF431 ( zrfC Δ), AF10 ( zrfA Δ zrfB Δ), and AF251 ( zrfA Δ zrfB Δ zrfC Δ) on both acid (SDAE; pH 4.5) and alkaline (SDNE; pH 7.5) zinc-limiting agar media not supplemented with Zn 2+ or supplemented with 1 to 1,000 μM Zn 2+ , as indicated at the top of each panel.

    Journal: Eukaryotic Cell

    Article Title: Aspergillus fumigatus Survival in Alkaline and Extreme Zinc-Limiting Environments Relies on the Induction of a Zinc Homeostasis System Encoded by the zrfC and aspf2 Genes ▿ Genes ▿ †

    doi: 10.1128/EC.00348-09

    Figure Lengend Snippet: Construction and phenotypic analysis of zrfC Δ strains. (A) A 1.86-kb NheI-XmnI DNA genomic fragment containing the complete coding sequence of zrfC in the CEA17 and AF15 strains (delimited with triangles) was replaced by the lacI-pyrG-lacI cassette (gray arrow flanked by dotted arrows) in the zrfC Δ (AF431) and zrfA Δ zrfB Δ zrfC Δ (AF251) strains by the use of a 7.22-kb DNA fragment (delimited with closed squares) obtained from plasmid pZRF35 as transforming DNA. The thinner arrows indicate putative open reading frames surrounding the zrfC gene. The pyrG gene of AF251 was removed by spontaneous intrachromosomal recombination to generate the uridine-uracil-auxotrophic strain AF2511, in which the zrfC coding sequence had been replaced by only one lacI fragment (dotted arrow delimited with triangles). All strains harbored the correct integration event at the zrfC locus, as verified by Southern blotting analyses, using as a probe a DNA fragment obtained by PCR with the oligonucleotide pair JA8 and JA26 and plasmid pZRF35 as the template. Only relevant restriction sites are indicated. The source of the genomic DNA, the restriction enzymes used in the digestions, and the sizes of the fragments detected that match the expected sizes are specifically indicated in each panel. (B) Growth of A. fumigatus strains AF431 ( zrfC Δ), AF10 ( zrfA Δ zrfB Δ), and AF251 ( zrfA Δ zrfB Δ zrfC Δ) on both acid (SDAE; pH 4.5) and alkaline (SDNE; pH 7.5) zinc-limiting agar media not supplemented with Zn 2+ or supplemented with 1 to 1,000 μM Zn 2+ , as indicated at the top of each panel.

    Article Snippet: The cDNA of zrfC was obtained by reverse transcription (RT)-PCR using oligonucleotides JA299 (for retrotranscription) and JA60 and JA54 (both for PCR), and a 1.6-kb cDNA fragment was subcloned into pGEM-T Easy (Promega) to generate plasmid pZRF30 and sequenced.

    Techniques: Sequencing, Plasmid Preparation, Southern Blot, Polymerase Chain Reaction

    A. thaliana rRNA genes are > 99% repressed relative to A. arenosa rRNA genes in the allotetraploid hybrid, A. suecica. ( a ) Ethidium-bromide stained agarose gel showing genomic DNA (lanes 1–3) or reverse-transcribed (RT) total RNA (lanes 4–6) amplified by PCR using primers flanking internal transcribed spacer 1 (ITS1; see diagram) and then subjected to digestion with Hha I. An extra Hha I site in ITS1 of A. arenosa rRNA genes allows A. arenosa (A.a.) and A. thaliana (A.t.) genes and their transcripts to be discriminated in A. suecica (A.s). Both progenitors' rRNA genes are present in A. suecica (lane 3; note that A. arenosa -specific bands are under-represented when in competition with A. thaliana ), but only the transcripts from the A. arenosa rRNA genes are abundant in the hybrid (lane 6). RNA samples from which reverse transcriptase was omitted before PCR show that RNA samples are free of contaminating DNA (lanes 7–9). ( b ) Titration experiment to determine the sensitivity of the RT-PCR assay for detection of under-dominant A. thaliana rRNA gene transcripts. RT product resulting from 100 ng of input total RNA was subjected to PCR in each reaction, but the ratio of A. thaliana to A. arenosa RT product was changed by symmetrical serial dilution. Conditions were identical to those used in a . Note that A. thaliana transcripts are detectable at levels as low as 1:525 relative to A. arenosa rRNA transcripts.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Restricted chromosomal silencing in nucleolar dominance

    doi: 10.1073/pnas.251424098

    Figure Lengend Snippet: A. thaliana rRNA genes are > 99% repressed relative to A. arenosa rRNA genes in the allotetraploid hybrid, A. suecica. ( a ) Ethidium-bromide stained agarose gel showing genomic DNA (lanes 1–3) or reverse-transcribed (RT) total RNA (lanes 4–6) amplified by PCR using primers flanking internal transcribed spacer 1 (ITS1; see diagram) and then subjected to digestion with Hha I. An extra Hha I site in ITS1 of A. arenosa rRNA genes allows A. arenosa (A.a.) and A. thaliana (A.t.) genes and their transcripts to be discriminated in A. suecica (A.s). Both progenitors' rRNA genes are present in A. suecica (lane 3; note that A. arenosa -specific bands are under-represented when in competition with A. thaliana ), but only the transcripts from the A. arenosa rRNA genes are abundant in the hybrid (lane 6). RNA samples from which reverse transcriptase was omitted before PCR show that RNA samples are free of contaminating DNA (lanes 7–9). ( b ) Titration experiment to determine the sensitivity of the RT-PCR assay for detection of under-dominant A. thaliana rRNA gene transcripts. RT product resulting from 100 ng of input total RNA was subjected to PCR in each reaction, but the ratio of A. thaliana to A. arenosa RT product was changed by symmetrical serial dilution. Conditions were identical to those used in a . Note that A. thaliana transcripts are detectable at levels as low as 1:525 relative to A. arenosa rRNA transcripts.

    Article Snippet: Reverse transcription (RT)-PCR was performed by using RNA that had been treated with RQ1 DNase I (Promega) to eliminate any contaminating genomic DNA.

    Techniques: Staining, Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Titration, Reverse Transcription Polymerase Chain Reaction, Serial Dilution

    A. thaliana locus F6N15.13 is expressed in A. suecica . PCR using genomic DNA (lanes 1–7) or reverse-transcribed poly(A) + RNA (lanes 8–13) was performed by using a primer pair that amplifies a single exon. Hha I cuts the A. thaliana (A.t.) PCR products but not the A. arenosa (A.a) products, allowing the progenitors' genes and transcripts to be discriminated in A. suecica (A.s) (lanes 5–7). Note that A. thaliana and A. arenosa transcripts are both detected in A. suecica (lane 12). RNAs not subjected to RT served as negative control in lanes 8 and 9.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Restricted chromosomal silencing in nucleolar dominance

    doi: 10.1073/pnas.251424098

    Figure Lengend Snippet: A. thaliana locus F6N15.13 is expressed in A. suecica . PCR using genomic DNA (lanes 1–7) or reverse-transcribed poly(A) + RNA (lanes 8–13) was performed by using a primer pair that amplifies a single exon. Hha I cuts the A. thaliana (A.t.) PCR products but not the A. arenosa (A.a) products, allowing the progenitors' genes and transcripts to be discriminated in A. suecica (A.s) (lanes 5–7). Note that A. thaliana and A. arenosa transcripts are both detected in A. suecica (lane 12). RNAs not subjected to RT served as negative control in lanes 8 and 9.

    Article Snippet: Reverse transcription (RT)-PCR was performed by using RNA that had been treated with RQ1 DNase I (Promega) to eliminate any contaminating genomic DNA.

    Techniques: Polymerase Chain Reaction, Negative Control

    Downregulation of endogenous p52 reduces AR activity in C4-2 cells. A, C4-2 cells were cotransfected with p52 or control shRNAs and pGL3-PSA-Luc in FBS or CS-FBS, and luciferase activity was measured after 48 h. Columns, mean; bars, SD. Downregulation of endogenous p52 expression in C4-2 cells reduced the transactivation of PSA promoter. C4-2 cells were transfected with p52 or control shRNAs. B, PSA qRT-PCR: total RNAs were analyzed by qRT-PCR for PSA mRNA. Results are expressed as fold change in expression. PSA ChIP: extracts were analyzed by ChIP assay using PSA-AREIII primers. Recruitment of AR to AREIII was abolished when endogenous p52 expression was downregulated. C, NKX3.1 qRT-PCR: cDNAs were also analyzed for the expression of NKX3.1 mRNA by qRT-PCR. Results are expressed as fold change in expression. NKX3.1 ChIP: extracts were analyzed by ChIP assay using primers against NKX3.1-ARE site. Constitutive recruitment of AR to the ARE was dependent on endogenous p52 expression in C4-2 cells. D, immunoblotting of above cell lysates with p52 and AR antibodies.

    Journal: Cancer research

    Article Title: Aberrant Activation of the Androgen Receptor by NF-?B2/p52 in Prostate Cancer Cells

    doi: 10.1158/0008-5472.CAN-09-3703

    Figure Lengend Snippet: Downregulation of endogenous p52 reduces AR activity in C4-2 cells. A, C4-2 cells were cotransfected with p52 or control shRNAs and pGL3-PSA-Luc in FBS or CS-FBS, and luciferase activity was measured after 48 h. Columns, mean; bars, SD. Downregulation of endogenous p52 expression in C4-2 cells reduced the transactivation of PSA promoter. C4-2 cells were transfected with p52 or control shRNAs. B, PSA qRT-PCR: total RNAs were analyzed by qRT-PCR for PSA mRNA. Results are expressed as fold change in expression. PSA ChIP: extracts were analyzed by ChIP assay using PSA-AREIII primers. Recruitment of AR to AREIII was abolished when endogenous p52 expression was downregulated. C, NKX3.1 qRT-PCR: cDNAs were also analyzed for the expression of NKX3.1 mRNA by qRT-PCR. Results are expressed as fold change in expression. NKX3.1 ChIP: extracts were analyzed by ChIP assay using primers against NKX3.1-ARE site. Constitutive recruitment of AR to the ARE was dependent on endogenous p52 expression in C4-2 cells. D, immunoblotting of above cell lysates with p52 and AR antibodies.

    Article Snippet: The cDNAs were subjected to real-time reverse transcription-PCR (RT-PCR) using SYBR Green iQ Supermix (Bio-Rad) according to the manufacturer’s instructions.

    Techniques: Activity Assay, Luciferase, Expressing, Transfection, Quantitative RT-PCR, Chromatin Immunoprecipitation

    Heatmap showing the 28 microRNAs (miRNAs) found to be differentially expressed in skeletal muscle of non‐small cell lung cancer (NSCLC) patients with cachexia (P, n = 8) in comparison with age‐matched healthy controls (C, n = 8). The expression of miRNAs was measured by TaqMan® Array Human MicroRNA in vastus lateralis muscle biopsies as described in the Methods section. Red: up‐regulated miRNAs; blue: down‐regulated miRNAs. Only those miRNAs with a P

    Journal: Journal of Cachexia, Sarcopenia and Muscle

    Article Title: Identification of microRNAs in skeletal muscle associated with lung cancer cachexia) Identification of microRNAs in skeletal muscle associated with lung cancer cachexia

    doi: 10.1002/jcsm.12512

    Figure Lengend Snippet: Heatmap showing the 28 microRNAs (miRNAs) found to be differentially expressed in skeletal muscle of non‐small cell lung cancer (NSCLC) patients with cachexia (P, n = 8) in comparison with age‐matched healthy controls (C, n = 8). The expression of miRNAs was measured by TaqMan® Array Human MicroRNA in vastus lateralis muscle biopsies as described in the Methods section. Red: up‐regulated miRNAs; blue: down‐regulated miRNAs. Only those miRNAs with a P

    Article Snippet: TaqMan® Array Human MicroRNA Single‐stranded cDNA was synthesized from total RNA samples using the TaqMan MicroRNA Reverse Transcription (RT) Kit (Applied Biosystems) and the Megaplex™ RT primers (Human Pool Set v3.0; Applied Biosystems).

    Techniques: Expressing

    Fragmented Golgi structures are involved in secretory trafficking and cholesterol homeostasis. (A) DLD1 and DLD1/shAPC cells were stably transfected with pSELECT-zeo-SEAP plasmid, and the secretion of secreted embryonic alkaline phosphatase (SEAP) was monitored over time. SEAP secretion in DLD1/shAPC cells were also monitored in the presence of 1 μg/ml nocodazole or 5 μg/ml brefeldin A (BFA). (B) HCT116 and HCT116/A1309 cells were coimmunostained with Sec31 (red) and GM130 (green). RGB plot profiles illustrate the fluorescence intensity along the white line (see Fig. S7 in the supplemental material). (C) SRE promoter assay in DLD1/shAPC and its isogenic cells expressing A400, A900, and A1309 in response to 2.5 μM TASIN-1 or 5 μM simvastatin treatment for 24 h. (D to H) Quantitative RT-PCR analysis for SREBP2 target genes in DLD1 isogenic cell lines treated with or without 2.5 μM TASIN-1 or 5 μM simvastatin for 24 h. (I to K) Quantitative RT-PCR analysis for SREBP2 target genes in DLD1 (I), DLD1/shAPC (J), and DLD1/APC-2,3 (K) cell lines after incubation in low-serum media (−Serum) for 18 h. mRNA expression levels in low-serum media were normalized with the levels in high-serum media (+Serum) in each cell type. (L) Biosynthesis of cholesterol was measured using 14 ) in DLD1, DLD1/shAPC, and DLD1/shAPC+A1309 cells under low-serum conditions (0.2% serum).

    Journal: Molecular and Cellular Biology

    Article Title: Truncated Adenomatous Polyposis Coli Mutation Induces Asef-Activated Golgi Fragmentation

    doi: 10.1128/MCB.00135-18

    Figure Lengend Snippet: Fragmented Golgi structures are involved in secretory trafficking and cholesterol homeostasis. (A) DLD1 and DLD1/shAPC cells were stably transfected with pSELECT-zeo-SEAP plasmid, and the secretion of secreted embryonic alkaline phosphatase (SEAP) was monitored over time. SEAP secretion in DLD1/shAPC cells were also monitored in the presence of 1 μg/ml nocodazole or 5 μg/ml brefeldin A (BFA). (B) HCT116 and HCT116/A1309 cells were coimmunostained with Sec31 (red) and GM130 (green). RGB plot profiles illustrate the fluorescence intensity along the white line (see Fig. S7 in the supplemental material). (C) SRE promoter assay in DLD1/shAPC and its isogenic cells expressing A400, A900, and A1309 in response to 2.5 μM TASIN-1 or 5 μM simvastatin treatment for 24 h. (D to H) Quantitative RT-PCR analysis for SREBP2 target genes in DLD1 isogenic cell lines treated with or without 2.5 μM TASIN-1 or 5 μM simvastatin for 24 h. (I to K) Quantitative RT-PCR analysis for SREBP2 target genes in DLD1 (I), DLD1/shAPC (J), and DLD1/APC-2,3 (K) cell lines after incubation in low-serum media (−Serum) for 18 h. mRNA expression levels in low-serum media were normalized with the levels in high-serum media (+Serum) in each cell type. (L) Biosynthesis of cholesterol was measured using 14 ) in DLD1, DLD1/shAPC, and DLD1/shAPC+A1309 cells under low-serum conditions (0.2% serum).

    Article Snippet: The mRNA levels were measured by quantitative reverse transcription-PCR (RT-PCR) using SsoFast EvaGreen Supermix (Bio-Rad), and the relative expression of each gene transcript was calculated and normalized to the GAPDH transcript level.

    Techniques: Stable Transfection, Transfection, Plasmid Preparation, Fluorescence, Promoter Assay, Expressing, Quantitative RT-PCR, Incubation

    Schematic representation of the experimental approach. (A) Mosquitoes were exposed to an infectious blood meal containing 10 8 TCID 50 /mL of either one of two DENV-1 isolates (Gen. IV and Gen. I). (B) Fully blood fed mosquitoes were sorted and incubated individually with permanent access to strips of filter paper soaked in 10 per cent sucrose solution to collect saliva samples non-sacrificially. Strips of filter paper were collected and replaced at 7, 10, 13, and 14 days after exposure. (C) Strips of filter paper were pooled by virus isolate and time point. Total RNA was extracted and purified from the pooled saliva samples and from the blood-meal samples, and the amount of viral RNA was estimated by real-time RT-PCR. The remainder of RNA was subjected to library preparation and high-throughput sequencing. (D) Sequencing reads from the blood-meal samples were trimmed according to their quality and used to assemble full-length viral genomes de novo . (E) After quality control, sequencing reads from the pooled saliva samples were aligned to the previously assembled reference genome. (F) For each nucleotide position of the reference viral genome, variants were called based on sequencing quality and depth. For each sample, variants with a frequency > 50 per cent would become the consensus sequence.

    Journal: Virus Evolution

    Article Title: Full-genome dengue virus sequencing in mosquito saliva shows lack of convergent positive selection during transmission by Aedesaegypti

    doi: 10.1093/ve/vex031

    Figure Lengend Snippet: Schematic representation of the experimental approach. (A) Mosquitoes were exposed to an infectious blood meal containing 10 8 TCID 50 /mL of either one of two DENV-1 isolates (Gen. IV and Gen. I). (B) Fully blood fed mosquitoes were sorted and incubated individually with permanent access to strips of filter paper soaked in 10 per cent sucrose solution to collect saliva samples non-sacrificially. Strips of filter paper were collected and replaced at 7, 10, 13, and 14 days after exposure. (C) Strips of filter paper were pooled by virus isolate and time point. Total RNA was extracted and purified from the pooled saliva samples and from the blood-meal samples, and the amount of viral RNA was estimated by real-time RT-PCR. The remainder of RNA was subjected to library preparation and high-throughput sequencing. (D) Sequencing reads from the blood-meal samples were trimmed according to their quality and used to assemble full-length viral genomes de novo . (E) After quality control, sequencing reads from the pooled saliva samples were aligned to the previously assembled reference genome. (F) For each nucleotide position of the reference viral genome, variants were called based on sequencing quality and depth. For each sample, variants with a frequency > 50 per cent would become the consensus sequence.

    Article Snippet: Viral RNA was detected by real-time reverse transcription polymerase chain reaction (RT-PCR) on a CFX96 Touch Real-Time PCR Detection System instrument using iScript One-Step RT-PCR Kit for Probes (Bio-Rad Laboratories, France) with primers and probe as described previously ( ; ).

    Techniques: Incubation, Purification, Quantitative RT-PCR, Next-Generation Sequencing, Sequencing

    Epigenetic modification of the STM locus. (A) RT-PCR with primers amplifying the REV , STM , or ACT2 coding regions on cDNAs generated from mRNA isolated from Col-0 wild-type inflorescence, stem, mature leaf (without the leaf axil region), root, and petal, respectively. ACT2 was used as a loading control. (B and C) Results of ChIP-qPCR performed on IP with antibodies against H3K27me3 (B) and H3K4me2/3 (C) on chromatin samples extracted from Col-0 wild-type inflorescences and mature leaves. The a-e regions (indicated as in Fig 5B ) were assayed. Error bars indicate SD. More controls are shown in (D). (D) ChIP enrichment test by PCR with an anti-H3K27me3 antibody and an anti-H3K4me2/3 antibody using Col-0 wild-type inflorescences and mature leaves, together with total DNA input (input) and no-antibody (mock) controls. An ACT2 promoter region was used as a negative control. (E) Up-regulation of STM expression in mutants affecting PRC1 and PRC2 requires REV . RT-qPCR analysis of STM in whole seedlings of Col-0 wild type and mutants affecting PRC1 and PRC2 w/ or w/o rev-6 . The vertical axis indicates relative mRNA amount compared with the amount in wild-type plants. Error bars indicate SD.

    Journal: PLoS Genetics

    Article Title: Two-Step Regulation of a Meristematic Cell Population Acting in Shoot Branching in Arabidopsis

    doi: 10.1371/journal.pgen.1006168

    Figure Lengend Snippet: Epigenetic modification of the STM locus. (A) RT-PCR with primers amplifying the REV , STM , or ACT2 coding regions on cDNAs generated from mRNA isolated from Col-0 wild-type inflorescence, stem, mature leaf (without the leaf axil region), root, and petal, respectively. ACT2 was used as a loading control. (B and C) Results of ChIP-qPCR performed on IP with antibodies against H3K27me3 (B) and H3K4me2/3 (C) on chromatin samples extracted from Col-0 wild-type inflorescences and mature leaves. The a-e regions (indicated as in Fig 5B ) were assayed. Error bars indicate SD. More controls are shown in (D). (D) ChIP enrichment test by PCR with an anti-H3K27me3 antibody and an anti-H3K4me2/3 antibody using Col-0 wild-type inflorescences and mature leaves, together with total DNA input (input) and no-antibody (mock) controls. An ACT2 promoter region was used as a negative control. (E) Up-regulation of STM expression in mutants affecting PRC1 and PRC2 requires REV . RT-qPCR analysis of STM in whole seedlings of Col-0 wild type and mutants affecting PRC1 and PRC2 w/ or w/o rev-6 . The vertical axis indicates relative mRNA amount compared with the amount in wild-type plants. Error bars indicate SD.

    Article Snippet: Reverse transcription quantitative PCR (RT-qPCR) was performed on a Bio-Rad CFX96 real-time PCR detection system with the KAPA SYBR FAST qPCR kit (KAPA Biosystems).

    Techniques: Modification, Reverse Transcription Polymerase Chain Reaction, Generated, Isolation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Expressing, Quantitative RT-PCR

    Direct up-regulation of STM expression by REV. (A) RT-qPCR analysis of STM expression in pREV :: REV-GR-HA rev-6 vegetative shoot apex tissues (with leaves removed) before and after simultaneous Dex and CHX treatment. The vertical axis indicates relative mRNA amount compared with the amount before treatment. Error bars indicate SD. (B) Schematic diagram of the STM genomic region. Vertical red lines indicate the sites containing the consensus REV binding sequence (ATGAT box). ATG denotes the translation start site. The underlying lines represent the DNA fragments amplified in ChIP assays, or used for plant protoplast assays. (C and D) ChIP enrichment test by PCR shows binding of REV-GR-HA to the ATGAT box-containing regions, especially the ones near the start site, in vegetative shoot apex tissues enriched with leaf axil (C) but not mature leaves ( > P 10 ) without the leaf axil region from 30-d old plants (D) of pREV :: REV-GR-HA rev-6 plants. A paired design was used, in which each measurement was paired with a corresponding control without antibody. Error bars indicate SD. More controls are shown in S4I Fig . (E) Transcriptional activity assays in Arabidopsis protoplasts. A p35S :: GFP empty vector was the negative control, and a p35S :: GUS line was the internal control. Relative LUC reporter gene expression is shown in the lower panel. The p1-p5 regions (indicated as in B) were assayed. Data are mean ± SD. Error bars are derived from three independent biological experiments, each run in triplicate.

    Journal: PLoS Genetics

    Article Title: Two-Step Regulation of a Meristematic Cell Population Acting in Shoot Branching in Arabidopsis

    doi: 10.1371/journal.pgen.1006168

    Figure Lengend Snippet: Direct up-regulation of STM expression by REV. (A) RT-qPCR analysis of STM expression in pREV :: REV-GR-HA rev-6 vegetative shoot apex tissues (with leaves removed) before and after simultaneous Dex and CHX treatment. The vertical axis indicates relative mRNA amount compared with the amount before treatment. Error bars indicate SD. (B) Schematic diagram of the STM genomic region. Vertical red lines indicate the sites containing the consensus REV binding sequence (ATGAT box). ATG denotes the translation start site. The underlying lines represent the DNA fragments amplified in ChIP assays, or used for plant protoplast assays. (C and D) ChIP enrichment test by PCR shows binding of REV-GR-HA to the ATGAT box-containing regions, especially the ones near the start site, in vegetative shoot apex tissues enriched with leaf axil (C) but not mature leaves ( > P 10 ) without the leaf axil region from 30-d old plants (D) of pREV :: REV-GR-HA rev-6 plants. A paired design was used, in which each measurement was paired with a corresponding control without antibody. Error bars indicate SD. More controls are shown in S4I Fig . (E) Transcriptional activity assays in Arabidopsis protoplasts. A p35S :: GFP empty vector was the negative control, and a p35S :: GUS line was the internal control. Relative LUC reporter gene expression is shown in the lower panel. The p1-p5 regions (indicated as in B) were assayed. Data are mean ± SD. Error bars are derived from three independent biological experiments, each run in triplicate.

    Article Snippet: Reverse transcription quantitative PCR (RT-qPCR) was performed on a Bio-Rad CFX96 real-time PCR detection system with the KAPA SYBR FAST qPCR kit (KAPA Biosystems).

    Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Sequencing, Amplification, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Activity Assay, Plasmid Preparation, Negative Control, Derivative Assay