ampin1c  (Qiagen)

 
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    Name:
    QIAquick PCR Purification Kit
    Description:
    For purification of up to 10 μg PCR products 100 bp to 10 kb Kit contents Qiagen QIAquick PCR Purification Kit 50 rxns 30L Elution Volume 10g Binding Capacity Tube Format Manual Processing Silica Technology 100 bp to 10 kb Fragment 40mers Fragments Removed Ideal for Sequencing Microarray Analysis Ligation and Transformation Restriction Digestion Labeling Microinjection PCR and in vitro Transcription For Purification of up to 10μg PCR Products Includes 50 QIAquick Spin Columns Buffers 2mL Collection Tubes Benefits Up to 95 recovery of ready to use DNA Cleanup of DNA up to 10 kb in three easy steps Gel loading dye for convenient sample analysis
    Catalog Number:
    28104
    Price:
    117
    Category:
    QIAquick PCR Purification Kit
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    Structured Review

    Qiagen ampin1c
    QIAquick PCR Purification Kit
    For purification of up to 10 μg PCR products 100 bp to 10 kb Kit contents Qiagen QIAquick PCR Purification Kit 50 rxns 30L Elution Volume 10g Binding Capacity Tube Format Manual Processing Silica Technology 100 bp to 10 kb Fragment 40mers Fragments Removed Ideal for Sequencing Microarray Analysis Ligation and Transformation Restriction Digestion Labeling Microinjection PCR and in vitro Transcription For Purification of up to 10μg PCR Products Includes 50 QIAquick Spin Columns Buffers 2mL Collection Tubes Benefits Up to 95 recovery of ready to use DNA Cleanup of DNA up to 10 kb in three easy steps Gel loading dye for convenient sample analysis
    https://www.bioz.com/result/ampin1c/product/Qiagen
    Average 91 stars, based on 27667 article reviews
    Price from $9.99 to $1999.99
    ampin1c - by Bioz Stars, 2020-11
    91/100 stars

    Images

    1) Product Images from "Generation of shape complexity through tissue conflict resolution"

    Article Title: Generation of shape complexity through tissue conflict resolution

    Journal: eLife

    doi: 10.7554/eLife.20156

    Expression patterns of AmPIN1 genes. ( A-L ) Expression patterns of AmPIN1 genes at 8 DAI ( A-C ), 10 DAI ( D-F ), 12 DAI ( G-I ) and 14 DAI ( J-L ), determined by RNA in situ hybridisation of alternative sections hybridised with AmPIN1a (left panels, A , D , G , J ), AmPIN1b (middle panels, B , E , H , K ) and AmPIN1c (right panels, C , F , I , L ) probes. The AmPIN1 genes show similar expression patterns in the vascular tissue of petal primordia ( A-F ), with some localised differences particularly at the tips of the petal primordia ( D-F and close-ups of red boxes in respective D-F images), in particular for AmPIN1c which shows stronger epidermal expression at the petal tips. At 12 DAI, the AmPIN1 genes continue to be expressed in the vascular tissue ( G-I ) but are additionally detected at the epidermis of the palate and lip regions, particularly strongly for AmPIN1c (white arrows in close-ups of blue boxes in respective G-I images). At 13 DAI, the epidermal Am PIN1 expression starts to decrease ( J-L ) in comparison with the vascular tissue signal which continues to be similar to previous stages (close-ups of green squares in respective J-L images). The upper and lower limits of the palate and lip domains are marked by pink dashed lines. The diagram in the upper right corner of every left panel represents the orientation of the section (white line) relative to the five petals (red-dorsal, orange -lateral and yellow -ventral). V: ventral petal; D: dorsal petal; st: stamen; ca: carpel. Scale bars, 100 μm. DOI: http://dx.doi.org/10.7554/eLife.20156.028
    Figure Legend Snippet: Expression patterns of AmPIN1 genes. ( A-L ) Expression patterns of AmPIN1 genes at 8 DAI ( A-C ), 10 DAI ( D-F ), 12 DAI ( G-I ) and 14 DAI ( J-L ), determined by RNA in situ hybridisation of alternative sections hybridised with AmPIN1a (left panels, A , D , G , J ), AmPIN1b (middle panels, B , E , H , K ) and AmPIN1c (right panels, C , F , I , L ) probes. The AmPIN1 genes show similar expression patterns in the vascular tissue of petal primordia ( A-F ), with some localised differences particularly at the tips of the petal primordia ( D-F and close-ups of red boxes in respective D-F images), in particular for AmPIN1c which shows stronger epidermal expression at the petal tips. At 12 DAI, the AmPIN1 genes continue to be expressed in the vascular tissue ( G-I ) but are additionally detected at the epidermis of the palate and lip regions, particularly strongly for AmPIN1c (white arrows in close-ups of blue boxes in respective G-I images). At 13 DAI, the epidermal Am PIN1 expression starts to decrease ( J-L ) in comparison with the vascular tissue signal which continues to be similar to previous stages (close-ups of green squares in respective J-L images). The upper and lower limits of the palate and lip domains are marked by pink dashed lines. The diagram in the upper right corner of every left panel represents the orientation of the section (white line) relative to the five petals (red-dorsal, orange -lateral and yellow -ventral). V: ventral petal; D: dorsal petal; st: stamen; ca: carpel. Scale bars, 100 μm. DOI: http://dx.doi.org/10.7554/eLife.20156.028

    Techniques Used: Expressing, In Situ, Hybridization

    2) Product Images from "Targeted mutagenesis in a human-parasitic nematode"

    Article Title: Targeted mutagenesis in a human-parasitic nematode

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006675

    CRISPR-mediated mutagenesis of Ss-unc-22 results in putative deletion of the target locus. ( A ) Representative gel of wild-type iL3s (top) or unc F 1 iL3s from RNP injections at site #3 (bottom). Genomic DNA from each iL3 was split into two reactions: ctrl. = control reaction amplifying 416 bp of the first exon of the Ss-act-2 gene to confirm the presence of genomic DNA; u22 = reaction amplifying 660 bp around site #3. Size markers = 1.5 kb, 1 kb, and 500 bp from top to bottom. ( B ) The Ss-unc-22 region is significantly depleted in unc F 1 iL3s. Left: relative quantity analysis of PCR products. All control bands and all u22 bands were quantified relative to their respective reference bands, denoted by asterisks in A . Values > 1 indicate more PCR product than the reference while values
    Figure Legend Snippet: CRISPR-mediated mutagenesis of Ss-unc-22 results in putative deletion of the target locus. ( A ) Representative gel of wild-type iL3s (top) or unc F 1 iL3s from RNP injections at site #3 (bottom). Genomic DNA from each iL3 was split into two reactions: ctrl. = control reaction amplifying 416 bp of the first exon of the Ss-act-2 gene to confirm the presence of genomic DNA; u22 = reaction amplifying 660 bp around site #3. Size markers = 1.5 kb, 1 kb, and 500 bp from top to bottom. ( B ) The Ss-unc-22 region is significantly depleted in unc F 1 iL3s. Left: relative quantity analysis of PCR products. All control bands and all u22 bands were quantified relative to their respective reference bands, denoted by asterisks in A . Values > 1 indicate more PCR product than the reference while values

    Techniques Used: CRISPR, Mutagenesis, Activated Clotting Time Assay, Polymerase Chain Reaction

    3) Product Images from "Regulation of renin expression by the orphan nuclear receptors Nr2f2 and Nr2f6"

    Article Title: Regulation of renin expression by the orphan nuclear receptors Nr2f2 and Nr2f6

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00362.2011

    Binding of Nr2f2 and Nr2f6 to the endogenous renin enhancer. Chromatin immunoprecipitation (ChIP) analysis was performed using increasing amounts of Nr2f2 or Nr2f6 antibody. Immunoprecipitated DNA was PCR amplified using primers targeting the HRE or a
    Figure Legend Snippet: Binding of Nr2f2 and Nr2f6 to the endogenous renin enhancer. Chromatin immunoprecipitation (ChIP) analysis was performed using increasing amounts of Nr2f2 or Nr2f6 antibody. Immunoprecipitated DNA was PCR amplified using primers targeting the HRE or a

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Amplification

    4) Product Images from "Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination"

    Article Title: Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0247-4

    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using PCR. b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses
    Figure Legend Snippet: TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using PCR. b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses

    Techniques Used: Cell Culture, Purification, Immunoprecipitation, Polymerase Chain Reaction

    5) Product Images from "Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits"

    Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-58586-3

    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).
    Figure Legend Snippet: Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Purification

    6) Product Images from "Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins"

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0148023

    Comparison of chromatin fragmentation by ultrasound alone or in combination with benzonase digestion. (A) Comparison of sonication efficiency at the L and H power outputs over time. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for various times (as indicated) in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The “Quick” lanes contain samples sonicated for 20 min and reverse cross-linked for 1 h at +60°C. (B) Coomassie Blue staining of protein fractions generated during the sonication time course shown in panel A. Note the absence of high molecular weight proteins after 20 min of ultrasound treatment. (C) Titration of benzonase (0.2U to 90U) to fragment chromatin solubilized by 2 min L-power sonication. Following the digest, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. See Material and Methods section for detailed reaction conditions. (D) Combination of brief sonication (2 min at L-power) and benzonase digestion (0.7U to 90U) preserves the integrity of large proteins.
    Figure Legend Snippet: Comparison of chromatin fragmentation by ultrasound alone or in combination with benzonase digestion. (A) Comparison of sonication efficiency at the L and H power outputs over time. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for various times (as indicated) in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The “Quick” lanes contain samples sonicated for 20 min and reverse cross-linked for 1 h at +60°C. (B) Coomassie Blue staining of protein fractions generated during the sonication time course shown in panel A. Note the absence of high molecular weight proteins after 20 min of ultrasound treatment. (C) Titration of benzonase (0.2U to 90U) to fragment chromatin solubilized by 2 min L-power sonication. Following the digest, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. See Material and Methods section for detailed reaction conditions. (D) Combination of brief sonication (2 min at L-power) and benzonase digestion (0.7U to 90U) preserves the integrity of large proteins.

    Techniques Used: Sonication, Concentration Assay, Size-exclusion Chromatography, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Generated, Molecular Weight, Titration

    Systematic optimization of sonication conditions. Part 2. (A) The effect of sample position on sonication efficiency at low power setting. 500 μL cell suspensions were loaded into positions L1, L7, R1, R7, all other positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Left panel: Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. Right panel: Afterwards, the samples were sonicated for an additional 9 min and reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 . p -values for selected T-tests are shown on the graph. (B) The effect of pulse time on sonication efficiency. 500 μL cell suspensions were loaded into position R1; vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 2 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), with variable pulse times (as indicated), no rotation and no ice. Following sonication, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. (C) The effect of buffer composition on sonication efficiency. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 10 min in 1:4 ELB:H 2 O supplemented with varying concentrations of SDS and Triton X-100 (as indicated), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. (D) The effect of water level and sample volume on sonication efficiency. Cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. Analysis of variance p -values are shown on the graph for each sample volume.
    Figure Legend Snippet: Systematic optimization of sonication conditions. Part 2. (A) The effect of sample position on sonication efficiency at low power setting. 500 μL cell suspensions were loaded into positions L1, L7, R1, R7, all other positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Left panel: Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. Right panel: Afterwards, the samples were sonicated for an additional 9 min and reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 . p -values for selected T-tests are shown on the graph. (B) The effect of pulse time on sonication efficiency. 500 μL cell suspensions were loaded into position R1; vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 2 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), with variable pulse times (as indicated), no rotation and no ice. Following sonication, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. (C) The effect of buffer composition on sonication efficiency. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 10 min in 1:4 ELB:H 2 O supplemented with varying concentrations of SDS and Triton X-100 (as indicated), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. (D) The effect of water level and sample volume on sonication efficiency. Cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. Analysis of variance p -values are shown on the graph for each sample volume.

    Techniques Used: Sonication, Concentration Assay, Size-exclusion Chromatography, Standard Deviation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Systematic optimization of sonication conditions. Part 1. (A) Schematic of the Bioruptor XL water bath with tube positions numbered from 1 to 12 in the left (L) and right (R) carousels; red arrows indicate the alignment marks for carousel assembly and positioning. (B) The effect of sample volume on sonication efficiency at low power setting. Cell suspensions of variable volume (100–700 μL, as indicated) were loaded into positions L3, L4, L5, L9, L10 and L11. Vacant L-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 8 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 , between all samples 1.4×10 −6 . p -values for selected T-tests are shown on the graph. (C) Reproducibility of sample sonication across positions L1–L12. Sonication was carried out for 40 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Samples were reverse cross-linked overnight and the resulting DNA was purified using the Qiagen PCR clean-up kit followed by 1.1% agarose gel analysis. (D) The effect of sample position and power setting on sonication efficiency. 500 μL cell suspensions were loaded into positions R10-R4 and vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 3.6×10 −9 (L-power) and 8.2×10 −10 (H-power). p -values for selected T-tests are shown on the graph.
    Figure Legend Snippet: Systematic optimization of sonication conditions. Part 1. (A) Schematic of the Bioruptor XL water bath with tube positions numbered from 1 to 12 in the left (L) and right (R) carousels; red arrows indicate the alignment marks for carousel assembly and positioning. (B) The effect of sample volume on sonication efficiency at low power setting. Cell suspensions of variable volume (100–700 μL, as indicated) were loaded into positions L3, L4, L5, L9, L10 and L11. Vacant L-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 8 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 , between all samples 1.4×10 −6 . p -values for selected T-tests are shown on the graph. (C) Reproducibility of sample sonication across positions L1–L12. Sonication was carried out for 40 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Samples were reverse cross-linked overnight and the resulting DNA was purified using the Qiagen PCR clean-up kit followed by 1.1% agarose gel analysis. (D) The effect of sample position and power setting on sonication efficiency. 500 μL cell suspensions were loaded into positions R10-R4 and vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 3.6×10 −9 (L-power) and 8.2×10 −10 (H-power). p -values for selected T-tests are shown on the graph.

    Techniques Used: Sonication, Concentration Assay, Size-exclusion Chromatography, Standard Deviation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    7) Product Images from "Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes"

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes

    Journal: bioRxiv

    doi: 10.1101/2020.08.05.237768

    Modular engineering of the PE Sox1(+35) immediately upstream of the Gria1 -TSS. a, Strategy used to insert the PE Sox1(+35)TFBS and PE Sox1(+35)TFBS+CGI modules 380 bp upstream of the Gria1 TSS. The upper left panel shows a close-up view of the epigenomic and genetic features of the PE Sox1(+35) . The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. The lower left panel shows the two combinations of PE Sox1(+35) modules (i.e. PE Sox1(+35)TFBS and PE Sox1(+35)TFBS CGI) inserted 380 bp upstream of the Gria1 TSS. The right panel shows the TAD in which Gria1 is included (i.e. Gria1 -TAD) according to publically available Hi-C data 33 , 65 ; TAD boundaries are denoted with dotted lines; H3K27me3 ChIP-seq signals in mESC are shown in green 9 ; CGIs are indicated as green rectangles; CTCF binding sites 35 are indicated as black rectangles; the yellow triangle indicates the integration site of the PE Sox1(+35) modules, 380 bp upstream of the Gria1 TSS. b, For the identification of mESC clonal lines with the desired insertion of the different PE Sox1(+35) modules, primer pairs flanking the insertion borders (1+3 and 4+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for WT mESC or two mESC clonal lines with homozygous insertions of the different PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)TFBS+CGI ) 380 bp upstream of the Gria1 TSS are shown. c, Independent biological replicate for the data presented in Fig. 4f. The expression of Gria1 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) modules 380 bp upstream of the Gria1 TSS (TFBS (blue); TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ).
    Figure Legend Snippet: Modular engineering of the PE Sox1(+35) immediately upstream of the Gria1 -TSS. a, Strategy used to insert the PE Sox1(+35)TFBS and PE Sox1(+35)TFBS+CGI modules 380 bp upstream of the Gria1 TSS. The upper left panel shows a close-up view of the epigenomic and genetic features of the PE Sox1(+35) . The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. The lower left panel shows the two combinations of PE Sox1(+35) modules (i.e. PE Sox1(+35)TFBS and PE Sox1(+35)TFBS CGI) inserted 380 bp upstream of the Gria1 TSS. The right panel shows the TAD in which Gria1 is included (i.e. Gria1 -TAD) according to publically available Hi-C data 33 , 65 ; TAD boundaries are denoted with dotted lines; H3K27me3 ChIP-seq signals in mESC are shown in green 9 ; CGIs are indicated as green rectangles; CTCF binding sites 35 are indicated as black rectangles; the yellow triangle indicates the integration site of the PE Sox1(+35) modules, 380 bp upstream of the Gria1 TSS. b, For the identification of mESC clonal lines with the desired insertion of the different PE Sox1(+35) modules, primer pairs flanking the insertion borders (1+3 and 4+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for WT mESC or two mESC clonal lines with homozygous insertions of the different PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)TFBS+CGI ) 380 bp upstream of the Gria1 TSS are shown. c, Independent biological replicate for the data presented in Fig. 4f. The expression of Gria1 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) modules 380 bp upstream of the Gria1 TSS (TFBS (blue); TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ).

    Techniques Used: Hi-C, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Standard Deviation

    Engineering of a PE Sox1(+35 ) construct with the TFBS module and an artificial CGI. a, Strategy used to insert the PE Sox1(+35)TFBS alone of together with an artificial CGI (aCGI; see Methods) into the Gata6 -TAD. The upper left panel shows a close-up view of the epigenomic and genetic features of the PE Sox1(+35) (Vert. Cons.= vertebrate PhastCons). The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. The lower left panel shows the two combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)TFBS+aCGI ) inserted into the Gata6 TAD. The right panel shows the TAD in which Gata6 is included (i.e. Gata6 -TAD) according to publically available Hi-C data 33 , 34 ; TAD boundaries are denoted with dotted lines; H3K27me3 ChIP-seq signals in mESC are shown in green 9 ; CGIs are indicated as green rectangles; CTCF binding sites 35 are indicated as black rectangles; the red triangle indicates the integration site of the PE Sox1(+35) modules, approximately 100 Kb downstream of Gata6 . b, For the identification of the PE Sox1(+35)TFBS+aCGI insertion, primer pairs flanking the insertion borders (1+3 and 4+2), amplifying potential duplications (4+3 and 4+4) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for two mESC clonal lines with homozygous insertions of PE Sox1(+35)TFBS+aCGI in the Gata6- TAD are shown. c, Independent biological replicate for the data presented in Fig. 2e. The expression of Gata6 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the PE Sox1(+35)TFBS (blue) or PE Sox1(+35)TFBS+aCGI (red) insertions. For the cells with the PE insertions, two different clonal cell lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two house-keeping genes ( Eef1a and Hprt ).
    Figure Legend Snippet: Engineering of a PE Sox1(+35 ) construct with the TFBS module and an artificial CGI. a, Strategy used to insert the PE Sox1(+35)TFBS alone of together with an artificial CGI (aCGI; see Methods) into the Gata6 -TAD. The upper left panel shows a close-up view of the epigenomic and genetic features of the PE Sox1(+35) (Vert. Cons.= vertebrate PhastCons). The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. The lower left panel shows the two combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)TFBS+aCGI ) inserted into the Gata6 TAD. The right panel shows the TAD in which Gata6 is included (i.e. Gata6 -TAD) according to publically available Hi-C data 33 , 34 ; TAD boundaries are denoted with dotted lines; H3K27me3 ChIP-seq signals in mESC are shown in green 9 ; CGIs are indicated as green rectangles; CTCF binding sites 35 are indicated as black rectangles; the red triangle indicates the integration site of the PE Sox1(+35) modules, approximately 100 Kb downstream of Gata6 . b, For the identification of the PE Sox1(+35)TFBS+aCGI insertion, primer pairs flanking the insertion borders (1+3 and 4+2), amplifying potential duplications (4+3 and 4+4) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for two mESC clonal lines with homozygous insertions of PE Sox1(+35)TFBS+aCGI in the Gata6- TAD are shown. c, Independent biological replicate for the data presented in Fig. 2e. The expression of Gata6 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the PE Sox1(+35)TFBS (blue) or PE Sox1(+35)TFBS+aCGI (red) insertions. For the cells with the PE insertions, two different clonal cell lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two house-keeping genes ( Eef1a and Hprt ).

    Techniques Used: Construct, Hi-C, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Standard Deviation

    Modular engineering of the PE Sox1(+35) within the FoxA2 -TAD and of the PE Wnt8b(+21) within the Gata6 -TAD. a, Strategy used to insert the PE Sox1(+35) components into the Foxa2 -TAD. The upper left panel shows a close-up view of the epigenomic and genetic features of the PE Sox1(+35) (Vert. Cons.= vertebrate PhastCons). The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. The lower left panel shows the three combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) inserted into the FoxA2 -TAD. The right panel shows the TAD in which Foxa2 is included (i.e. Foxa2 -TAD) according to publically available Hi-C data 33 , 65 ; TAD boundaries are denoted with dotted lines; H3K27me3 ChIP-seq signals in mESC are shown in green 9 ; CGIs are indicated as green rectangles; the red triangle indicates the integration site of the PE Sox1(+35) modules, approximately 100 Kb downstream of Foxa2 . b, Strategy used to insert the PE Wnt8b(+21) components into the Gata6- TAD as described in (a). c-d, For identifying the successful insertion of the different PE Sox1(+35) (c) or PE Wnt8b(+21) (d) modules, primer pairs flanking the insertion borders (1+3 and 4+2; 1+5 and 6+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1; or 6+5, 5+2 and 6+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for two mESC clonal lines with homozygous insertions for each of the three different combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) or PE Wnt8b(+21) modules (i.e. (i) PE Wnt8b(+21)TFBS ; (ii) PE Wnt8b(+21)CGI ; (iii) PE Wnt8b(+21)TFBS+CGI ) in the Foxa2- TAD (c) or Gata6 -TAD (d), respectively, are shown. e-f, Independent biological replicate for the data shown in Fig. 2c (e) and Fig. 2d (f). The expression of Foxa2 (e), Gata6 (f), Sox1 (e) and Wnt8b (f) was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) (e) or Wnt8b(+21) (e) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal cell lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two house-keeping genes ( Eef1a and Hprt ).
    Figure Legend Snippet: Modular engineering of the PE Sox1(+35) within the FoxA2 -TAD and of the PE Wnt8b(+21) within the Gata6 -TAD. a, Strategy used to insert the PE Sox1(+35) components into the Foxa2 -TAD. The upper left panel shows a close-up view of the epigenomic and genetic features of the PE Sox1(+35) (Vert. Cons.= vertebrate PhastCons). The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. The lower left panel shows the three combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) inserted into the FoxA2 -TAD. The right panel shows the TAD in which Foxa2 is included (i.e. Foxa2 -TAD) according to publically available Hi-C data 33 , 65 ; TAD boundaries are denoted with dotted lines; H3K27me3 ChIP-seq signals in mESC are shown in green 9 ; CGIs are indicated as green rectangles; the red triangle indicates the integration site of the PE Sox1(+35) modules, approximately 100 Kb downstream of Foxa2 . b, Strategy used to insert the PE Wnt8b(+21) components into the Gata6- TAD as described in (a). c-d, For identifying the successful insertion of the different PE Sox1(+35) (c) or PE Wnt8b(+21) (d) modules, primer pairs flanking the insertion borders (1+3 and 4+2; 1+5 and 6+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1; or 6+5, 5+2 and 6+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for two mESC clonal lines with homozygous insertions for each of the three different combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) or PE Wnt8b(+21) modules (i.e. (i) PE Wnt8b(+21)TFBS ; (ii) PE Wnt8b(+21)CGI ; (iii) PE Wnt8b(+21)TFBS+CGI ) in the Foxa2- TAD (c) or Gata6 -TAD (d), respectively, are shown. e-f, Independent biological replicate for the data shown in Fig. 2c (e) and Fig. 2d (f). The expression of Foxa2 (e), Gata6 (f), Sox1 (e) and Wnt8b (f) was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) (e) or Wnt8b(+21) (e) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal cell lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two house-keeping genes ( Eef1a and Hprt ).

    Techniques Used: Hi-C, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Standard Deviation

    PE Sox1(+35)CGI recruits PcG and contributes to the cis-regulatory function of PE Sox1(+35) . a, For the identification of the PE Sox1(+35)CGI deletion, primer pairs flanking each of the deletion breakpoints (1+3 and 4+2), located within the deleted region (5+6) or amplifying a large or small fragment depending on the absence or presence of the deletion (1+2) were used. The PCR results obtained for WT ESC and for two mESC clonal lines with homozygous deletions of the PE Sox1(+35)CGI ( PE Sox1(+35)CGI -/ ) are shown. b, H3K27me3 levels at PE Sox1(+35) were measured by ChIP-qPCR in WT mESC (grey), and in two PE Sox1(+35)CG I-/- mESCs clones using primers adjacent to the deleted region. ChIP-qPCR signals were normalized against two negative regions (Supplementary Data 1). Error bars correspond to standard deviations from technical triplicates. c, Independent biological replicate for the data presented in Fig. 1d. The expression of Sox1 was investigated by RT-qPCR in mESCs (left panel) and AntNPC (right panel) that were either WT (grey), homozygous for a deletion of the PE Sox1(+35) CGI ( PE Sox1 CGI -/- ; red) or homozygous for the complete PE Sox1(+35) deletion 9 ( PE Sox1 -/- ; black). Two different PE Sox1 CGI -/- mESC clones (circles and diamonds) and one PE Sox1 -/- clone were studied. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values of each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ).
    Figure Legend Snippet: PE Sox1(+35)CGI recruits PcG and contributes to the cis-regulatory function of PE Sox1(+35) . a, For the identification of the PE Sox1(+35)CGI deletion, primer pairs flanking each of the deletion breakpoints (1+3 and 4+2), located within the deleted region (5+6) or amplifying a large or small fragment depending on the absence or presence of the deletion (1+2) were used. The PCR results obtained for WT ESC and for two mESC clonal lines with homozygous deletions of the PE Sox1(+35)CGI ( PE Sox1(+35)CGI -/ ) are shown. b, H3K27me3 levels at PE Sox1(+35) were measured by ChIP-qPCR in WT mESC (grey), and in two PE Sox1(+35)CG I-/- mESCs clones using primers adjacent to the deleted region. ChIP-qPCR signals were normalized against two negative regions (Supplementary Data 1). Error bars correspond to standard deviations from technical triplicates. c, Independent biological replicate for the data presented in Fig. 1d. The expression of Sox1 was investigated by RT-qPCR in mESCs (left panel) and AntNPC (right panel) that were either WT (grey), homozygous for a deletion of the PE Sox1(+35) CGI ( PE Sox1 CGI -/- ; red) or homozygous for the complete PE Sox1(+35) deletion 9 ( PE Sox1 -/- ; black). Two different PE Sox1 CGI -/- mESC clones (circles and diamonds) and one PE Sox1 -/- clone were studied. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values of each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ).

    Techniques Used: Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Clone Assay, Expressing, Quantitative RT-PCR, Standard Deviation

    Generation of mESC lines with structural variants within the Six3/Six2 locus. a, For the identification of mESC lines with the Six3/Six2 TAD boundary deletion, primer pairs flanking the deleted region (1+3 and 4+2), amplifying the deleted fragment (5+6) and amplifying a large or small fragment depending on the absence or presence of the deletion (1+2), respectively, were used. The PCR results obtained for two mESC clonal lines with 36Kb homozygous deletions ( del36 ) are shown. b, For the identification of mESC lines with the Six3/Six2 inversion, primer pairs flanking the inverted region (1+3, 4+2, 1+4 and 3+2) and amplifying potential duplications (4+3, 3+3 and 4+4) were used. The PCR results obtained for two mESC clonal lines with 110 Kb homozygous inversions ( inv110 ) are shown. c, 4C-seq experiments were performed using the PE Six3(-133) (upper panels) or the Six2 promoter (lower panels) as viewpoints in mESCs that were either WT (blue) or homozygous for the del36 deletion (red).
    Figure Legend Snippet: Generation of mESC lines with structural variants within the Six3/Six2 locus. a, For the identification of mESC lines with the Six3/Six2 TAD boundary deletion, primer pairs flanking the deleted region (1+3 and 4+2), amplifying the deleted fragment (5+6) and amplifying a large or small fragment depending on the absence or presence of the deletion (1+2), respectively, were used. The PCR results obtained for two mESC clonal lines with 36Kb homozygous deletions ( del36 ) are shown. b, For the identification of mESC lines with the Six3/Six2 inversion, primer pairs flanking the inverted region (1+3, 4+2, 1+4 and 3+2) and amplifying potential duplications (4+3, 3+3 and 4+4) were used. The PCR results obtained for two mESC clonal lines with 110 Kb homozygous inversions ( inv110 ) are shown. c, 4C-seq experiments were performed using the PE Six3(-133) (upper panels) or the Six2 promoter (lower panels) as viewpoints in mESCs that were either WT (blue) or homozygous for the del36 deletion (red).

    Techniques Used: Polymerase Chain Reaction

    Epigenetic characterization of the PE Sox1(+35) modules engineered within the Gata6-TAD. a , Detailed view of the bisulfite sequencing data presented in Fig. 3a , in which mESC (Day0) and AntNPC (Day5) differentiated from cell lines with the PE Sox1(+35)TFBS (left panel) or PE Sox1(+35)TFBS+CGI modules (right panel) inserted in the Gata6- TAD were analyzed. DNA methylation levels were measured using a forward bisulfite primer upstream of the insertion site and a reverse primer inside the TFBS module (Methods). The circles in the plots correspond to individual CpG dinucleotides located within the TFBS module. Unmethylated CpGs are shown in white, methylated CpGs in black and not-covered CpGs are shown in gray. 10 alleles (rows) were analyzed for each differentiation stage and cell line. b , Chromatin accessibility at the endogenous PE Sox1(+35) , the Gata6 TAD insertion site (primer pairs P1 and P2) and the Gata6 promoter were measured by FAIRE-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (gray) or homozygous for the insertions of the different PE Sox1(+35) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). FAIRE-qPCR signals were normalized against two negative control regions (Supplementary Data 1). Error bars correspond to standard deviations from technical triplicates. The location of the primer pairs P1 and P2 around the Gata6- TAD insertion site is represented as red arrows in the diagram shown to the right. c , DNA methylation and nucleosome occupancy at the TFBS module were simultaneously analyzed by NOME-PCR in ESC lines with the PE Sox1(+35)TFBS (left panel) or PE Sox1(+35)TFBS+CGI modules (right panel) inserted in the Gata6- TAD. In the upper panels, the black and white circles represent methylated or unmethylated CpG sites, respectively. In the lower panels, the blue or white circles represent accessible or inaccessible GpC sites for the GpC methyltransferase, respectively. Red bars represent regions large enough to accommodate a nucleosome and that are considered as inaccessible. The dotted line represents the region where the TFBS sequence starts. The primers used to amplify the TFBS sequences are shown as red arrows in the schematic diagrams, with one of the primers being located within the inserted TFBS and the other one immediately outside. The grey shaded area represent a nucleosome depleted region. d , Scatter plots showing population-averaged nucleosome occupancy (red) and DNA methylation (black) levels within the TFBS sequence in cells with either the PE Sox1(+35)TFBS (left panel) or PE Sox1(+35)TFBS+CGI (right panel) modules inserted within the Gata6 -TAD. The grey shaded area represent a nucleosome depleted region.
    Figure Legend Snippet: Epigenetic characterization of the PE Sox1(+35) modules engineered within the Gata6-TAD. a , Detailed view of the bisulfite sequencing data presented in Fig. 3a , in which mESC (Day0) and AntNPC (Day5) differentiated from cell lines with the PE Sox1(+35)TFBS (left panel) or PE Sox1(+35)TFBS+CGI modules (right panel) inserted in the Gata6- TAD were analyzed. DNA methylation levels were measured using a forward bisulfite primer upstream of the insertion site and a reverse primer inside the TFBS module (Methods). The circles in the plots correspond to individual CpG dinucleotides located within the TFBS module. Unmethylated CpGs are shown in white, methylated CpGs in black and not-covered CpGs are shown in gray. 10 alleles (rows) were analyzed for each differentiation stage and cell line. b , Chromatin accessibility at the endogenous PE Sox1(+35) , the Gata6 TAD insertion site (primer pairs P1 and P2) and the Gata6 promoter were measured by FAIRE-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (gray) or homozygous for the insertions of the different PE Sox1(+35) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). FAIRE-qPCR signals were normalized against two negative control regions (Supplementary Data 1). Error bars correspond to standard deviations from technical triplicates. The location of the primer pairs P1 and P2 around the Gata6- TAD insertion site is represented as red arrows in the diagram shown to the right. c , DNA methylation and nucleosome occupancy at the TFBS module were simultaneously analyzed by NOME-PCR in ESC lines with the PE Sox1(+35)TFBS (left panel) or PE Sox1(+35)TFBS+CGI modules (right panel) inserted in the Gata6- TAD. In the upper panels, the black and white circles represent methylated or unmethylated CpG sites, respectively. In the lower panels, the blue or white circles represent accessible or inaccessible GpC sites for the GpC methyltransferase, respectively. Red bars represent regions large enough to accommodate a nucleosome and that are considered as inaccessible. The dotted line represents the region where the TFBS sequence starts. The primers used to amplify the TFBS sequences are shown as red arrows in the schematic diagrams, with one of the primers being located within the inserted TFBS and the other one immediately outside. The grey shaded area represent a nucleosome depleted region. d , Scatter plots showing population-averaged nucleosome occupancy (red) and DNA methylation (black) levels within the TFBS sequence in cells with either the PE Sox1(+35)TFBS (left panel) or PE Sox1(+35)TFBS+CGI (right panel) modules inserted within the Gata6 -TAD. The grey shaded area represent a nucleosome depleted region.

    Techniques Used: Methylation Sequencing, DNA Methylation Assay, Methylation, Real-time Polymerase Chain Reaction, Negative Control, Polymerase Chain Reaction, Gel Permeation Chromatography, Sequencing

    Generation and characterization of cell lines with engineered PE Sox1(+35) modules within the Gria1-TAD . a, For the identification of mESC clonal lines with the desired insertion of the different PE Sox1(+35) modules, primer pairs flanking the insertion borders (1+3 and 4+2; 1+5 and 6+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1; or 6+5, 5+2 and 6+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for WT mESC or two mESC clonal lines with homozygous insertions of the different PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) in the Gria1- TAD are shown. b, Independent biological replicate for the data presented in Fig. 4b. The expression of Gria1 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) modules (TFBS (blue), CGI (yellow), TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ). c, Bisulfite sequencing analyses of ESC lines with the PE Sox1(+35) TFBS or PE Sox1(+35)TFBS+CGI modules inserted in the Gria1- TAD. DNA methylation levels were measured using a forward bisulfite primer upstream of the insertion site and a reverse primer inside the TFBS module (see Methods). The circles shown in the left plots correspond to individual CpG dinucleotides located within the TFBS module: unmethylated CpGs are shown in white, methylated CpGs in black and not-covered CpGs are shown in gray. 10 alleles (rows) were analyzed for each differentiation stage and cell line. The plot on the right summarizes the DNA methylation levels measured within the TFBS in the mESC lines containing the PE Sox1(+35)TFBS and PE Sox1(+35)TFBS+CGI mESC inserts within the Gria1 -TAD. d-e, RNAP2 (d), MED1 (d), H3K27me3 (e) and H2AK119ub (e) levels at the endogenous PE Sox1(+35) , the Gria1- TAD insertion site (primer pairs P1 and P2) and the Gria1 promoter were measured by ChIP-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (gray) or homozygous for the insertions of the different PE Sox1(+35) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). ChIP-qPCR signals were normalized against two negative control regions (Supplementary Data 1). Error bars correspond to standard deviations from technical triplicates. The location of the primers P1 and P2 around the Gria1- TAD insertion site is represented as red arrows in the diagram shown to the right.
    Figure Legend Snippet: Generation and characterization of cell lines with engineered PE Sox1(+35) modules within the Gria1-TAD . a, For the identification of mESC clonal lines with the desired insertion of the different PE Sox1(+35) modules, primer pairs flanking the insertion borders (1+3 and 4+2; 1+5 and 6+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1; or 6+5, 5+2 and 6+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for WT mESC or two mESC clonal lines with homozygous insertions of the different PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) in the Gria1- TAD are shown. b, Independent biological replicate for the data presented in Fig. 4b. The expression of Gria1 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) modules (TFBS (blue), CGI (yellow), TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ). c, Bisulfite sequencing analyses of ESC lines with the PE Sox1(+35) TFBS or PE Sox1(+35)TFBS+CGI modules inserted in the Gria1- TAD. DNA methylation levels were measured using a forward bisulfite primer upstream of the insertion site and a reverse primer inside the TFBS module (see Methods). The circles shown in the left plots correspond to individual CpG dinucleotides located within the TFBS module: unmethylated CpGs are shown in white, methylated CpGs in black and not-covered CpGs are shown in gray. 10 alleles (rows) were analyzed for each differentiation stage and cell line. The plot on the right summarizes the DNA methylation levels measured within the TFBS in the mESC lines containing the PE Sox1(+35)TFBS and PE Sox1(+35)TFBS+CGI mESC inserts within the Gria1 -TAD. d-e, RNAP2 (d), MED1 (d), H3K27me3 (e) and H2AK119ub (e) levels at the endogenous PE Sox1(+35) , the Gria1- TAD insertion site (primer pairs P1 and P2) and the Gria1 promoter were measured by ChIP-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (gray) or homozygous for the insertions of the different PE Sox1(+35) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). ChIP-qPCR signals were normalized against two negative control regions (Supplementary Data 1). Error bars correspond to standard deviations from technical triplicates. The location of the primers P1 and P2 around the Gria1- TAD insertion site is represented as red arrows in the diagram shown to the right.

    Techniques Used: Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Standard Deviation, Methylation Sequencing, DNA Methylation Assay, Methylation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    Modular engineering of the PE Sox1(+35) within the Gata6 -TAD. a, Genome-browser view of the epigenomic and genomic features of two previously characterized PEs 9 (left: PE Six3-(133) ; Right: PE Lmx1b(+59) ) in which the oCGIs overlap with conserved sequences bound by P300 and, thus, likely to represent TFBS. The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. Vert. Cons.= vertebrate PhastCons. b, For the identification of the different PE Sox1(+35) module insertions, primer pairs flanking the insertion borders (1+3 and 4+2; 1+5 and 6+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1; or 6+5, 5+2 and 6+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for WT mESC and for two mESC clonal lines with homozygous insertions for each of the three different combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) inserted in the Gata6- TAD are shown. c, Independent biological replicate for the data presented in Fig. 2b. The expression of Gata6 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal cell lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ).
    Figure Legend Snippet: Modular engineering of the PE Sox1(+35) within the Gata6 -TAD. a, Genome-browser view of the epigenomic and genomic features of two previously characterized PEs 9 (left: PE Six3-(133) ; Right: PE Lmx1b(+59) ) in which the oCGIs overlap with conserved sequences bound by P300 and, thus, likely to represent TFBS. The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. Vert. Cons.= vertebrate PhastCons. b, For the identification of the different PE Sox1(+35) module insertions, primer pairs flanking the insertion borders (1+3 and 4+2; 1+5 and 6+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1; or 6+5, 5+2 and 6+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for WT mESC and for two mESC clonal lines with homozygous insertions for each of the three different combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) inserted in the Gata6- TAD are shown. c, Independent biological replicate for the data presented in Fig. 2b. The expression of Gata6 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal cell lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ).

    Techniques Used: Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Standard Deviation

    8) Product Images from "Gene-specific Transcriptional Activation Mediated by the p150 Subunit of the Chromatin Assembly Factor 1 *Gene-specific Transcriptional Activation Mediated by the p150 Subunit of the Chromatin Assembly Factor 1 * S⃞"

    Article Title: Gene-specific Transcriptional Activation Mediated by the p150 Subunit of the Chromatin Assembly Factor 1 *Gene-specific Transcriptional Activation Mediated by the p150 Subunit of the Chromatin Assembly Factor 1 * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M901833200

    N-terminal 244 residues harbor the transcriptional activation domain of p150. A, N-terminal residues 1–244 of p150 are essential for MIEP activation. H1299 cells were transfected with pMIEP–733/+2 together with vector ( lane 1 ) or plasmid encoding the indicated p150 fragment tagged with V5 at the C terminus. Cell lysates were harvested 24 h post-transfection and analyzed for luciferase activity ( upper panel ) and protein expression ( lower panel ). The expression of both the endogenous and transfected p150 was analyzed by anti-p150 antibody. The asterisk indicates the band corresponding to the endogenous p150. The lower panel shows a schematic diagram of the p150 deletion constructs used and the result of MIEP activation. PEST , domain enriched in proline ( P ), glutamic acid ( E ), serine ( S ), and threonine ( T ); KER, domain enriched in lysine ( K ), glutamic acid ( E ), and arginine ( R ); ED, domain enriched in glutamic acid ( E ) and aspartic acid ( D ). B, wild-type p150, but not p150 lacking N-terminal aa 1–310, increases the association of RNA polymerase II with MIEP. H1299 cells were transfected with pMIEP–733/+2 together with vector or plasmid encoding C-terminally V5-tagged full-length p150 ( 1–938-V5 ) or p150 fragment from aa 311–938 ( 311–938-V5 ), followed by ChIP analysis. Polymerase II-associated DNAs were immunoprecipitated by anti-polymerase II antibody and analyzed on a LightCycler 480 with primers targeting the MIEP region from –245 to +2 bp, which contains the TATAA box. All real time PCR values were normalized to that of the input control. C, N-terminal aa 1–296 fused to the DNA binding domain of Gal4 protein (GalDBD) up-regulate the GalDBD-responsive promoter activity. H1299 cells were transfected with the Renilla reporter driven by the thymidine kinase promoter and the firefly luciferase reporter driven by the thymidine kinase promoter containing five additional Gal4-responsive elements, together with vector or the plasmid encoding the indicated GalDBD-fused p150 fragment, and analyzed for luciferase activity ( upper panel ) and protein expression ( lower panel ). All luciferase values were normalized to that of Renilla .
    Figure Legend Snippet: N-terminal 244 residues harbor the transcriptional activation domain of p150. A, N-terminal residues 1–244 of p150 are essential for MIEP activation. H1299 cells were transfected with pMIEP–733/+2 together with vector ( lane 1 ) or plasmid encoding the indicated p150 fragment tagged with V5 at the C terminus. Cell lysates were harvested 24 h post-transfection and analyzed for luciferase activity ( upper panel ) and protein expression ( lower panel ). The expression of both the endogenous and transfected p150 was analyzed by anti-p150 antibody. The asterisk indicates the band corresponding to the endogenous p150. The lower panel shows a schematic diagram of the p150 deletion constructs used and the result of MIEP activation. PEST , domain enriched in proline ( P ), glutamic acid ( E ), serine ( S ), and threonine ( T ); KER, domain enriched in lysine ( K ), glutamic acid ( E ), and arginine ( R ); ED, domain enriched in glutamic acid ( E ) and aspartic acid ( D ). B, wild-type p150, but not p150 lacking N-terminal aa 1–310, increases the association of RNA polymerase II with MIEP. H1299 cells were transfected with pMIEP–733/+2 together with vector or plasmid encoding C-terminally V5-tagged full-length p150 ( 1–938-V5 ) or p150 fragment from aa 311–938 ( 311–938-V5 ), followed by ChIP analysis. Polymerase II-associated DNAs were immunoprecipitated by anti-polymerase II antibody and analyzed on a LightCycler 480 with primers targeting the MIEP region from –245 to +2 bp, which contains the TATAA box. All real time PCR values were normalized to that of the input control. C, N-terminal aa 1–296 fused to the DNA binding domain of Gal4 protein (GalDBD) up-regulate the GalDBD-responsive promoter activity. H1299 cells were transfected with the Renilla reporter driven by the thymidine kinase promoter and the firefly luciferase reporter driven by the thymidine kinase promoter containing five additional Gal4-responsive elements, together with vector or the plasmid encoding the indicated GalDBD-fused p150 fragment, and analyzed for luciferase activity ( upper panel ) and protein expression ( lower panel ). All luciferase values were normalized to that of Renilla .

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Construct, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    p150 binds to a specific region of MIEP in vivo and in vitro . A, p150-mediated activation requires the MIEP region from –733 to –541 bp. H1299 cells were transfected with vector ( white bar ) or plasmid encoding HA-tagged p150 ( black bar ) together with pMIEP–733/+2 ( lanes 1 and 2 ), pMIEP–540/+2 ( lanes 3 and 4 ), or pMIEP–235/+2 ( lanes 5 and 6 ) and then harvested for luciferase activity analysis ( top panel ) and protein expression ( middle panel ). The bottom panel shows a schematic diagram of the MIEP deletion constructs and the result of HA-p150-mediated MIEP activation. B, ChIP assays indicate that the exogenously added HA-p150 specifically associates with the MIEP from –733 to –537 bp in cells. H1299 cells were transfected with vector or plasmid encoding HA-p150 together with pMIEP-733/+2, followed by ChIP analysis. HA-p150-associated DNAs were immunoprecipitated by anti-HA antibody and analyzed on a LightCycler 480 with primers targeting to a specific region on the MIEP ( black bars ) or three different regions within the luciferase gene ( white, gray, and hatched bars ). All real time PCR values were normalized to that of the input control. IgG was used for a background control. C, endogenous p150 binds to the MIEP. H1299 cells were transfected with vector or plasmid encoding the HA-tagged C-terminal residues (aa 641–938) of p150 (HA-p150C) together with the reporter plasmid pMIEP-733/+2 and followed by ChIP analysis similar to B, except here an antibody against the N terminus of p150 was used to precipitate the endogenous p150-associated DNAs. The precipitated DNAs were analyzed on a LightCycler 480 with primers targeting to –733 to –537 bp of MIEP. All real time PCR values were normalized to that of the input control. D, p150, but not p60, binds to MIEP from –733 to –537 bp in vitro . DAPA was performed to analyze the direct binding of p150 with MIEP. The in vitro translated 35 S-labeled p150 or p60 was incubated with streptavidin beads carrying biotin-labeled MIEP fragment from –733 to –537 bp. The 10% of input ( lane 1 ) and pulled down proteins on DNA/beads ( lane 2 ) was separated by SDS-PAGE and analyzed by autoradiography. E, minimum p150-responsive element of MIEP is from –593 to –574 bp. H1299 cells were transfected with vector ( white bar ) or plasmid encoding HA-tagged p150 ( black bar ) together with pMIEP–733/+2 ( lanes 1 and 2 ), or the plasmid encoding the firefly luciferase gene driven by serially deleted MIEP derivative as indicated ( lanes 3–20 ), and then harvested for luciferase activity analysis ( upper panel ) and protein expression ( lower panel ). Actin was used as a loading control. F, MIEP region from –593 to –574 bp is critical for p150 binding to MIEP in vitro . The protein lysates of H1299 cells were incubated with streptavidin beads carrying the indicated biotin-labeled MIEP fragment. The pulled down proteins on DNA/beads were then separated with 10% SDS-PAGE, followed by Western analysis with anti-p150 antibody.
    Figure Legend Snippet: p150 binds to a specific region of MIEP in vivo and in vitro . A, p150-mediated activation requires the MIEP region from –733 to –541 bp. H1299 cells were transfected with vector ( white bar ) or plasmid encoding HA-tagged p150 ( black bar ) together with pMIEP–733/+2 ( lanes 1 and 2 ), pMIEP–540/+2 ( lanes 3 and 4 ), or pMIEP–235/+2 ( lanes 5 and 6 ) and then harvested for luciferase activity analysis ( top panel ) and protein expression ( middle panel ). The bottom panel shows a schematic diagram of the MIEP deletion constructs and the result of HA-p150-mediated MIEP activation. B, ChIP assays indicate that the exogenously added HA-p150 specifically associates with the MIEP from –733 to –537 bp in cells. H1299 cells were transfected with vector or plasmid encoding HA-p150 together with pMIEP-733/+2, followed by ChIP analysis. HA-p150-associated DNAs were immunoprecipitated by anti-HA antibody and analyzed on a LightCycler 480 with primers targeting to a specific region on the MIEP ( black bars ) or three different regions within the luciferase gene ( white, gray, and hatched bars ). All real time PCR values were normalized to that of the input control. IgG was used for a background control. C, endogenous p150 binds to the MIEP. H1299 cells were transfected with vector or plasmid encoding the HA-tagged C-terminal residues (aa 641–938) of p150 (HA-p150C) together with the reporter plasmid pMIEP-733/+2 and followed by ChIP analysis similar to B, except here an antibody against the N terminus of p150 was used to precipitate the endogenous p150-associated DNAs. The precipitated DNAs were analyzed on a LightCycler 480 with primers targeting to –733 to –537 bp of MIEP. All real time PCR values were normalized to that of the input control. D, p150, but not p60, binds to MIEP from –733 to –537 bp in vitro . DAPA was performed to analyze the direct binding of p150 with MIEP. The in vitro translated 35 S-labeled p150 or p60 was incubated with streptavidin beads carrying biotin-labeled MIEP fragment from –733 to –537 bp. The 10% of input ( lane 1 ) and pulled down proteins on DNA/beads ( lane 2 ) was separated by SDS-PAGE and analyzed by autoradiography. E, minimum p150-responsive element of MIEP is from –593 to –574 bp. H1299 cells were transfected with vector ( white bar ) or plasmid encoding HA-tagged p150 ( black bar ) together with pMIEP–733/+2 ( lanes 1 and 2 ), or the plasmid encoding the firefly luciferase gene driven by serially deleted MIEP derivative as indicated ( lanes 3–20 ), and then harvested for luciferase activity analysis ( upper panel ) and protein expression ( lower panel ). Actin was used as a loading control. F, MIEP region from –593 to –574 bp is critical for p150 binding to MIEP in vitro . The protein lysates of H1299 cells were incubated with streptavidin beads carrying the indicated biotin-labeled MIEP fragment. The pulled down proteins on DNA/beads were then separated with 10% SDS-PAGE, followed by Western analysis with anti-p150 antibody.

    Techniques Used: In Vivo, In Vitro, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Construct, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Labeling, Incubation, SDS Page, Autoradiography, Western Blot

    N-terminal residues 1-310 of p150 are essential for the recruitment of p300 to MIEP. H1299 cells were transfected with pMIEP–733/+2 together with vector or plasmid encoding C-terminally V5-tagged full-length p150 ( 1–938-V5 ) or aa 311–938 of p150 ( 311–938-V5 ), followed by ChIP analysis. Acetylated histone H3-associated ( top panel ) or H4-associated ( middle panel ) or p300-associated DNAs were immunoprecipitated ( IP ) by corresponding antibody and analyzed on a LightCycler 480 with primers targeting to the MIEP region from –733 to 537 bp. All real time PCR values were normalized to that of the input control.
    Figure Legend Snippet: N-terminal residues 1-310 of p150 are essential for the recruitment of p300 to MIEP. H1299 cells were transfected with pMIEP–733/+2 together with vector or plasmid encoding C-terminally V5-tagged full-length p150 ( 1–938-V5 ) or aa 311–938 of p150 ( 311–938-V5 ), followed by ChIP analysis. Acetylated histone H3-associated ( top panel ) or H4-associated ( middle panel ) or p300-associated DNAs were immunoprecipitated ( IP ) by corresponding antibody and analyzed on a LightCycler 480 with primers targeting to the MIEP region from –733 to 537 bp. All real time PCR values were normalized to that of the input control.

    Techniques Used: Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    9) Product Images from "Selection-Driven Accumulation of Suppressor Mutants in Bacillus subtilis: The Apparent High Mutation Frequency of the Cryptic gudB Gene and the Rapid Clonal Expansion of gudB+ Suppressors Are Due to Growth under Selection"

    Article Title: Selection-Driven Accumulation of Suppressor Mutants in Bacillus subtilis: The Apparent High Mutation Frequency of the Cryptic gudB Gene and the Rapid Clonal Expansion of gudB+ Suppressors Are Due to Growth under Selection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0066120

    Direct visualization of the emergence and clonal expansion of the decryptified gudB + allele in B. subtilis . (A) Decryptification of the gudB CR allele and clonal expansion of the gudB + mutants over time in complex medium. Exposure time, 0.6 s; scale bar, 5 µm. The state of the DR in the gudB CR allele in strain BP22 ( ΔrocG gfp-gudB CR ) was analyzed by colony PCR and the DNA species were visualized by PAA gel electrophoresis ( Figure S2 ). Strain BP23 ( ΔrocG gfp-gudB + ) expressing the active gudB + allele served as the positive control. (B) Emergence of gfp-gudB + suppressor mutants in a developing colony of strain BP22 ( ΔrocG gfp-gudB CR ) on rich medium. The white arrows indicate late suppressors. Exposure time, 2 s; scale bar, 2 mm.
    Figure Legend Snippet: Direct visualization of the emergence and clonal expansion of the decryptified gudB + allele in B. subtilis . (A) Decryptification of the gudB CR allele and clonal expansion of the gudB + mutants over time in complex medium. Exposure time, 0.6 s; scale bar, 5 µm. The state of the DR in the gudB CR allele in strain BP22 ( ΔrocG gfp-gudB CR ) was analyzed by colony PCR and the DNA species were visualized by PAA gel electrophoresis ( Figure S2 ). Strain BP23 ( ΔrocG gfp-gudB + ) expressing the active gudB + allele served as the positive control. (B) Emergence of gfp-gudB + suppressor mutants in a developing colony of strain BP22 ( ΔrocG gfp-gudB CR ) on rich medium. The white arrows indicate late suppressors. Exposure time, 2 s; scale bar, 2 mm.

    Techniques Used: Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Expressing, Positive Control

    Stabilities of DRs present in the native gudB CR and in the gudB CR Sac I -gfp alleles. (A) In addition to the native gudB CR allele, a second gudB CR -gfp fusion that could be potentially mutated during growth of a B. subtilis ΔrocG mutant under selective pressure was introduced into the amyE locus on the chromosome. (B) DNA species comprising the 9 bp DR were amplified by colony PCR using gudB -specific oligonucleotides (see Materials and Methods). To distinguish the DNA species derived from the two gudB CR alleles, a Sac I site was introduced into the gudB CR -gfp allele by exchanging G at position 402 by C. (C) Schematic illustration of the fragment pattern of DNA species obtained from cells collected prior to selective growth and after selection. The same samples were treated with Sac I. The emergence of a 147 bp DNA species shown by red letters would indicate the decryptification of the gudB CR -gfp allele. (D) Fragment pattern of DNA species obtained from real samples.
    Figure Legend Snippet: Stabilities of DRs present in the native gudB CR and in the gudB CR Sac I -gfp alleles. (A) In addition to the native gudB CR allele, a second gudB CR -gfp fusion that could be potentially mutated during growth of a B. subtilis ΔrocG mutant under selective pressure was introduced into the amyE locus on the chromosome. (B) DNA species comprising the 9 bp DR were amplified by colony PCR using gudB -specific oligonucleotides (see Materials and Methods). To distinguish the DNA species derived from the two gudB CR alleles, a Sac I site was introduced into the gudB CR -gfp allele by exchanging G at position 402 by C. (C) Schematic illustration of the fragment pattern of DNA species obtained from cells collected prior to selective growth and after selection. The same samples were treated with Sac I. The emergence of a 147 bp DNA species shown by red letters would indicate the decryptification of the gudB CR -gfp allele. (D) Fragment pattern of DNA species obtained from real samples.

    Techniques Used: Mutagenesis, Amplification, Polymerase Chain Reaction, Derivative Assay, Selection

    10) Product Images from "Dosage Compensation in the Mouse Balances Up-Regulation and Silencing of X-Linked GenesSex, Dose, and Equality"

    Article Title: Dosage Compensation in the Mouse Balances Up-Regulation and Silencing of X-Linked GenesSex, Dose, and Equality

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0050326

    Allele-Specific Analysis of X-Linked Gene Expression during Differentiation of Hybrid ( 129 × castaneous ) Female ES Cells (A) Electrophoretic separation of cDNA restriction fragments from the three cluster 4 genes Acsl4 , Jarid1c , and GM784 reveals allele-specific expression. PCR fragments that are specific for each gene were prepared from cDNAs from undifferentiated (day 0) or differentiated (day 12) 3F1 hybrid ES cells. Fragments were uncut (U) or cut (C) with restriction enzymes, so as to reveal SNPs that distinguish m.m.domesticus ( 129 , black arrows) and m.m.castaneous (white arrows) alleles. Small double arrows show where bands of the same size are generated from 129 and castaneous . For all three genes, the expected 129 product is missing from both undifferentiated (day 0) and differentiated (day 12) cells, showing that expression is exclusively from the castaneous allele. cDNA from the male CCE/R ES cell line provided the 129 control. Details of primers, restriction enzymes, and expected fragment sizes are given in Table S5 . (B) Electrophoretic separation of cDNA restriction fragments from four X-linked genes Ogt , Brodl , Pctk1 , and Pgr15l to reveal allele-specific expression in 3F1 hybrid female ES cells at different days of differentiation (days 0–15 as indicated); labelling as for (A). (C) In undifferentiated 16.6 hybrid female ES cells, cluster 4 gene Acsl4 (upper gel) shows monoallelic expression ( castaneous allele only) while Jarid1c (lower gel) is expressed from both alleles; labelling as for (A).
    Figure Legend Snippet: Allele-Specific Analysis of X-Linked Gene Expression during Differentiation of Hybrid ( 129 × castaneous ) Female ES Cells (A) Electrophoretic separation of cDNA restriction fragments from the three cluster 4 genes Acsl4 , Jarid1c , and GM784 reveals allele-specific expression. PCR fragments that are specific for each gene were prepared from cDNAs from undifferentiated (day 0) or differentiated (day 12) 3F1 hybrid ES cells. Fragments were uncut (U) or cut (C) with restriction enzymes, so as to reveal SNPs that distinguish m.m.domesticus ( 129 , black arrows) and m.m.castaneous (white arrows) alleles. Small double arrows show where bands of the same size are generated from 129 and castaneous . For all three genes, the expected 129 product is missing from both undifferentiated (day 0) and differentiated (day 12) cells, showing that expression is exclusively from the castaneous allele. cDNA from the male CCE/R ES cell line provided the 129 control. Details of primers, restriction enzymes, and expected fragment sizes are given in Table S5 . (B) Electrophoretic separation of cDNA restriction fragments from four X-linked genes Ogt , Brodl , Pctk1 , and Pgr15l to reveal allele-specific expression in 3F1 hybrid female ES cells at different days of differentiation (days 0–15 as indicated); labelling as for (A). (C) In undifferentiated 16.6 hybrid female ES cells, cluster 4 gene Acsl4 (upper gel) shows monoallelic expression ( castaneous allele only) while Jarid1c (lower gel) is expressed from both alleles; labelling as for (A).

    Techniques Used: Expressing, Polymerase Chain Reaction, Generated

    Expression of X-Linked Genes in Male and Female ICM (A) Gel showing presence or absence of PCR products derived from the Sry gene in trophectodermal material from single blastocysts used for ICM cDNA preparation. Lane 1, 123 bp size markers; lane 2, male embryo; lane 3, female embryo; lane 4 male ES cell line CCE/R. (B) Correlation in expression levels of X-linked genes between female ( y -axis) and male ( x -axis) ICMs. The value for each gene is expressed relative to the expression of autosomal genes (X:A ratio, log 2 scale). Pearson product moment correlation coefficient ( r value) and FDR-corrected probability of chance correlation ( p ) are shown. (C) Box plot showing expression of X-linked genes (X:A ratio) in male and female ICMs. The box encompasses the 25th–75th percentile, and the upper and lower lines represent the 10th–90th percentile. Outlying values are shown as individual dots. Female values are from two biological replicates and male values from two technical replicates. (D) Expression of the Y-encoded, male-specific antigen Eif2s3y and Xist RNA, relative to autosomal genes (log 2 ratio), in ICMs from female and male embryos.
    Figure Legend Snippet: Expression of X-Linked Genes in Male and Female ICM (A) Gel showing presence or absence of PCR products derived from the Sry gene in trophectodermal material from single blastocysts used for ICM cDNA preparation. Lane 1, 123 bp size markers; lane 2, male embryo; lane 3, female embryo; lane 4 male ES cell line CCE/R. (B) Correlation in expression levels of X-linked genes between female ( y -axis) and male ( x -axis) ICMs. The value for each gene is expressed relative to the expression of autosomal genes (X:A ratio, log 2 scale). Pearson product moment correlation coefficient ( r value) and FDR-corrected probability of chance correlation ( p ) are shown. (C) Box plot showing expression of X-linked genes (X:A ratio) in male and female ICMs. The box encompasses the 25th–75th percentile, and the upper and lower lines represent the 10th–90th percentile. Outlying values are shown as individual dots. Female values are from two biological replicates and male values from two technical replicates. (D) Expression of the Y-encoded, male-specific antigen Eif2s3y and Xist RNA, relative to autosomal genes (log 2 ratio), in ICMs from female and male embryos.

    Techniques Used: Expressing, Polymerase Chain Reaction, Derivative Assay

    11) Product Images from "Tip60 functions as a potential corepressor of KLF4 in regulation of HDC promoter activity"

    Article Title: Tip60 functions as a potential corepressor of KLF4 in regulation of HDC promoter activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm656

    KLF4, Tip60 and HDAC7 bind to HDC promoter by chromatin immunoprecipitation assay. (A) Overnight starved AGSE cells were treated with 10 −8 M gastrin for 2 h. Cells were then fixed with formaldehyde followed by protein extraction as described in Materials and Methods section. Antibodies against KLF4, Tip60, HDAC7, acetyl-H4 plus control IgG were used to precipitate DNA–protein complexes. Purified DNA samples from precipitated DNA–protein complexes were used as template for PCR to amplify a fragment of HDC promoter, intron 1 and intron 2. ( B ) Gastrin treatment increased 107HDC promoter activity. Vector (pGL2), 107HDC reporter and 107HDCM reporter were transfected into AGSE cells. Before harvesting, cells were treated with 10 −8 M gastrin from 2 h. Then, luciferase assays were performed as described. ( C ) mRNA levels of KLF4, HDC, Tip60 and HDAC7 after 10 −8 M gastrin treatment were shown. Total RNA was extracted after gastrin treatment at different time points. Reverse transcription and follow-up quantitative RT-PCR was performed. Relative mRNA level was calculated as described in Materials and Methods section. Means ± SD for three independent experiments were shown, and statistical difference ( P
    Figure Legend Snippet: KLF4, Tip60 and HDAC7 bind to HDC promoter by chromatin immunoprecipitation assay. (A) Overnight starved AGSE cells were treated with 10 −8 M gastrin for 2 h. Cells were then fixed with formaldehyde followed by protein extraction as described in Materials and Methods section. Antibodies against KLF4, Tip60, HDAC7, acetyl-H4 plus control IgG were used to precipitate DNA–protein complexes. Purified DNA samples from precipitated DNA–protein complexes were used as template for PCR to amplify a fragment of HDC promoter, intron 1 and intron 2. ( B ) Gastrin treatment increased 107HDC promoter activity. Vector (pGL2), 107HDC reporter and 107HDCM reporter were transfected into AGSE cells. Before harvesting, cells were treated with 10 −8 M gastrin from 2 h. Then, luciferase assays were performed as described. ( C ) mRNA levels of KLF4, HDC, Tip60 and HDAC7 after 10 −8 M gastrin treatment were shown. Total RNA was extracted after gastrin treatment at different time points. Reverse transcription and follow-up quantitative RT-PCR was performed. Relative mRNA level was calculated as described in Materials and Methods section. Means ± SD for three independent experiments were shown, and statistical difference ( P

    Techniques Used: Chromatin Immunoprecipitation, Protein Extraction, Purification, Polymerase Chain Reaction, Activity Assay, Plasmid Preparation, Transfection, Luciferase, Quantitative RT-PCR

    12) Product Images from "Identification of two regulatory binding sites which confer myotube specific expression of the mono-ADP-ribosyltransferase ART1 gene"

    Article Title: Identification of two regulatory binding sites which confer myotube specific expression of the mono-ADP-ribosyltransferase ART1 gene

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-9-91

    Myogenin and MEF-2 bind to the ART1 promoter in myotubes, but not in myoblasts in vivo . The chromatin from C2C12 cells (myoblasts and differentiated myotubes) was cross-linked with formalin. Cells were lysed, the nuclear extracts were prepared and sonicated (four times 15 strokes; output, 70%; duty cycle, 60%; Bandelin Sonopuls GM70, Bandelin, Berlin, Germany). After precipitation with antibodies against myogenin (αMG), MEF-2 (αMEF-2) or with an unspecific IgG isotype antibody (Iso) as a control, the ART1 promoter region containing the E box und the A/T rich element was amplified by PCR from the precipitated DNA. Lanes are input DNA (Input) in dilutions: (1/100) 1:100, (1/10) 1:10 und undiluted (1), no antibody (no), water control (H 2 O) and DNA ladder (M). The PCR amplification of the desmin gene promoter enhancer [ 43 ] which contains a functional E box and a functional MEF-2 binding site served as a positive control whereas the amplification of the proximately part of the HPRT-1 housekeeping gene promoter served as negative control.
    Figure Legend Snippet: Myogenin and MEF-2 bind to the ART1 promoter in myotubes, but not in myoblasts in vivo . The chromatin from C2C12 cells (myoblasts and differentiated myotubes) was cross-linked with formalin. Cells were lysed, the nuclear extracts were prepared and sonicated (four times 15 strokes; output, 70%; duty cycle, 60%; Bandelin Sonopuls GM70, Bandelin, Berlin, Germany). After precipitation with antibodies against myogenin (αMG), MEF-2 (αMEF-2) or with an unspecific IgG isotype antibody (Iso) as a control, the ART1 promoter region containing the E box und the A/T rich element was amplified by PCR from the precipitated DNA. Lanes are input DNA (Input) in dilutions: (1/100) 1:100, (1/10) 1:10 und undiluted (1), no antibody (no), water control (H 2 O) and DNA ladder (M). The PCR amplification of the desmin gene promoter enhancer [ 43 ] which contains a functional E box and a functional MEF-2 binding site served as a positive control whereas the amplification of the proximately part of the HPRT-1 housekeeping gene promoter served as negative control.

    Techniques Used: In Vivo, Sonication, Amplification, Polymerase Chain Reaction, Functional Assay, Binding Assay, Positive Control, Negative Control

    13) Product Images from "IRF1 Negatively Regulates Oncogenic KPNA2 Expression Under Growth Stimulation and Hypoxia in Lung Cancer Cells"

    Article Title: IRF1 Negatively Regulates Oncogenic KPNA2 Expression Under Growth Stimulation and Hypoxia in Lung Cancer Cells

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S221832

    IRF1 binds to the promoter region of KPNA2 and negatively modulates KPNA2 transcription. ( A ) Diagram depicting the regions of predicted IRF1 binding sites (highlighted with blue and green boxes) within the KPNA2 promoter region (NC_000017.11, ranging from 68,035,602 to 68,046,859) based on two software packages, PROMO and TFBIND. The red boxes indicate the location of primer pairs used in the ChIP assay. ( B – C ) The ChIP assay detected positive binding of IRF1 to the KPNA2 promoter sequence. Fragmented chromatin prepared from A549 ( B ) and CL1-5 cells ( C ) was immunoprecipitated with IgG, IRF1 or E2F1 antibodies, as indicated. An anti-E2F1 antibody was used as a positive control. DNA isolated from the immunoprecipitated material was amplified by PCR, and the resulting fragments were analyzed by agarose gel electrophoresis. ( D – E ) IRF1 knockdown increased the mRNA level of KPNA2 in lung ADC cells. A549 or CL1-5 cells were transfected with negative control (NC) or IRF1 siRNA for 24 h, and total RNA was purified and subjected to qRT-PCR using IRF1 and KPNA2 primers. mRNA levels were calculated as a ratio relative to the control treatment. The data are presented as the mean ± SD from three independent experiments. * p
    Figure Legend Snippet: IRF1 binds to the promoter region of KPNA2 and negatively modulates KPNA2 transcription. ( A ) Diagram depicting the regions of predicted IRF1 binding sites (highlighted with blue and green boxes) within the KPNA2 promoter region (NC_000017.11, ranging from 68,035,602 to 68,046,859) based on two software packages, PROMO and TFBIND. The red boxes indicate the location of primer pairs used in the ChIP assay. ( B – C ) The ChIP assay detected positive binding of IRF1 to the KPNA2 promoter sequence. Fragmented chromatin prepared from A549 ( B ) and CL1-5 cells ( C ) was immunoprecipitated with IgG, IRF1 or E2F1 antibodies, as indicated. An anti-E2F1 antibody was used as a positive control. DNA isolated from the immunoprecipitated material was amplified by PCR, and the resulting fragments were analyzed by agarose gel electrophoresis. ( D – E ) IRF1 knockdown increased the mRNA level of KPNA2 in lung ADC cells. A549 or CL1-5 cells were transfected with negative control (NC) or IRF1 siRNA for 24 h, and total RNA was purified and subjected to qRT-PCR using IRF1 and KPNA2 primers. mRNA levels were calculated as a ratio relative to the control treatment. The data are presented as the mean ± SD from three independent experiments. * p

    Techniques Used: Binding Assay, Software, Chromatin Immunoprecipitation, Sequencing, Immunoprecipitation, Positive Control, Isolation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transfection, Negative Control, Purification, Quantitative RT-PCR

    14) Product Images from "Phenotypic and Transcriptomic Analyses Demonstrate Interactions between the Transcriptional Regulators CtsR and Sigma B in Listeria monocytogenes ▿ ▿ †"

    Article Title: Phenotypic and Transcriptomic Analyses Demonstrate Interactions between the Transcriptional Regulators CtsR and Sigma B in Listeria monocytogenes ▿ ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01085-07

    Transcript levels for clpC (A), gadA (B), prfA (C), plcA (D), and inlA (E) in the parent strain (10403S) and the Δ ctsR , Δ sigB , and Δ ctsR Δ sigB strains. Bacteria were either (i) grown to log phase in BHI at 37°C (BHI) or (ii) grown to log phase, followed by exposure to BHI with 0.3 M NaCl for 10 min at 37°C (BHI-NaCl). Transcript levels are expressed as log cDNA copy numbers normalized to the geometric mean of cDNA copy numbers for the housekeeping genes rpoB and gap {i.e., log 10 target gene − [(log 10 rpoB + log 10 gap )/2]}; indicated as “log 10 normalized copy no.” on the y axes). The values shown represent the averages of qRT-PCR assays performed on three independent RNA collections; the error bars show standard deviations. NS indicates that neither the ctsR deletion, the sigB deletion, nor the interaction between the ctsR and sigB deletions showed a significant effect using a linear model for statistical analyses. If the linear model showed a significant effect of either the ctsR deletion or the sigB deletion on transcript levels for a given gene for cells grown under a given condition (BHI or BHI-NaCl), the respective P value is given on the graph (see Table S2 in the supplemental material for all results of these statistical analyses). Tukey's multiple-comparison procedure was used to determine whether transcript levels for a given gene differed between specific strains; the bars labeled with different letters (a and b) indicate transcript levels that differed significantly ( P
    Figure Legend Snippet: Transcript levels for clpC (A), gadA (B), prfA (C), plcA (D), and inlA (E) in the parent strain (10403S) and the Δ ctsR , Δ sigB , and Δ ctsR Δ sigB strains. Bacteria were either (i) grown to log phase in BHI at 37°C (BHI) or (ii) grown to log phase, followed by exposure to BHI with 0.3 M NaCl for 10 min at 37°C (BHI-NaCl). Transcript levels are expressed as log cDNA copy numbers normalized to the geometric mean of cDNA copy numbers for the housekeeping genes rpoB and gap {i.e., log 10 target gene − [(log 10 rpoB + log 10 gap )/2]}; indicated as “log 10 normalized copy no.” on the y axes). The values shown represent the averages of qRT-PCR assays performed on three independent RNA collections; the error bars show standard deviations. NS indicates that neither the ctsR deletion, the sigB deletion, nor the interaction between the ctsR and sigB deletions showed a significant effect using a linear model for statistical analyses. If the linear model showed a significant effect of either the ctsR deletion or the sigB deletion on transcript levels for a given gene for cells grown under a given condition (BHI or BHI-NaCl), the respective P value is given on the graph (see Table S2 in the supplemental material for all results of these statistical analyses). Tukey's multiple-comparison procedure was used to determine whether transcript levels for a given gene differed between specific strains; the bars labeled with different letters (a and b) indicate transcript levels that differed significantly ( P

    Techniques Used: Quantitative RT-PCR, Labeling

    15) Product Images from "Androgen Receptor Antagonism By Divalent Ethisterone Conjugates In Castrate-Resistant Prostate Cancer Cells"

    Article Title: Androgen Receptor Antagonism By Divalent Ethisterone Conjugates In Castrate-Resistant Prostate Cancer Cells

    Journal: ACS chemical biology

    doi: 10.1021/cb300332w

    Conjugates 1 and 2 disrupt co-activator peptide recruitment and DNA binding. Conjugate 2 also induces cell cycle arrest. ( A ) In vitro time-resolved fluorescence resonance energy transfer (TR-FRET) analysis of the interaction between purified GST-tagged AR-LBD, terbium-labeled AR-specific anti-GST antibody and fluorescein-labeled AR FxxLF co-activator peptide (increasing concentrations of DHT, Bicalutamide (Bic.), and Conjugate 1 or 2 were evaluated). The TR-FRET signal intensity between terbium-labeled antibody and labeled FxxLF-motif peptide is established by co-activator recruitment to the AR-LBD (520:495 nm emission ratio after excitation at 340 nm). Data presented as mean ± SD of triplicates. ( B ) Chromatin immunoprecipitation analysis of AR intreated LNCaP-abl cells. Real-time PCR quantification of immunoprecipitated PSA enhancer is shown (R-1881, 10 nM; Conjugate 1 or 2 , 1 μM). Data presented as mean + SD of triplicates. ( C ) Fluorescence-activated cell sorting analysis of treated LNCaP-abl cells (Veh., EtOH treated cells; Conjugate 1 or 2 , 1 μM).
    Figure Legend Snippet: Conjugates 1 and 2 disrupt co-activator peptide recruitment and DNA binding. Conjugate 2 also induces cell cycle arrest. ( A ) In vitro time-resolved fluorescence resonance energy transfer (TR-FRET) analysis of the interaction between purified GST-tagged AR-LBD, terbium-labeled AR-specific anti-GST antibody and fluorescein-labeled AR FxxLF co-activator peptide (increasing concentrations of DHT, Bicalutamide (Bic.), and Conjugate 1 or 2 were evaluated). The TR-FRET signal intensity between terbium-labeled antibody and labeled FxxLF-motif peptide is established by co-activator recruitment to the AR-LBD (520:495 nm emission ratio after excitation at 340 nm). Data presented as mean ± SD of triplicates. ( B ) Chromatin immunoprecipitation analysis of AR intreated LNCaP-abl cells. Real-time PCR quantification of immunoprecipitated PSA enhancer is shown (R-1881, 10 nM; Conjugate 1 or 2 , 1 μM). Data presented as mean + SD of triplicates. ( C ) Fluorescence-activated cell sorting analysis of treated LNCaP-abl cells (Veh., EtOH treated cells; Conjugate 1 or 2 , 1 μM).

    Techniques Used: Binding Assay, In Vitro, Fluorescence, Förster Resonance Energy Transfer, Purification, Labeling, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, FACS

    16) Product Images from "Mediator subunit MED25 links the jasmonate receptor to transcriptionally active chromatin"

    Article Title: Mediator subunit MED25 links the jasmonate receptor to transcriptionally active chromatin

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

    doi: 10.1073/pnas.1710885114

    Enrichment of H3K9ac on the chromatin of JAZ8 and ERF1 . ( A ) Schematic diagrams of JAZ8 , ERF1 , and the PCR amplicons indicated as letters A–D used for ChIP-qPCR. ( B ) ChIP-qPCR shows the enrichment of H3K9ac on the chromatin of JAZ8 and ERF1 . The
    Figure Legend Snippet: Enrichment of H3K9ac on the chromatin of JAZ8 and ERF1 . ( A ) Schematic diagrams of JAZ8 , ERF1 , and the PCR amplicons indicated as letters A–D used for ChIP-qPCR. ( B ) ChIP-qPCR shows the enrichment of H3K9ac on the chromatin of JAZ8 and ERF1 . The

    Techniques Used: Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Enrichment of COI1 and MED25 on the promoters of JAZ8 and ERF1 . ( A ) Schematic diagrams of JAZ8 , ERF1 , and PCR amplicons indicated as letters A–D used for ChIP-qPCR. ( B ) ChIP-qPCR showing the enrichment of COI1 on the chromatin of JAZ8 and ERF1
    Figure Legend Snippet: Enrichment of COI1 and MED25 on the promoters of JAZ8 and ERF1 . ( A ) Schematic diagrams of JAZ8 , ERF1 , and PCR amplicons indicated as letters A–D used for ChIP-qPCR. ( B ) ChIP-qPCR showing the enrichment of COI1 on the chromatin of JAZ8 and ERF1

    Techniques Used: Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    COI1 and MED25 affect each other’s enrichment on the promoters of JAZ8 and ERF1 . ( A ) Schematic diagrams of JAZ8 , ERF1 , and the PCR amplicons indicated as letters A–D used for ChIP-qPCR. ( B ) Sequential ChIP analysis showing that COI1 and
    Figure Legend Snippet: COI1 and MED25 affect each other’s enrichment on the promoters of JAZ8 and ERF1 . ( A ) Schematic diagrams of JAZ8 , ERF1 , and the PCR amplicons indicated as letters A–D used for ChIP-qPCR. ( B ) Sequential ChIP analysis showing that COI1 and

    Techniques Used: Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Depletion of COI1 or MED25 impairs the function of HAC1 on the promoters of JAZ8 and ERF1 . ( A ) Schematic diagrams of JAZ8 , ERF1 , and PCR amplicons indicated as letters A–D used for ChIP-qPCR. ( B ) ChIP-qPCR assays showing that coi1-2 impairs the
    Figure Legend Snippet: Depletion of COI1 or MED25 impairs the function of HAC1 on the promoters of JAZ8 and ERF1 . ( A ) Schematic diagrams of JAZ8 , ERF1 , and PCR amplicons indicated as letters A–D used for ChIP-qPCR. ( B ) ChIP-qPCR assays showing that coi1-2 impairs the

    Techniques Used: Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    17) Product Images from "Transcriptional repression of cancer stem cell marker CD133 by tumor suppressor p53"

    Article Title: Transcriptional repression of cancer stem cell marker CD133 by tumor suppressor p53

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.313

    p53 directly regulates CD133 transcription. ( a ) Transcript variants produced by the alternate activity of five promoters (promoter 1–5) were identified in five cell lines by nested PCR. ( b ) p53-mediated repression of CD133 promoter activity in NTERA2, NCCIT, and H1299 cells. ( c ) Activity of the control CD133 promoter and two mutated CD133 promoters in NTERA2 cells. ( d ) Activity of the control CD133 promoter and mutated CD133 promoter in H1299 cell lines. Empty or p53 expression vectors were co-transfected with the reporter gene vector. ( e ) ChIP analysis of p53-binding to CD133 promoter DNA in NTERA2, U2OS, and H1299 cells that had been transfected with either control or p53 expression vector. Protein levels of ectopically expressed p53 were examined in total ChIP lysates. ( f ) ChIP analysis of p53-binding to CD133 promoter DNA in NCCIT cells treated with Dox or DMSO. The error bars represent the mean±S.D. n =3. * P
    Figure Legend Snippet: p53 directly regulates CD133 transcription. ( a ) Transcript variants produced by the alternate activity of five promoters (promoter 1–5) were identified in five cell lines by nested PCR. ( b ) p53-mediated repression of CD133 promoter activity in NTERA2, NCCIT, and H1299 cells. ( c ) Activity of the control CD133 promoter and two mutated CD133 promoters in NTERA2 cells. ( d ) Activity of the control CD133 promoter and mutated CD133 promoter in H1299 cell lines. Empty or p53 expression vectors were co-transfected with the reporter gene vector. ( e ) ChIP analysis of p53-binding to CD133 promoter DNA in NTERA2, U2OS, and H1299 cells that had been transfected with either control or p53 expression vector. Protein levels of ectopically expressed p53 were examined in total ChIP lysates. ( f ) ChIP analysis of p53-binding to CD133 promoter DNA in NCCIT cells treated with Dox or DMSO. The error bars represent the mean±S.D. n =3. * P

    Techniques Used: Produced, Activity Assay, Nested PCR, Expressing, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Binding Assay

    18) Product Images from "A Functional Screen for Myc-Responsive Genes Reveals Serine Hydroxymethyltransferase, a Major Source of the One-Carbon Unit for Cell Metabolism"

    Article Title: A Functional Screen for Myc-Responsive Genes Reveals Serine Hydroxymethyltransferase, a Major Source of the One-Carbon Unit for Cell Metabolism

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.16.5793-5800.2002

    c-Myc binds to the promoters of mSHMT and cSHMT genes in vivo. (A) Schematic presentation of human, mouse, and rat mSHMT and cSHMT promoters. Open boxes represent exons. Myc binding sites and ATG codons are shown. Arrows indicate the positions and direction of primers used in PCR. (B and C) HO15.19 cells infected with empty vector (+vector lanes), HO15.19 cells infected with c- myc -expression vector (+myc lanes), HEK 293 cells, or Raji cells were cross-linked and lysed, and chromatin was immunoprecipitated with c-Myc-specific (M lanes) or Gal4-specific (G lanes) antibodies, followed by the reversal of the cross-linking and DNA isolation. Isolated DNA was used in PCR with radiolabeled oligonucleotides flanking Myc binding sites in the promoters of the genes indicated to the right of the blots. For negative controls, two types of PCR were performed. The first one was carried out with oligonucleotides flanking CACGTG sites that do not interact with c-Myc in the promoter of the rat glucokinase gene ( GLU ) (C). Another PCR was performed with oligonucleotides flanking a DNA region containing no CACGTG sites in the rat PCNA gene (B) or in the third intron of a silent human β-globin gene (C). Quantitation of the PCR products was performed with the Molecular Dynamics IhoshphorImaging system. A number below the a blot indicates the fold difference in the signal intensity, determined by dividing a given PCR signal by a corresponding PCNA-specific (B) or β-globin-specific (C) signal and by the ratio of these signals in the corresponding Gal4 lane.
    Figure Legend Snippet: c-Myc binds to the promoters of mSHMT and cSHMT genes in vivo. (A) Schematic presentation of human, mouse, and rat mSHMT and cSHMT promoters. Open boxes represent exons. Myc binding sites and ATG codons are shown. Arrows indicate the positions and direction of primers used in PCR. (B and C) HO15.19 cells infected with empty vector (+vector lanes), HO15.19 cells infected with c- myc -expression vector (+myc lanes), HEK 293 cells, or Raji cells were cross-linked and lysed, and chromatin was immunoprecipitated with c-Myc-specific (M lanes) or Gal4-specific (G lanes) antibodies, followed by the reversal of the cross-linking and DNA isolation. Isolated DNA was used in PCR with radiolabeled oligonucleotides flanking Myc binding sites in the promoters of the genes indicated to the right of the blots. For negative controls, two types of PCR were performed. The first one was carried out with oligonucleotides flanking CACGTG sites that do not interact with c-Myc in the promoter of the rat glucokinase gene ( GLU ) (C). Another PCR was performed with oligonucleotides flanking a DNA region containing no CACGTG sites in the rat PCNA gene (B) or in the third intron of a silent human β-globin gene (C). Quantitation of the PCR products was performed with the Molecular Dynamics IhoshphorImaging system. A number below the a blot indicates the fold difference in the signal intensity, determined by dividing a given PCR signal by a corresponding PCNA-specific (B) or β-globin-specific (C) signal and by the ratio of these signals in the corresponding Gal4 lane.

    Techniques Used: In Vivo, Binding Assay, Polymerase Chain Reaction, Infection, Plasmid Preparation, Expressing, Immunoprecipitation, DNA Extraction, Isolation, Quantitation Assay

    19) Product Images from "A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy"

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy

    Journal: Nature medicine

    doi: 10.1038/nm.4179

    Chaer regulates cardiac hypertrophy ( a ) RNA reads of mouse Chaer and genomic structure. Three reading frames are shown with stop codon labeled by black lines and the longest open reading frames labeled in red. ( b ) Northern blot analysis for Chaer in adult mouse tissues. Gapdh , Glyceraldehyde 3-phosphate dehydrogenase; SKM, skeletal muscle. ( c ) In vitro translation assay for Chaer , HOX transcript antisense RNA ( Hotair ) and GFP. ( d ) Schematic of Chaer knockout in mouse genome using CRISPR-cas9 system showing two guide RNA sequences used. ( e ) Wild-type (WT) and Chaer knockout (KO) alleles detected by genomic DNA PCR. ( f ) Effect of Chaer KO on heart weight and myofilament cross-section areas 4 weeks after trans-aortic constriction (TAC) surgery, Data were mean ± s.e.m. Sample numbers were labeled on bars. *** P
    Figure Legend Snippet: Chaer regulates cardiac hypertrophy ( a ) RNA reads of mouse Chaer and genomic structure. Three reading frames are shown with stop codon labeled by black lines and the longest open reading frames labeled in red. ( b ) Northern blot analysis for Chaer in adult mouse tissues. Gapdh , Glyceraldehyde 3-phosphate dehydrogenase; SKM, skeletal muscle. ( c ) In vitro translation assay for Chaer , HOX transcript antisense RNA ( Hotair ) and GFP. ( d ) Schematic of Chaer knockout in mouse genome using CRISPR-cas9 system showing two guide RNA sequences used. ( e ) Wild-type (WT) and Chaer knockout (KO) alleles detected by genomic DNA PCR. ( f ) Effect of Chaer KO on heart weight and myofilament cross-section areas 4 weeks after trans-aortic constriction (TAC) surgery, Data were mean ± s.e.m. Sample numbers were labeled on bars. *** P

    Techniques Used: Labeling, Northern Blot, In Vitro, Knock-Out, CRISPR, Polymerase Chain Reaction

    20) Product Images from "Identification of the GATA Factor TRPS1 as a Repressor of the Osteocalcin Promoter *"

    Article Title: Identification of the GATA Factor TRPS1 as a Repressor of the Osteocalcin Promoter *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.052316

    Identification of proteins bound to the 6OSE2 DNA sequence. A , IgCα and OSE2 DNA sequences used in the DNA pull-down assays. Runx2 binding sites are underlined. B , ethidium bromide-stained agarose gel showing biotinylated PCR products. Ctrl , a
    Figure Legend Snippet: Identification of proteins bound to the 6OSE2 DNA sequence. A , IgCα and OSE2 DNA sequences used in the DNA pull-down assays. Runx2 binding sites are underlined. B , ethidium bromide-stained agarose gel showing biotinylated PCR products. Ctrl , a

    Techniques Used: Sequencing, Binding Assay, Staining, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Expression and DNA binding activity of TRPS1 in differentiated bone marrow stromal cells. A , relative Trps1 , osteocalcin, and Runx2 mRNA levels, determined by qRT-PCR, in undifferentiated bone marrow stromal cells and bone marrow stromal cells cultured
    Figure Legend Snippet: Expression and DNA binding activity of TRPS1 in differentiated bone marrow stromal cells. A , relative Trps1 , osteocalcin, and Runx2 mRNA levels, determined by qRT-PCR, in undifferentiated bone marrow stromal cells and bone marrow stromal cells cultured

    Techniques Used: Expressing, Binding Assay, Activity Assay, Quantitative RT-PCR, Cell Culture

    21) Product Images from "p53 isoforms regulate premature aging in human cells"

    Article Title: p53 isoforms regulate premature aging in human cells

    Journal: Oncogene

    doi: 10.1038/s41388-017-0101-3

    Δ133p53 expression results in decreased DNA double-strand break (DSB) foci in HGPS fibroblasts HGPS fibroblasts approaching replicative senescence AG11513 at passage 10 and HGADFN188 at passage 12 were transduced with Δ133p53- or control- lentiviral vectors. Control- and Δ133p53-expressing AG11513 were analyzed at passage 12 and 14, respectively. Control- and Δ133p53-expressing HGADFN188 were analyzed at passage 14 and 16, respectively. (A) Quantitative real time PCR of RAD51 , a DNA repair factor involved in homologous recombination (HR). (B) Immunoblot of Δ133p53 and RAD51. Quantification performed with Image J is shown below. Data are mean ± s.d. from three independent experiments. ** P
    Figure Legend Snippet: Δ133p53 expression results in decreased DNA double-strand break (DSB) foci in HGPS fibroblasts HGPS fibroblasts approaching replicative senescence AG11513 at passage 10 and HGADFN188 at passage 12 were transduced with Δ133p53- or control- lentiviral vectors. Control- and Δ133p53-expressing AG11513 were analyzed at passage 12 and 14, respectively. Control- and Δ133p53-expressing HGADFN188 were analyzed at passage 14 and 16, respectively. (A) Quantitative real time PCR of RAD51 , a DNA repair factor involved in homologous recombination (HR). (B) Immunoblot of Δ133p53 and RAD51. Quantification performed with Image J is shown below. Data are mean ± s.d. from three independent experiments. ** P

    Techniques Used: Expressing, Transduction, Real-time Polymerase Chain Reaction, Homologous Recombination

    Δ133p53 dominant-negative inhibition of FLp53 results in increased RAD51 expression ( A ) 041−/− fibroblasts (p53-null) were transduced with control- or Δ133p53- lentiviral supernatant. RAD51 mRNA expression was analyzed by quantitative RT-PCR. No significant change in RAD51 mRNA expression was detected in Δ133p53-expressing cells. Expression of Δ133p53 was confirmed by western blot. GAPDH was used for normalization. (B-E) AG11513 (HGPS) fibroblasts stably expressing a control or Δ133p53-lentiviral vector at passage 12-14 were transfected with siRNA targeting p53 (sip53), the transcription factor E2F1 (siE2F1) or a negative control (siNC). Cell pellets were collected 5 days after siRNA transfection. (B) Knockdown of FLp53, which does not change the expression Δ133p53, was confirmed by Western Blot. RAD51 protein expression was analyzed in the siRNA-transfected samples. Quantification was performed using Image J. β-actin was used as loading control. Relative expression of the indicated proteins in each siRNA-transfected sample was normalized to that of control-transduced HGPS fibroblasts transfected with negative control siRNA (siNC). (C-F) mRNA expression of the (C) senescence-associated gene p21/CDKN1A , (D,F) DNA repair factor RAD51 and (E) transcription factor E2F1 was analyzed by quantitative RT-PCR. GAPDH was used for normalization. Relative expression was normalized to control-transduced HGPS fibroblasts transfected with negative control siRNA (siNC). The data are mean ± s.d. from three independent experiments. ** P
    Figure Legend Snippet: Δ133p53 dominant-negative inhibition of FLp53 results in increased RAD51 expression ( A ) 041−/− fibroblasts (p53-null) were transduced with control- or Δ133p53- lentiviral supernatant. RAD51 mRNA expression was analyzed by quantitative RT-PCR. No significant change in RAD51 mRNA expression was detected in Δ133p53-expressing cells. Expression of Δ133p53 was confirmed by western blot. GAPDH was used for normalization. (B-E) AG11513 (HGPS) fibroblasts stably expressing a control or Δ133p53-lentiviral vector at passage 12-14 were transfected with siRNA targeting p53 (sip53), the transcription factor E2F1 (siE2F1) or a negative control (siNC). Cell pellets were collected 5 days after siRNA transfection. (B) Knockdown of FLp53, which does not change the expression Δ133p53, was confirmed by Western Blot. RAD51 protein expression was analyzed in the siRNA-transfected samples. Quantification was performed using Image J. β-actin was used as loading control. Relative expression of the indicated proteins in each siRNA-transfected sample was normalized to that of control-transduced HGPS fibroblasts transfected with negative control siRNA (siNC). (C-F) mRNA expression of the (C) senescence-associated gene p21/CDKN1A , (D,F) DNA repair factor RAD51 and (E) transcription factor E2F1 was analyzed by quantitative RT-PCR. GAPDH was used for normalization. Relative expression was normalized to control-transduced HGPS fibroblasts transfected with negative control siRNA (siNC). The data are mean ± s.d. from three independent experiments. ** P

    Techniques Used: Dominant Negative Mutation, Inhibition, Expressing, Transduction, Quantitative RT-PCR, Western Blot, Stable Transfection, Plasmid Preparation, Transfection, Negative Control

    22) Product Images from "A Case of Lipoprotein Glomerulopathy with apoE Chicago and apoE (Glu3Lys) Treated with Fenofibrate"

    Article Title: A Case of Lipoprotein Glomerulopathy with apoE Chicago and apoE (Glu3Lys) Treated with Fenofibrate

    Journal: Case Reports in Nephrology and Dialysis

    doi: 10.1159/000478902

    a ApoE phenotype determined by isoelectric focusing polyacrylamide gel electrophoresis (IEF). Lane 1 (E2/2), lane 2 (E3/3, wild-type), and lane 3 (E4/4) are samples; lane 4 (E3/3) is the patient; lane 5 (E4/4) is the mother, lanes 6 and 7 (E3/4) are the 2 brothers; and lane 8 (E2/4) is a re-analysis of a previous patient. b apoE genotype examined by restriction fragment length polymorphism (RFLP). PCR-amplified DNA of apoE , including codons 112 and 158, was digested with Hha I. Lane 1 shows the DNA size marker (100-bp DNA ladder); lane 2 (ε3/3, wild-type), lane 3 (ε2/2), and lane 4 (ε3/4) are samples; lane 5 (ε3/3) is the patient, lanes 6, 7, and 8 (ε3/4) are the mother and the 2 brothers; lane 9 (ε2/3) is a re-analysis of a previous patient. c , d Sequence analysis of PCR-amplified DNA of apoE in the patient. c In exon 3, the normal allele contained the sequence GAG coding for amino acid 3, glutamine. The mutant allele contained the substituted sequence AAG, coding for amino acid 3, lysine. d In exon 4, the normal allele contained the sequence CGG coding for amino 147, arginine. The mutation allele contained the substituted sequence CCG, coding for amino acid 147, proline. e The patient's family tree. Since 3 apoE mutations expressed in the patient's mother were observed in the patient (E-Glu3Lys and E-Chicago [Arg147Pro]) and the 2 brothers (E4 [Cys112Arg]), we confirmed that E-Glu3Lys and E-Chicago were on one allele, and E4 (Cys112Arg) was on the other allele.
    Figure Legend Snippet: a ApoE phenotype determined by isoelectric focusing polyacrylamide gel electrophoresis (IEF). Lane 1 (E2/2), lane 2 (E3/3, wild-type), and lane 3 (E4/4) are samples; lane 4 (E3/3) is the patient; lane 5 (E4/4) is the mother, lanes 6 and 7 (E3/4) are the 2 brothers; and lane 8 (E2/4) is a re-analysis of a previous patient. b apoE genotype examined by restriction fragment length polymorphism (RFLP). PCR-amplified DNA of apoE , including codons 112 and 158, was digested with Hha I. Lane 1 shows the DNA size marker (100-bp DNA ladder); lane 2 (ε3/3, wild-type), lane 3 (ε2/2), and lane 4 (ε3/4) are samples; lane 5 (ε3/3) is the patient, lanes 6, 7, and 8 (ε3/4) are the mother and the 2 brothers; lane 9 (ε2/3) is a re-analysis of a previous patient. c , d Sequence analysis of PCR-amplified DNA of apoE in the patient. c In exon 3, the normal allele contained the sequence GAG coding for amino acid 3, glutamine. The mutant allele contained the substituted sequence AAG, coding for amino acid 3, lysine. d In exon 4, the normal allele contained the sequence CGG coding for amino 147, arginine. The mutation allele contained the substituted sequence CCG, coding for amino acid 147, proline. e The patient's family tree. Since 3 apoE mutations expressed in the patient's mother were observed in the patient (E-Glu3Lys and E-Chicago [Arg147Pro]) and the 2 brothers (E4 [Cys112Arg]), we confirmed that E-Glu3Lys and E-Chicago were on one allele, and E4 (Cys112Arg) was on the other allele.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Electrofocusing, Polymerase Chain Reaction, Amplification, Marker, Sequencing, Mutagenesis

    23) Product Images from "CXCL13 Activation of c-Myc Induce RANK Ligand Expression in Stromal/Preosteoblast Cells in the Oral Squamous Cell Carcinoma Tumor-Bone Microenvironment"

    Article Title: CXCL13 Activation of c-Myc Induce RANK Ligand Expression in Stromal/Preosteoblast Cells in the Oral Squamous Cell Carcinoma Tumor-Bone Microenvironment

    Journal: Oncogene

    doi: 10.1038/onc.2012.24

    CXCL13 induces c-Myc activation in human bone marrow derived stromal cells (SAKA-T) and murine preosteoblast (MC3T3-E1) cells. (A) MC3T3-E1 cells were stimulated with CXCL13 (15 ng/ml) for 6 h and c-Myc nuclear translocation was analyzed by confocal microscopy. (B) ChIP assay for c-Myc binding to hRANKL promoter region. SAKA-T cells were stimulated with and without CXCL13 (15 ng/ml) for 6 h and ChIP assay for c-Myc binding to hRANKL promoter was performed as described in methods. (C) Quantitative real-time PCR analysis of chromatin immune complexes for p-c-Myc binding to RANKL promoter region. The DNA amplification was normalized with respect to input. Data represent triplicate studies and mean ± SD (*p
    Figure Legend Snippet: CXCL13 induces c-Myc activation in human bone marrow derived stromal cells (SAKA-T) and murine preosteoblast (MC3T3-E1) cells. (A) MC3T3-E1 cells were stimulated with CXCL13 (15 ng/ml) for 6 h and c-Myc nuclear translocation was analyzed by confocal microscopy. (B) ChIP assay for c-Myc binding to hRANKL promoter region. SAKA-T cells were stimulated with and without CXCL13 (15 ng/ml) for 6 h and ChIP assay for c-Myc binding to hRANKL promoter was performed as described in methods. (C) Quantitative real-time PCR analysis of chromatin immune complexes for p-c-Myc binding to RANKL promoter region. The DNA amplification was normalized with respect to input. Data represent triplicate studies and mean ± SD (*p

    Techniques Used: Activation Assay, Derivative Assay, Translocation Assay, Confocal Microscopy, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Amplification

    24) Product Images from "A core viral protein binds host nucleosomes to sequester immune danger signals"

    Article Title: A core viral protein binds host nucleosomes to sequester immune danger signals

    Journal: Nature

    doi: 10.1038/nature18317

    Protein VII retains HMGB1 and HMGB2 in chromatin a , Western blot of adenovirus infected or doxycycline treated A549 cells showing the relative levels of protein VII expression. HMGB1 levels do not change upon infection or protein VII expression. Tubulin is shown as a loading control. b , Quantitative PCR analysis of mRNA transcripts of HMGB1 in various cell types as indicated (for A549, n biological =3, for THP-1, n biological =2, error bar=±s.d.). The levels of HMGB1 do not significantly change. c , Immunofluorescence analysis of a time-course of protein VII-HA (red) induction shown with HMGB1 (green) and DAPI (grey, blue in merge) in A549 cells. Expression of protein VII-HA results in a change to the HMGB1 distribution upon expression. d , HMGB1 (green) localization changes between 12 and 24 hpi of wild-type adenovirus in A549 cells, and adopts a pattern similar to protein VII as in Fig 1a . DBP (red) is shown as a marker of infection, DNA is stained with DAPI (blue in merge). e , Same as ( d ) showing HMGB2 adopts the same pattern as HMGB1 during Ad5 infection at 24 hpi. f , Multiple cells showing the same pattern of HMGB1 re-localization upon expressing VII-GFP as in Fig 3g . g , HMGB1 retention in the high salt fraction is conserved across Ad serotypes. Western blot analysis of HMGB1 from salt fractionated A549 cells infected with Ad5, Ad9 or Ad12 as shown.
    Figure Legend Snippet: Protein VII retains HMGB1 and HMGB2 in chromatin a , Western blot of adenovirus infected or doxycycline treated A549 cells showing the relative levels of protein VII expression. HMGB1 levels do not change upon infection or protein VII expression. Tubulin is shown as a loading control. b , Quantitative PCR analysis of mRNA transcripts of HMGB1 in various cell types as indicated (for A549, n biological =3, for THP-1, n biological =2, error bar=±s.d.). The levels of HMGB1 do not significantly change. c , Immunofluorescence analysis of a time-course of protein VII-HA (red) induction shown with HMGB1 (green) and DAPI (grey, blue in merge) in A549 cells. Expression of protein VII-HA results in a change to the HMGB1 distribution upon expression. d , HMGB1 (green) localization changes between 12 and 24 hpi of wild-type adenovirus in A549 cells, and adopts a pattern similar to protein VII as in Fig 1a . DBP (red) is shown as a marker of infection, DNA is stained with DAPI (blue in merge). e , Same as ( d ) showing HMGB2 adopts the same pattern as HMGB1 during Ad5 infection at 24 hpi. f , Multiple cells showing the same pattern of HMGB1 re-localization upon expressing VII-GFP as in Fig 3g . g , HMGB1 retention in the high salt fraction is conserved across Ad serotypes. Western blot analysis of HMGB1 from salt fractionated A549 cells infected with Ad5, Ad9 or Ad12 as shown.

    Techniques Used: Western Blot, Infection, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Marker, Staining

    Protein VII is necessary and sufficient for chromatin retention of HMGB1 in human and mouse cells a–b , Replication of Ad5-flox-VII virus on 293 or 293-Cre cells. Quantitative PCR analysis of viral genomic DNA over a time-course of infection ( a ) shows the DBP gene is increasing exponentially in 293 and 293-Cre cells when infected with Ad5-f lox-VII virus. In contrast, PCR for the protein VII gene ( b ) demonstrates deletion in 293-Cre cells (n biological =2, error bar=±s.d.). c , Salt fractionation of 293-Cre cells infected with wild-type Ad5 indicating that the Cre recombinase does not interfere with the ability of protein VII to retain HMGB1 in the high salt chromatin fraction. Protein VII is also necessary for the chromatin retention of HMGB2. d , THP-1 cells transduced to express protein VII-GFP results in chromatin distortion and HMGB1 retention in chromatin. Immunofluorescence of transduced PMA-treated THP-1 cells showing protein VII-GFP (green), HMGB1 (red) and DNA (grey, blue in merge). e , Transduction to express protein VII-GFP is sufficient to relocalize mouse HMGB1 in mouse embryonic fibroblast (MEF) cells. f , Salt fractionation of mouse embryonic fibroblast cells transduced to express protein VII-GFP. Human Ad5 protein VII is sufficient to retain mouse HMGB1 in the high salt fraction in MEF cells. The control vector expressing GFP alone does not have this effect.
    Figure Legend Snippet: Protein VII is necessary and sufficient for chromatin retention of HMGB1 in human and mouse cells a–b , Replication of Ad5-flox-VII virus on 293 or 293-Cre cells. Quantitative PCR analysis of viral genomic DNA over a time-course of infection ( a ) shows the DBP gene is increasing exponentially in 293 and 293-Cre cells when infected with Ad5-f lox-VII virus. In contrast, PCR for the protein VII gene ( b ) demonstrates deletion in 293-Cre cells (n biological =2, error bar=±s.d.). c , Salt fractionation of 293-Cre cells infected with wild-type Ad5 indicating that the Cre recombinase does not interfere with the ability of protein VII to retain HMGB1 in the high salt chromatin fraction. Protein VII is also necessary for the chromatin retention of HMGB2. d , THP-1 cells transduced to express protein VII-GFP results in chromatin distortion and HMGB1 retention in chromatin. Immunofluorescence of transduced PMA-treated THP-1 cells showing protein VII-GFP (green), HMGB1 (red) and DNA (grey, blue in merge). e , Transduction to express protein VII-GFP is sufficient to relocalize mouse HMGB1 in mouse embryonic fibroblast (MEF) cells. f , Salt fractionation of mouse embryonic fibroblast cells transduced to express protein VII-GFP. Human Ad5 protein VII is sufficient to retain mouse HMGB1 in the high salt fraction in MEF cells. The control vector expressing GFP alone does not have this effect.

    Techniques Used: Real-time Polymerase Chain Reaction, Infection, Polymerase Chain Reaction, Salting Out, Immunofluorescence, Transduction, Plasmid Preparation, Expressing

    25) Product Images from "Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A"

    Article Title: Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks770

    Intragenic methylation of PolgA in reprogrammed and mtDNA divergent ESCs. Bisulphite sequencing analysis of CpG methylation in ( A ) CC9 mus , ( B ) CC9 dunni , ( C ) iPS QS , ( D ) iPS NGFP2 ( E ) NT-ES, ( F ) iPS NFGPinj pluripotent stem cells. ( G ) The percentage CpG methylation per sequence determined by bisulphite sequencing (percentage mean ± SEM). ( H ) Real time PCR quantification of PolgA expression in cultured CC9 mus , CC9 spretus , CC9 dunni , iPS QS/R26 (iPS QS ), iPS NGFP , NT-ES, iPS NGFPin j pluripotent stem cells expressed relative to ESD3 cells. ( J ) Cumulative analysis of the relationship between DNA methylation levels ( Figures 1 L and 3 G) and the expression of PolgA ( Figures 2 A and 3 H) performed using Pearson correlation coefficient (R 2 ). ( K ) MtDNA copies/cell in cultured CC9 mus , CC9 spretus , CC9 dunni , iPS QS/R26 , iPS NGFP , NT-ES, iPS NGFPinj pluripotent stem cells. Values represent mean ± SEM and significant differences between cell types are: * P
    Figure Legend Snippet: Intragenic methylation of PolgA in reprogrammed and mtDNA divergent ESCs. Bisulphite sequencing analysis of CpG methylation in ( A ) CC9 mus , ( B ) CC9 dunni , ( C ) iPS QS , ( D ) iPS NGFP2 ( E ) NT-ES, ( F ) iPS NFGPinj pluripotent stem cells. ( G ) The percentage CpG methylation per sequence determined by bisulphite sequencing (percentage mean ± SEM). ( H ) Real time PCR quantification of PolgA expression in cultured CC9 mus , CC9 spretus , CC9 dunni , iPS QS/R26 (iPS QS ), iPS NGFP , NT-ES, iPS NGFPin j pluripotent stem cells expressed relative to ESD3 cells. ( J ) Cumulative analysis of the relationship between DNA methylation levels ( Figures 1 L and 3 G) and the expression of PolgA ( Figures 2 A and 3 H) performed using Pearson correlation coefficient (R 2 ). ( K ) MtDNA copies/cell in cultured CC9 mus , CC9 spretus , CC9 dunni , iPS QS/R26 , iPS NGFP , NT-ES, iPS NGFPinj pluripotent stem cells. Values represent mean ± SEM and significant differences between cell types are: * P

    Techniques Used: Methylation, Bisulfite Sequencing, CpG Methylation Assay, Sequencing, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, DNA Methylation Assay

    DNA methylation at the Exon 2 loci is associated with reduced RNApII transcriptional elongation. ( A ) Diagrammatic representation of the PolgA gene and primers sites used for ChIP. Numbers correspond to the centre nucleotide of each primer amplicon, relative to the transcription start site (TSS). Enrichment for (RNApII) and RNApII phosphorylated on serine 2 of the carboxy-terminal domain (RNApIIS2) was analysed by real time PCR at the exon 2 methylation site of PolgA and at downstream and upstream regions in: ( B ) CC9 mus ; ( C ) MEF; ( D ) NSC-CC9 mus and ( E ) heart samples. Values represent mean ± SEM. Significant differences between cell types are: * P
    Figure Legend Snippet: DNA methylation at the Exon 2 loci is associated with reduced RNApII transcriptional elongation. ( A ) Diagrammatic representation of the PolgA gene and primers sites used for ChIP. Numbers correspond to the centre nucleotide of each primer amplicon, relative to the transcription start site (TSS). Enrichment for (RNApII) and RNApII phosphorylated on serine 2 of the carboxy-terminal domain (RNApIIS2) was analysed by real time PCR at the exon 2 methylation site of PolgA and at downstream and upstream regions in: ( B ) CC9 mus ; ( C ) MEF; ( D ) NSC-CC9 mus and ( E ) heart samples. Values represent mean ± SEM. Significant differences between cell types are: * P

    Techniques Used: DNA Methylation Assay, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Methylation

    Analysis of PolgA and mtDNA enrichment in 5mC and 5hmC MeDIP of ESCs and somatic tissues. DNA samples from cultured CC9 mus , CC9 spretus and CC9 dunni cells; liver, spleen, heart and brain samples were immunoprecipitated using antibodies against ( A ) 5mC and ( B ) 5hmC, and analysed using real time PCR for PolgA (exon 2) enrichment. Bars represent means ± SEM. Significant differences between cell types are indicated (** P
    Figure Legend Snippet: Analysis of PolgA and mtDNA enrichment in 5mC and 5hmC MeDIP of ESCs and somatic tissues. DNA samples from cultured CC9 mus , CC9 spretus and CC9 dunni cells; liver, spleen, heart and brain samples were immunoprecipitated using antibodies against ( A ) 5mC and ( B ) 5hmC, and analysed using real time PCR for PolgA (exon 2) enrichment. Bars represent means ± SEM. Significant differences between cell types are indicated (** P

    Techniques Used: Methylated DNA Immunoprecipitation, Cell Culture, Immunoprecipitation, Real-time Polymerase Chain Reaction

    DNA Methylation of exon 2 correlates with reduced steady state mRNA levels of PolgA . ( A ) Real time PCR quantification of PolgA expression in cultured ESD3 and MEF cells, and in liver, spleen, heart, muscle, kidney and brain samples, expressed relative to ESD3. ( B ) The relationship between DNA methylation levels from Figure 1 L and the corresponding PolgA expression was determined using Pearson correlation coefficient (R 2 ). ( C ) MtDNA copies/cell in cultured ESD3 and MEF cells, and liver, spleen, heart, muscle, kidney and brain, as determined by real time PCR. ( D ) The relationship between the levels of DNA methylation from Figure 1 L and the corresponding mtDNA copies/cell was determined using Pearson correlation coefficient (R 2 ). Values represent mean ± SEM and significant differences between cell types are: * P
    Figure Legend Snippet: DNA Methylation of exon 2 correlates with reduced steady state mRNA levels of PolgA . ( A ) Real time PCR quantification of PolgA expression in cultured ESD3 and MEF cells, and in liver, spleen, heart, muscle, kidney and brain samples, expressed relative to ESD3. ( B ) The relationship between DNA methylation levels from Figure 1 L and the corresponding PolgA expression was determined using Pearson correlation coefficient (R 2 ). ( C ) MtDNA copies/cell in cultured ESD3 and MEF cells, and liver, spleen, heart, muscle, kidney and brain, as determined by real time PCR. ( D ) The relationship between the levels of DNA methylation from Figure 1 L and the corresponding mtDNA copies/cell was determined using Pearson correlation coefficient (R 2 ). Values represent mean ± SEM and significant differences between cell types are: * P

    Techniques Used: DNA Methylation Assay, Real-time Polymerase Chain Reaction, Expressing, Cell Culture

    26) Product Images from "Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo"

    Article Title: Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo

    Journal: RNA

    doi: 10.1261/rna.035097.112

    Validation of GIIIc reporters in vitro. ( A ) Western blots indicate that rat DT cells differentially express epithelial markers E-cadherin and pan-cytokeratin, and rat AT3 cells express mesenchymal markers fibronectin and vimetin. β-actin was used as a loading control. ( B ) RT-PCR and restriction digestion reveal that DT and AT3 cells express the endogenous FGFR2 IIIb and IIIc isoforms, respectively. (M) Mock digested RT-PCR amplicon; (AvaI) IIIb-specific restriction digest; (EcoRV) IIIc-specific restriction digest. ( C ) RT-PCR reveals that both DT and AT3 cells include IIIc in the pGIIIcI ΔΔ construct, but only DT cells robustly skip IIIc in the pGIIIcI 2 construct. Bars indicate the percent inclusion for each sample, and error bars represent standard deviations of the mean. ( D ) The GIIIcI ΔΔ and GIIIcI 2 reporters were stably transfected into DT and AT3 cells. Untransfected cells that did not receive both the reporter and the G418 selection gene were gated away using fluorescence activated cell sorting (FACS). As expected, epithelial-specific IIIc skipping of pGIIIcI 2 is observed in DT cells, but deletion of IAS2 and ISAR partially abrogates epithelial-specific control of IIIc.
    Figure Legend Snippet: Validation of GIIIc reporters in vitro. ( A ) Western blots indicate that rat DT cells differentially express epithelial markers E-cadherin and pan-cytokeratin, and rat AT3 cells express mesenchymal markers fibronectin and vimetin. β-actin was used as a loading control. ( B ) RT-PCR and restriction digestion reveal that DT and AT3 cells express the endogenous FGFR2 IIIb and IIIc isoforms, respectively. (M) Mock digested RT-PCR amplicon; (AvaI) IIIb-specific restriction digest; (EcoRV) IIIc-specific restriction digest. ( C ) RT-PCR reveals that both DT and AT3 cells include IIIc in the pGIIIcI ΔΔ construct, but only DT cells robustly skip IIIc in the pGIIIcI 2 construct. Bars indicate the percent inclusion for each sample, and error bars represent standard deviations of the mean. ( D ) The GIIIcI ΔΔ and GIIIcI 2 reporters were stably transfected into DT and AT3 cells. Untransfected cells that did not receive both the reporter and the G418 selection gene were gated away using fluorescence activated cell sorting (FACS). As expected, epithelial-specific IIIc skipping of pGIIIcI 2 is observed in DT cells, but deletion of IAS2 and ISAR partially abrogates epithelial-specific control of IIIc.

    Techniques Used: In Vitro, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Construct, Stable Transfection, Transfection, Selection, Fluorescence, FACS

    27) Product Images from "Gata6 promotes hair follicle progenitor cell renewal by genome maintenance during proliferation"

    Article Title: Gata6 promotes hair follicle progenitor cell renewal by genome maintenance during proliferation

    Journal: The EMBO Journal

    doi: 10.15252/embj.201694572

    Gata6 is up‐regulated in differentiating hair germ cells Experimental scheme for doxycycline chased K5tTA × pTRE‐H2B‐GFP mice used to obtain divided and un‐divided cell populations during the differentiating phase—telogen (T)–anagen (A) transition—and self‐renewing phase of HFSCs during early anagen, as previously described in Zhang et al ). Bulge cells were sorted as divided or un‐divided based on H2B‐GFP levels and high CD34 and α6‐integrin expression; hair germ cells at telogen–anagen transition were the divided cells with low CD34 levels and high expression of α6‐integrin. qRT–PCR confirmation of Gata6 expression in differentiating (hair germ, divided), self‐renewing (bulge, divided), or non‐dividing bulge stem cells (average ± SD, n = 3). Immunofluorescence image of the skin shows epidermis and infundibulum stained with Hoechst for DNA (blue), basal layer/ORS marker K14 (green), and Gata6 (red). Scale bar: 30 μm. Gel image of DNA genotyped for floxed Gata6 allele. Shown are genotypes from three mice: heterozygote (Gata6 fl/wt ), homozygous WT (Gata6 wt/wt ), and homozygous floxed (Gata6 fl/fl ).
    Figure Legend Snippet: Gata6 is up‐regulated in differentiating hair germ cells Experimental scheme for doxycycline chased K5tTA × pTRE‐H2B‐GFP mice used to obtain divided and un‐divided cell populations during the differentiating phase—telogen (T)–anagen (A) transition—and self‐renewing phase of HFSCs during early anagen, as previously described in Zhang et al ). Bulge cells were sorted as divided or un‐divided based on H2B‐GFP levels and high CD34 and α6‐integrin expression; hair germ cells at telogen–anagen transition were the divided cells with low CD34 levels and high expression of α6‐integrin. qRT–PCR confirmation of Gata6 expression in differentiating (hair germ, divided), self‐renewing (bulge, divided), or non‐dividing bulge stem cells (average ± SD, n = 3). Immunofluorescence image of the skin shows epidermis and infundibulum stained with Hoechst for DNA (blue), basal layer/ORS marker K14 (green), and Gata6 (red). Scale bar: 30 μm. Gel image of DNA genotyped for floxed Gata6 allele. Shown are genotypes from three mice: heterozygote (Gata6 fl/wt ), homozygous WT (Gata6 wt/wt ), and homozygous floxed (Gata6 fl/fl ).

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Marker

    28) Product Images from "A feedback loop comprising PRMT7 and miR-24-2 interplays with Oct4, Nanog, Klf4 and c-Myc to regulate stemness"

    Article Title: A feedback loop comprising PRMT7 and miR-24-2 interplays with Oct4, Nanog, Klf4 and c-Myc to regulate stemness

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw788

    PRMT7 and its catalytic activity are indispensable for the stemness of mESCs, and Oct4, Nanog, Klf4 and c-Myc protein levels are more reduced than their mRNA levels in PRMT7-depleted mESCs. ( A ) Microscopic and AP-staining images of shLuc-treated and PRMT7-depleted V6.5 mESCs. Five shPRMT7s (shPRMT7-4, -5, -6, -7 and -8) were used to deplete PRMT7. BF, bright field; AP, alkaline phosphatase; red scale bar, 100 μm. ( B ) The percentages of shLuc- or shPRMT7-treated V6.5 mESCs in G1, S and G2/M phase of the cell cycle. shLuc-treated cells were used as a control. ( C ) Rescue experiments of PRMT7-depleted mESCs with PRMT7 or its catalytic mutant (m.PRMT7). shPRMT7-7 or shPRMT7-8 plasmids were co-electroporated with expression plasmids encoding both GFP and either PRMT7 or its catalytic mutant. The GFP-expressing vector pCDH1-EF1-IRES-GFP (vector/GFP) was used to clone PRMT7 cDNA. Microscopic, green-fluorescent and AP staining images of indicated group of cells are shown (left panel). Western blotting was used to analyze the expression levels of PRMT7 or its mutant in eight different experimental conditions (right panel). ( D ) Analysis of Oct4, Nanog, Sox2, c-Myc and Klf4 mRNA levels in shLuc-treated and PRMT7-depleted V6.5 mESCs using quantitative RT-PCR. ( E ) The effect of ectopic expression of PRMT7 or its catalytic mutant (m.PRMT7) on Oct4, Nanog, Sox2, c-Myc, Klf4 and Prmt7 mRNA levels in PRMT7-depleted mESCs. Relative Oct4, Nanog, Sox2, c-Myc, Klf4 and Prmt7 mRNA levels were examined in eight different experimental conditions (see panel C for lane description). Red scale bar, 100 μm. ( F and G ) Western blot analysis of Oct4, Nanog, Sox2, c-Myc, Klf4 and PRMT7 levels in shLuc-treated and PRMT7-depleted V6.5 mESCs. Oct4, Nanog, Sox2, c-Myc, Klf4 and PRMT7 protein levels in shLuc-treated and PRMT7-depleted V6.5 mESCs were quantified (G). Data are presented as the mean ± SD of three independent experiments. P
    Figure Legend Snippet: PRMT7 and its catalytic activity are indispensable for the stemness of mESCs, and Oct4, Nanog, Klf4 and c-Myc protein levels are more reduced than their mRNA levels in PRMT7-depleted mESCs. ( A ) Microscopic and AP-staining images of shLuc-treated and PRMT7-depleted V6.5 mESCs. Five shPRMT7s (shPRMT7-4, -5, -6, -7 and -8) were used to deplete PRMT7. BF, bright field; AP, alkaline phosphatase; red scale bar, 100 μm. ( B ) The percentages of shLuc- or shPRMT7-treated V6.5 mESCs in G1, S and G2/M phase of the cell cycle. shLuc-treated cells were used as a control. ( C ) Rescue experiments of PRMT7-depleted mESCs with PRMT7 or its catalytic mutant (m.PRMT7). shPRMT7-7 or shPRMT7-8 plasmids were co-electroporated with expression plasmids encoding both GFP and either PRMT7 or its catalytic mutant. The GFP-expressing vector pCDH1-EF1-IRES-GFP (vector/GFP) was used to clone PRMT7 cDNA. Microscopic, green-fluorescent and AP staining images of indicated group of cells are shown (left panel). Western blotting was used to analyze the expression levels of PRMT7 or its mutant in eight different experimental conditions (right panel). ( D ) Analysis of Oct4, Nanog, Sox2, c-Myc and Klf4 mRNA levels in shLuc-treated and PRMT7-depleted V6.5 mESCs using quantitative RT-PCR. ( E ) The effect of ectopic expression of PRMT7 or its catalytic mutant (m.PRMT7) on Oct4, Nanog, Sox2, c-Myc, Klf4 and Prmt7 mRNA levels in PRMT7-depleted mESCs. Relative Oct4, Nanog, Sox2, c-Myc, Klf4 and Prmt7 mRNA levels were examined in eight different experimental conditions (see panel C for lane description). Red scale bar, 100 μm. ( F and G ) Western blot analysis of Oct4, Nanog, Sox2, c-Myc, Klf4 and PRMT7 levels in shLuc-treated and PRMT7-depleted V6.5 mESCs. Oct4, Nanog, Sox2, c-Myc, Klf4 and PRMT7 protein levels in shLuc-treated and PRMT7-depleted V6.5 mESCs were quantified (G). Data are presented as the mean ± SD of three independent experiments. P

    Techniques Used: Activity Assay, Staining, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Quantitative RT-PCR

    miR-24-3p and miR-24-2-5p target Prmt7 3′UTR and impede the stemness of mESCs. ( A ) Schematic representation of luciferase reporter constructs containing WT Prmt7 -3′UTR or its mutants. ( B ) Relative luciferase activities of reporter constructs containing Prmt7 -3′UTR or its mutants in HEK293T cells after transfection of miR-24-3p and miR-24-2-5p mimics. ( C and E ) Microscopic and AP staining images of V6.5 mESCs after treatment with miR-24-3p (C) or miR-24-2-5p (E) mimic. V6.5 mESCs were treated with miR-24-3p and miR-24-2-5p mimics and incubated for 2d or 4d. ( D and F ) Quantitative analysis of Oct4, Nanog, Sox2, Klf4, c-Myc and Prmt7 mRNA levels in V6.5 mESCs after treatment with miR-24-3p (D) or miR-24-2-5p (F) mimic. ( G ) Quantitative RT-PCR analysis of miR-24-3p and miR-24-2-5p levels in WT and differentiated V6.5 mESCs (EBs + RA). ( H and I ) The effect of LNA-miRNAs (a type of anti-sense microRNAs) against miR-24-3p or miR-24-2-5p on RA-induced mESC differentiation. mESCs were transfected with LNA-miRNAs (H). Oct4, Nanog, Sox2, c-Myc and Klf4 mRNA levels were measured using quantitative RT-PCR (I). In G−I, mESCs were induced to form EBs for 5 days and then treated with RA for another 5 days. Data are presented as the mean ± SD of three independent experiments. P
    Figure Legend Snippet: miR-24-3p and miR-24-2-5p target Prmt7 3′UTR and impede the stemness of mESCs. ( A ) Schematic representation of luciferase reporter constructs containing WT Prmt7 -3′UTR or its mutants. ( B ) Relative luciferase activities of reporter constructs containing Prmt7 -3′UTR or its mutants in HEK293T cells after transfection of miR-24-3p and miR-24-2-5p mimics. ( C and E ) Microscopic and AP staining images of V6.5 mESCs after treatment with miR-24-3p (C) or miR-24-2-5p (E) mimic. V6.5 mESCs were treated with miR-24-3p and miR-24-2-5p mimics and incubated for 2d or 4d. ( D and F ) Quantitative analysis of Oct4, Nanog, Sox2, Klf4, c-Myc and Prmt7 mRNA levels in V6.5 mESCs after treatment with miR-24-3p (D) or miR-24-2-5p (F) mimic. ( G ) Quantitative RT-PCR analysis of miR-24-3p and miR-24-2-5p levels in WT and differentiated V6.5 mESCs (EBs + RA). ( H and I ) The effect of LNA-miRNAs (a type of anti-sense microRNAs) against miR-24-3p or miR-24-2-5p on RA-induced mESC differentiation. mESCs were transfected with LNA-miRNAs (H). Oct4, Nanog, Sox2, c-Myc and Klf4 mRNA levels were measured using quantitative RT-PCR (I). In G−I, mESCs were induced to form EBs for 5 days and then treated with RA for another 5 days. Data are presented as the mean ± SD of three independent experiments. P

    Techniques Used: Luciferase, Construct, Transfection, Staining, Incubation, Quantitative RT-PCR

    PRMT7 downregulates the levels of miR-24-3p and miR-24-2-5p, which collectively target the 3′UTRs of Oct4, Nanog, Klf4 and c-Myc mRNAs. ( A ) Venn diagrams of miRNAs that were significantly upregulated or downregulated by two shPRMT7s (shPRMT7-7 and shPRMT7-8). ( B ) Comparison of cellular levels of multiple miRNAs between shLuc-treated and PRMT7-depleted V6.5 mESCs using quantitative, miRNA-specific PCR. ( C ) The effect of ectopic expression of PRMT7 or its catalytic mutant (m.PRMT7) on miR-24-3p and miR24-2-5p levels in PRMT7-depleted mESCs. ( D ) Schematic representation of luciferase reporter constructs containing Oct4 -3′UTR, Nanog -3′UTR, Sox2 -3′UTR, Klf4 -3′UTR or c-Myc -3′UTR. ( E ) Relative luciferase activities of reporter constructs containing Oct4 -3′UTR, Nanog -3′UTR, Sox2 -3′UTR, Klf4 -3′UTR or c-Myc -3′UTR in the absence or presence of miR24-2 expression. The reporter constructs, alone or together with a miR-24-2 expression plasmid encoding miR-24-3p and miR24-2-5p, were transfected into HEK293T cells. Firefly luciferase activities were normalized to the internal transfection control Renila luciferase. ( F and G ) Comparison of endogenous association of Ago2 with Oct4, Nanog, Sox2, Klf4 and c-Myc mRNAs between shLuc-treated cells and PRMT7-depleted cells. Ago2 IP was performed (F) and IP eluates were analyzed using quantitative RT-PCR (G). ( H − J ) Analysis of the association of Ago2 with Oct4, Nanog, Sox2, Klf4 and c-Myc mRNAs after transient transfection of control mimic, miR-24-3p mimic (H) or miR-24-2-5p mimic (I) in mESCs. Following transfection of mimic RNAs, cells were incubated for 48 h, lysed and used for Ago2 IP (J). IP eluates were analyzed using quantitative RT-PCR (H and I). Data are presented as the mean ± SD of three independent experiments. P
    Figure Legend Snippet: PRMT7 downregulates the levels of miR-24-3p and miR-24-2-5p, which collectively target the 3′UTRs of Oct4, Nanog, Klf4 and c-Myc mRNAs. ( A ) Venn diagrams of miRNAs that were significantly upregulated or downregulated by two shPRMT7s (shPRMT7-7 and shPRMT7-8). ( B ) Comparison of cellular levels of multiple miRNAs between shLuc-treated and PRMT7-depleted V6.5 mESCs using quantitative, miRNA-specific PCR. ( C ) The effect of ectopic expression of PRMT7 or its catalytic mutant (m.PRMT7) on miR-24-3p and miR24-2-5p levels in PRMT7-depleted mESCs. ( D ) Schematic representation of luciferase reporter constructs containing Oct4 -3′UTR, Nanog -3′UTR, Sox2 -3′UTR, Klf4 -3′UTR or c-Myc -3′UTR. ( E ) Relative luciferase activities of reporter constructs containing Oct4 -3′UTR, Nanog -3′UTR, Sox2 -3′UTR, Klf4 -3′UTR or c-Myc -3′UTR in the absence or presence of miR24-2 expression. The reporter constructs, alone or together with a miR-24-2 expression plasmid encoding miR-24-3p and miR24-2-5p, were transfected into HEK293T cells. Firefly luciferase activities were normalized to the internal transfection control Renila luciferase. ( F and G ) Comparison of endogenous association of Ago2 with Oct4, Nanog, Sox2, Klf4 and c-Myc mRNAs between shLuc-treated cells and PRMT7-depleted cells. Ago2 IP was performed (F) and IP eluates were analyzed using quantitative RT-PCR (G). ( H − J ) Analysis of the association of Ago2 with Oct4, Nanog, Sox2, Klf4 and c-Myc mRNAs after transient transfection of control mimic, miR-24-3p mimic (H) or miR-24-2-5p mimic (I) in mESCs. Following transfection of mimic RNAs, cells were incubated for 48 h, lysed and used for Ago2 IP (J). IP eluates were analyzed using quantitative RT-PCR (H and I). Data are presented as the mean ± SD of three independent experiments. P

    Techniques Used: Polymerase Chain Reaction, Expressing, Mutagenesis, Luciferase, Construct, Plasmid Preparation, Transfection, Quantitative RT-PCR, Incubation

    The Prmt7 gene is activated by Oct4, Nanog, c-Myc and Klf4. ( A ) Analysis of Oct4, Nanog, c-Myc and Klf4 levels at the Prmt7 promoter using the publicly available ChIP-Seq database. ( B ) Microscopic images of V6.5 mESCs treated with shPRMT7-7, shOct4, shc-Myc, shKlf4 or shNanog. Red scale bar, 100 μm. ( C−F ) Effects of individual knockdown of Oct4 (C), Nanog (D), c-Myc (E), and Klf4 (F) on Prmt7 mRNA levels. V6.5 mESCs were electroporated with shOct4, shc-Myc, shKlf4, or shNanog. Four days later, cells were harvested. Expression levels were analyzed using quantitative RT-PCR. ( G ) A proposed model depicting a role for PRMT7 in maintaining the self-renewal and pluripotency of mESCs. miR-24-3p can silence the expression of Oct4, Nanog, Klf4 and c-Myc , whereas miR-24-2-5p can target Klf4 and c-Myc 3′UTRs. In addition, miR-24-3p and miR-24-2-5p target Prmt7 3′UTR. In mESCs, PRMT7 represses the miR-24-2 gene encoding miR-24-3p and miR-24-2-5p via H4R3 methylation. Therefore, PRMT7 antagonizes the anti-pluripotent effects of miR-24-3p and miR-24-2-5p against Oct4, Nanog, Klf4 and c-Myc in mESCs and positively regulates Oct4, Nanog, Klf4, and c-Myc levels to maintain mESC stemness. During differentiation, increased miR-24-3p and miR-24-2-5p levels may reduce PRMT7, Oct4, Nanog, Klf4, and c-Myc levels, facilitating mESC differentiation. The regulatory loop involving PRMT7 and miR-24-3p/miR-24-2-5p is interactive with the major pluripotent system containing Oct4, Nanog, Klf4, and c-Myc to fine-tune mESC stemness.
    Figure Legend Snippet: The Prmt7 gene is activated by Oct4, Nanog, c-Myc and Klf4. ( A ) Analysis of Oct4, Nanog, c-Myc and Klf4 levels at the Prmt7 promoter using the publicly available ChIP-Seq database. ( B ) Microscopic images of V6.5 mESCs treated with shPRMT7-7, shOct4, shc-Myc, shKlf4 or shNanog. Red scale bar, 100 μm. ( C−F ) Effects of individual knockdown of Oct4 (C), Nanog (D), c-Myc (E), and Klf4 (F) on Prmt7 mRNA levels. V6.5 mESCs were electroporated with shOct4, shc-Myc, shKlf4, or shNanog. Four days later, cells were harvested. Expression levels were analyzed using quantitative RT-PCR. ( G ) A proposed model depicting a role for PRMT7 in maintaining the self-renewal and pluripotency of mESCs. miR-24-3p can silence the expression of Oct4, Nanog, Klf4 and c-Myc , whereas miR-24-2-5p can target Klf4 and c-Myc 3′UTRs. In addition, miR-24-3p and miR-24-2-5p target Prmt7 3′UTR. In mESCs, PRMT7 represses the miR-24-2 gene encoding miR-24-3p and miR-24-2-5p via H4R3 methylation. Therefore, PRMT7 antagonizes the anti-pluripotent effects of miR-24-3p and miR-24-2-5p against Oct4, Nanog, Klf4 and c-Myc in mESCs and positively regulates Oct4, Nanog, Klf4, and c-Myc levels to maintain mESC stemness. During differentiation, increased miR-24-3p and miR-24-2-5p levels may reduce PRMT7, Oct4, Nanog, Klf4, and c-Myc levels, facilitating mESC differentiation. The regulatory loop involving PRMT7 and miR-24-3p/miR-24-2-5p is interactive with the major pluripotent system containing Oct4, Nanog, Klf4, and c-Myc to fine-tune mESC stemness.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Quantitative RT-PCR, Methylation

    PRMT7 occupies the miR-24-2 gene and negatively regulates its expression via H4R3 methylation. ( A ) Schematic representation of the miR-24-2 gene. A set of arrow heads indicate a ChIP PCR amplicon. ( B ) Luciferase activities of the miR-24-2 promoter and its deletion mutants with or without ectopic expression of PRMT7 in V6.5 mESCs. V6.5 mESCs were transfected using Lipofectamine 3000 and incubated for 48 h. ( C ) Analysis of PRMT7 occupancy at the miR-24-2 promoter using quantitative ChIP. ( D−F ) Analysis of PRMT7 (D), H4R3me2s (E) and H4R3me2a (F) levels at the region ‘b’ in the miR-24-2 promoter after rescue experiments of PRMT7-depleted mESCs. Quantitative ChIP assays were performed using four groups of V6.5 mESCs: (i) shLuc-treated cells, (ii) PRMT7-depleted cells, (iii) PRMT7-depleted cells with ectopic expression of PRMT7 and (iv) PRMT7-depleted cells with ectopic expression of a catalytic mutant of PRMT7 (m.PRMT7).
    Figure Legend Snippet: PRMT7 occupies the miR-24-2 gene and negatively regulates its expression via H4R3 methylation. ( A ) Schematic representation of the miR-24-2 gene. A set of arrow heads indicate a ChIP PCR amplicon. ( B ) Luciferase activities of the miR-24-2 promoter and its deletion mutants with or without ectopic expression of PRMT7 in V6.5 mESCs. V6.5 mESCs were transfected using Lipofectamine 3000 and incubated for 48 h. ( C ) Analysis of PRMT7 occupancy at the miR-24-2 promoter using quantitative ChIP. ( D−F ) Analysis of PRMT7 (D), H4R3me2s (E) and H4R3me2a (F) levels at the region ‘b’ in the miR-24-2 promoter after rescue experiments of PRMT7-depleted mESCs. Quantitative ChIP assays were performed using four groups of V6.5 mESCs: (i) shLuc-treated cells, (ii) PRMT7-depleted cells, (iii) PRMT7-depleted cells with ectopic expression of PRMT7 and (iv) PRMT7-depleted cells with ectopic expression of a catalytic mutant of PRMT7 (m.PRMT7).

    Techniques Used: Expressing, Methylation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Luciferase, Transfection, Incubation, Mutagenesis

    29) Product Images from "CrfA, a Small Noncoding RNA Regulator of Adaptation to Carbon Starvation in Caulobacter crescentus ▿ ▿ †"

    Article Title: CrfA, a Small Noncoding RNA Regulator of Adaptation to Carbon Starvation in Caulobacter crescentus ▿ ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00343-10

    CrfA and a stem-loop in the 5′-UTR of CC3461 mRNA that is complementary to a region of CrfA are both required for carbon starvation to activate CC3461 mRNA levels during carbon starvation. (A) qRT-PCR was utilized to measure the steady-state levels
    Figure Legend Snippet: CrfA and a stem-loop in the 5′-UTR of CC3461 mRNA that is complementary to a region of CrfA are both required for carbon starvation to activate CC3461 mRNA levels during carbon starvation. (A) qRT-PCR was utilized to measure the steady-state levels

    Techniques Used: Quantitative RT-PCR

    30) Product Images from "Detection and Isolation of Ultrasmall Microorganisms from a 120,000-Year-Old Greenland Glacier Ice Core"

    Article Title: Detection and Isolation of Ultrasmall Microorganisms from a 120,000-Year-Old Greenland Glacier Ice Core

    Journal:

    doi: 10.1128/AEM.71.12.7806-7818.2005

    Results from PCR amplification of 16S-23S rRNA gene IGS regions, using DNAs extracted from anaerobic enrichment cultures. (A) Round I enrichments in MM1 plus acetate inoculated with either nonfiltered (lanes 1 to 3) or filtered (lanes 4 to 6) ice after
    Figure Legend Snippet: Results from PCR amplification of 16S-23S rRNA gene IGS regions, using DNAs extracted from anaerobic enrichment cultures. (A) Round I enrichments in MM1 plus acetate inoculated with either nonfiltered (lanes 1 to 3) or filtered (lanes 4 to 6) ice after

    Techniques Used: Polymerase Chain Reaction, Amplification

    31) Product Images from "Human Telomeres Are Hypersensitive to UV-Induced DNA Damage and Refractory to Repair"

    Article Title: Human Telomeres Are Hypersensitive to UV-Induced DNA Damage and Refractory to Repair

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1000926

    Absence of excision repair of CPDs in telomeres. (A) WI38 human diploid fibroblasts were irradiated with minimally-lethal doses of UVC (10 J/m 2 ) and returned to the incubator for varying lengths of time before harvesting (0–48 h). The DNA was then isolated, the IPoD technique was used to isolate damaged DNA, photolyase was used to remove CPD, and PCR was performed on region of interest: Telomere, mitochondrial CYTB gene (mtDNA), 28S ribosomal DNA, or p53 . For each time point, the integrated intensity of the band or lane containing the PCR–amplified IP pulldown fraction is normalized against the unamplified input DNA for that time point. The amount of DNA in the fraction pulled down by antibody to CPD decreases with time in the p53 and 28S genes, reflecting normal excision repair, but not in telomeres or mtDNA ( CYTB gene). (B) A similar experiment was performed in primary human skin fibroblasts UV-irradiated at 20 J/m 2 . In this experiment, CPD were removed from IPoD-immunoprecipitated CPD containing DNA using DNA photolyase before PCR amplification and repair in the two telomeric DNA strands was analyzed together and separately. Each experiment was performed in triplicate. P values are derived from the two-tailed heteroscadastic Student's t-test.
    Figure Legend Snippet: Absence of excision repair of CPDs in telomeres. (A) WI38 human diploid fibroblasts were irradiated with minimally-lethal doses of UVC (10 J/m 2 ) and returned to the incubator for varying lengths of time before harvesting (0–48 h). The DNA was then isolated, the IPoD technique was used to isolate damaged DNA, photolyase was used to remove CPD, and PCR was performed on region of interest: Telomere, mitochondrial CYTB gene (mtDNA), 28S ribosomal DNA, or p53 . For each time point, the integrated intensity of the band or lane containing the PCR–amplified IP pulldown fraction is normalized against the unamplified input DNA for that time point. The amount of DNA in the fraction pulled down by antibody to CPD decreases with time in the p53 and 28S genes, reflecting normal excision repair, but not in telomeres or mtDNA ( CYTB gene). (B) A similar experiment was performed in primary human skin fibroblasts UV-irradiated at 20 J/m 2 . In this experiment, CPD were removed from IPoD-immunoprecipitated CPD containing DNA using DNA photolyase before PCR amplification and repair in the two telomeric DNA strands was analyzed together and separately. Each experiment was performed in triplicate. P values are derived from the two-tailed heteroscadastic Student's t-test.

    Techniques Used: Irradiation, Isolation, Polymerase Chain Reaction, Amplification, Immunoprecipitation, Derivative Assay, Two Tailed Test

    Hypersensitivity of telomeres relative to coding regions. The frequency of CPD is ∼7 fold higher in human fibroblast telomeres than in a fragment of the p53 gene or 28S ribosomal DNA at 20 J/m 2 UVC. This sensitivity also holds for each telomere strand individually and is unaffected by precise correction for the percentage of dimerizable dipyrimidines in the various sequences. (Upper) Graphical representation of the quantification of the PCR–amplified bands or lanes. The ratio IP/input corrects for copy number. (Lower) Gels of PCR–amplified IP DNA. Each experiment was performed in triplicate. P values are derived from the two-tailed heteroscadastic Student's t-test.
    Figure Legend Snippet: Hypersensitivity of telomeres relative to coding regions. The frequency of CPD is ∼7 fold higher in human fibroblast telomeres than in a fragment of the p53 gene or 28S ribosomal DNA at 20 J/m 2 UVC. This sensitivity also holds for each telomere strand individually and is unaffected by precise correction for the percentage of dimerizable dipyrimidines in the various sequences. (Upper) Graphical representation of the quantification of the PCR–amplified bands or lanes. The ratio IP/input corrects for copy number. (Lower) Gels of PCR–amplified IP DNA. Each experiment was performed in triplicate. P values are derived from the two-tailed heteroscadastic Student's t-test.

    Techniques Used: Polymerase Chain Reaction, Amplification, Derivative Assay, Two Tailed Test

    The ImmunoPrecipitation of DNA Damage technique (IPoD). (A) Schematic representation of the technique. Damaged or control DNA is sonicated to 500–1,000 bp fragments, denatured, and immunoprecipitated (IPd) using a DNA lesion-specific antibody and staph A beads. (B) Linearity of the IPoD signal at minimally-lethal doses. UV-induced cyclobutane pyrimidine dimers (CPD) in human diploid fibroblasts were assayed at low UV doses in the p53 gene, the 28S ribosomal DNA gene, and the telomeres. For each sample, the integrated intensity of the band or lane containing the PCR–amplified IP pulldown fraction is normalized against the unamplified input DNA for that sample. For comparison between doses, the pulldown percentages at 0 and 20 J/m 2 were assigned a value of 0% and 100%, respectively. The dose response is linear for each genome region analyzed and this linearity was not affected by removing CPD before PCR using photolyase. Each result depicted in (B) is derived from triplicate experiments.
    Figure Legend Snippet: The ImmunoPrecipitation of DNA Damage technique (IPoD). (A) Schematic representation of the technique. Damaged or control DNA is sonicated to 500–1,000 bp fragments, denatured, and immunoprecipitated (IPd) using a DNA lesion-specific antibody and staph A beads. (B) Linearity of the IPoD signal at minimally-lethal doses. UV-induced cyclobutane pyrimidine dimers (CPD) in human diploid fibroblasts were assayed at low UV doses in the p53 gene, the 28S ribosomal DNA gene, and the telomeres. For each sample, the integrated intensity of the band or lane containing the PCR–amplified IP pulldown fraction is normalized against the unamplified input DNA for that sample. For comparison between doses, the pulldown percentages at 0 and 20 J/m 2 were assigned a value of 0% and 100%, respectively. The dose response is linear for each genome region analyzed and this linearity was not affected by removing CPD before PCR using photolyase. Each result depicted in (B) is derived from triplicate experiments.

    Techniques Used: Immunoprecipitation, Sonication, Polymerase Chain Reaction, Amplification, Derivative Assay

    32) Product Images from "Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins"

    Article Title: Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins

    Journal: BMC Biology

    doi: 10.1186/1741-7007-2-21

    ChIP analysis of histone modification at the SYBL1 promoter region. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using an XhoI polymorphism in the 5' UTR of the SYBL1 gene in the Xq28 pseudoautosomal region that is also present on the Y chromosome. In normal male cells, the Y-linked locus is inactivated, hypermethylated, and late-replicating as is the inactive X allele in female cells [23,31,49]. To determine if this region has abnormal histone modifications on the X-inactivated but DNA hypomethylated ICF female X, ChIP assays were performed for acetylated histone H3 (acH3) and K4-methylated histone H3 (mK4) in normal male lymphoblasts and in PT3 ICF female fibroblasts; the ethidium bromide-stained gels of each sample are shown before and after digestion with XhoI. The undigested alleles (XhoI+) are 268 bp; the digested alleles (XhoI-) result in fragments of 108 and 260 bp. The ChIP assay for acetylated histone H3 (acH3) shows that only the XhoI-digested allele (XhoI+) is hyperacetylated in a normal male lymphoblast (NMLB1), and this corresponds to the active X allele (Xa) by RT-PCR (data not shown). An hTERT-transformed clone of PT3 ICF fibroblasts was also analyzed by ChIP. This clone has normal monoallelic expression of SYBL1 even though the promoter region is extremely hypomethylated as determined by bisulfite methylation analysis of DNA. The inactive X allele in the PT3 clone is hypoacetylated at histone H3 and hypomethylated at H3K4 because only the active X allele (XhoI-) is immunoprecipitated with either the acetylated or K4-methylated histone H3 antibodies (although a small portion of the inactive X also appears to have been precipitated by the acetylated H3 antibody).
    Figure Legend Snippet: ChIP analysis of histone modification at the SYBL1 promoter region. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using an XhoI polymorphism in the 5' UTR of the SYBL1 gene in the Xq28 pseudoautosomal region that is also present on the Y chromosome. In normal male cells, the Y-linked locus is inactivated, hypermethylated, and late-replicating as is the inactive X allele in female cells [23,31,49]. To determine if this region has abnormal histone modifications on the X-inactivated but DNA hypomethylated ICF female X, ChIP assays were performed for acetylated histone H3 (acH3) and K4-methylated histone H3 (mK4) in normal male lymphoblasts and in PT3 ICF female fibroblasts; the ethidium bromide-stained gels of each sample are shown before and after digestion with XhoI. The undigested alleles (XhoI+) are 268 bp; the digested alleles (XhoI-) result in fragments of 108 and 260 bp. The ChIP assay for acetylated histone H3 (acH3) shows that only the XhoI-digested allele (XhoI+) is hyperacetylated in a normal male lymphoblast (NMLB1), and this corresponds to the active X allele (Xa) by RT-PCR (data not shown). An hTERT-transformed clone of PT3 ICF fibroblasts was also analyzed by ChIP. This clone has normal monoallelic expression of SYBL1 even though the promoter region is extremely hypomethylated as determined by bisulfite methylation analysis of DNA. The inactive X allele in the PT3 clone is hypoacetylated at histone H3 and hypomethylated at H3K4 because only the active X allele (XhoI-) is immunoprecipitated with either the acetylated or K4-methylated histone H3 antibodies (although a small portion of the inactive X also appears to have been precipitated by the acetylated H3 antibody).

    Techniques Used: Chromatin Immunoprecipitation, Modification, Methylation, Staining, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Expressing, Immunoprecipitation

    33) Product Images from "The X Protein of Hepatitis B Virus Inhibits Apoptosis in Hepatoma Cells through Enhancing the Methionine Adenosyltransferase 2A Gene Expression and Reducing S-Adenosylmethionine Production *"

    Article Title: The X Protein of Hepatitis B Virus Inhibits Apoptosis in Hepatoma Cells through Enhancing the Methionine Adenosyltransferase 2A Gene Expression and Reducing S-Adenosylmethionine Production *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.167783

    Analyses of effects of HBx on binding of CREB and NF -κ B to the MAT2A promoter. A , analyses of the effect of HBx on the binding of CREB to the MAT2A promoter by EMSA. EMSA was performed with nuclear extracts of HepG2 cells transfected with control plasmid ( lane 1 ) or pcDNA-HBx at different concentrations ( lanes 2–7 ). CREB probe was generated by annealing single-stranded and end-labeled oligonucleotides containing the cognate MAT2A promoter region (nucleotides −385/−365). Mutated oligonucleotide ( lane 2 ), nonspecific competitor ( lane 6 ), or specific competitor (unlabeled CREB probe, lane 7 ) were used as a control. For supershift, antibody to CREB ( lane 4 ) was incubated with nuclear extracts before being added to the reaction. B , analyses of the effect of HBx on the binding of NF -κ B to the MAT2A promoter by EMSA. EMSA was performed with nuclear extracts of HepG2 cells transfected with pcDNA-HBx. NF -κ B probe was generated by annealing single-stranded and end-labeled oligonucleotides containing the cognate MAT2A promoter region (nucleotides −296/−284). Mutated oligonucleotide ( lane 2 ), nonspecific competitor ( lane 6 ), or specific competitor (unlabeled NF -κ B probe, lane 7 ) were used as a control. For supershift, antibody to p65 ( lane 4 ) was incubated with nuclear extracts before being added to the reaction. Samples were electrophoresed on 5% nondenaturing polyacrylamide gels and visualized by autoradiography. Arrows indicate the shift bands or supershifted protein-DNA complexes or free probes. C , determination of the role of HBx in the binding of CREB and NF -κ B to the MAT2A promoter by ChIP assays. HepG2 cells transfected with pcDNA-HBx (+) or control vector (−) were lysed and subjected to ChIP assays. The exact locations of PCR products of Chip1 and Chip2 underlie the simplified genomic structures of the MAT2A gene promoter. The results are representatives of four independent experiments.
    Figure Legend Snippet: Analyses of effects of HBx on binding of CREB and NF -κ B to the MAT2A promoter. A , analyses of the effect of HBx on the binding of CREB to the MAT2A promoter by EMSA. EMSA was performed with nuclear extracts of HepG2 cells transfected with control plasmid ( lane 1 ) or pcDNA-HBx at different concentrations ( lanes 2–7 ). CREB probe was generated by annealing single-stranded and end-labeled oligonucleotides containing the cognate MAT2A promoter region (nucleotides −385/−365). Mutated oligonucleotide ( lane 2 ), nonspecific competitor ( lane 6 ), or specific competitor (unlabeled CREB probe, lane 7 ) were used as a control. For supershift, antibody to CREB ( lane 4 ) was incubated with nuclear extracts before being added to the reaction. B , analyses of the effect of HBx on the binding of NF -κ B to the MAT2A promoter by EMSA. EMSA was performed with nuclear extracts of HepG2 cells transfected with pcDNA-HBx. NF -κ B probe was generated by annealing single-stranded and end-labeled oligonucleotides containing the cognate MAT2A promoter region (nucleotides −296/−284). Mutated oligonucleotide ( lane 2 ), nonspecific competitor ( lane 6 ), or specific competitor (unlabeled NF -κ B probe, lane 7 ) were used as a control. For supershift, antibody to p65 ( lane 4 ) was incubated with nuclear extracts before being added to the reaction. Samples were electrophoresed on 5% nondenaturing polyacrylamide gels and visualized by autoradiography. Arrows indicate the shift bands or supershifted protein-DNA complexes or free probes. C , determination of the role of HBx in the binding of CREB and NF -κ B to the MAT2A promoter by ChIP assays. HepG2 cells transfected with pcDNA-HBx (+) or control vector (−) were lysed and subjected to ChIP assays. The exact locations of PCR products of Chip1 and Chip2 underlie the simplified genomic structures of the MAT2A gene promoter. The results are representatives of four independent experiments.

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Generated, Labeling, Incubation, Autoradiography, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    34) Product Images from "Inheritance of CENP-A nucleosomes during DNA replication requires HJURP"

    Article Title: Inheritance of CENP-A nucleosomes during DNA replication requires HJURP

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2018.09.003

    CENP-A deposition proteins are associated with centromeres during DNA replication. (A) Schematic representation of the experimental approach used in B. Cells were blocked at the G1/S boundary or early S phase by addition of thymidine, and allowed to under S phase following thymidine removal. (B) Cells expressing BirA*-fused proteins under doxycycline inducible promoter were treated as shown in A. Biotinylated proteins were isolated by streptavidin purification following by immunoblot analysis with an HJURP antibody. The experiment was conducted twice, each experiment was conducted using 0.9*10 7 cells. Input and pull down fractions in the experiment were run on independent gels. (C) Schematic representation of the DLD1-Tir1 cell line where HJURP was endogenously tagged at both alleles with AID-YFP. ChiP from HJURP-AID-YFP cells (left) or YFP-CENP-A cells (right) at indicated time points. ChIP was performed using anti-GFP antibody or normal rabbit IgG. RT-PCR was performed using primers specific for α-satellite DNA of chromosome 7. The graph represents an average of two independent experiments, +/− SEM. (D) FACS profiles of cells used as an input for ChIP.
    Figure Legend Snippet: CENP-A deposition proteins are associated with centromeres during DNA replication. (A) Schematic representation of the experimental approach used in B. Cells were blocked at the G1/S boundary or early S phase by addition of thymidine, and allowed to under S phase following thymidine removal. (B) Cells expressing BirA*-fused proteins under doxycycline inducible promoter were treated as shown in A. Biotinylated proteins were isolated by streptavidin purification following by immunoblot analysis with an HJURP antibody. The experiment was conducted twice, each experiment was conducted using 0.9*10 7 cells. Input and pull down fractions in the experiment were run on independent gels. (C) Schematic representation of the DLD1-Tir1 cell line where HJURP was endogenously tagged at both alleles with AID-YFP. ChiP from HJURP-AID-YFP cells (left) or YFP-CENP-A cells (right) at indicated time points. ChIP was performed using anti-GFP antibody or normal rabbit IgG. RT-PCR was performed using primers specific for α-satellite DNA of chromosome 7. The graph represents an average of two independent experiments, +/− SEM. (D) FACS profiles of cells used as an input for ChIP.

    Techniques Used: Expressing, Isolation, Purification, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, FACS

    35) Product Images from "CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes"

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2017.04.005

    The Recruitment of CHD4 to Oxidative DNA Damage Sites Depends on OGG1 (A) CoIPs of lysates from SW480 cells untreated or treated with 2 mM H 2 O 2 for 30 min were performed with the indicated antibodies. (B) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer. The immunoprecipitated samples were detected by western blot analyses using the antibodies indicated. (C) After SW480 OGG1 KO cells were transfected with pCMV-Taq or pCMV-OGG1 for 48 hr, the cells were untreated or treated with 2mMH 2 O 2 for 30 min. Whole-cell extracts and the tight chromatin fractions were analyzed by immunoblotting with the indicated antibodies. (D) Whole-cell extracts and the tight chromatin fractions from SW480 CHD4 KD cells untreated (Un) or treated with 2 mM H 2 O 2 for 30 min were analyzed by immunoblotting as in (C). (E) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer with or without 8-OHdG oligonucleotide. The immuno-precipitated samples were detected by western blot analyses using the antibodies indicated. (F) Biotin labeled 8-OHdG oligonucleotide incubated with OGG1 and Flag-CHD4 was pulled down using streptavidin beads. Bound proteins were eluted and analyzed by immunoblotting with the indicated antibodies. (G) SW480 OGG1 KO cells were untreated or treated with 2mMH 2 O 2 for 30 min followed by ChIP for control IgG, 8-OHdG, and CHD4 at the promoter CpG islands of eight representative genes and analyzed by real-time RT-PCR. Data are represented as mean ± SEM for triplicate experiments. (H) Cells were untreated or treated with 2 mM H 2 O 2 for 30 min. Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG at the promoter CpG islands of eight TSGs. Data are represented as mean ± SEM for triplicate experiments. (I) Cells were untreated or treated with 2mMH 2 O 2 for 30 min, and nascent RNA was labeled concurrently. Real-time RT-PCR data are presented as mean ± SEM of the treated over untreated values for triplicate experiments. (J) Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG or epigenetic silencing proteins at the promoter CpG islands of eight representative TSGs in fresh frozen human CRC tissues (n = 20) and normal colon epithelial tissues (n = 6). Data are represented as mean ± SEM. .
    Figure Legend Snippet: The Recruitment of CHD4 to Oxidative DNA Damage Sites Depends on OGG1 (A) CoIPs of lysates from SW480 cells untreated or treated with 2 mM H 2 O 2 for 30 min were performed with the indicated antibodies. (B) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer. The immunoprecipitated samples were detected by western blot analyses using the antibodies indicated. (C) After SW480 OGG1 KO cells were transfected with pCMV-Taq or pCMV-OGG1 for 48 hr, the cells were untreated or treated with 2mMH 2 O 2 for 30 min. Whole-cell extracts and the tight chromatin fractions were analyzed by immunoblotting with the indicated antibodies. (D) Whole-cell extracts and the tight chromatin fractions from SW480 CHD4 KD cells untreated (Un) or treated with 2 mM H 2 O 2 for 30 min were analyzed by immunoblotting as in (C). (E) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer with or without 8-OHdG oligonucleotide. The immuno-precipitated samples were detected by western blot analyses using the antibodies indicated. (F) Biotin labeled 8-OHdG oligonucleotide incubated with OGG1 and Flag-CHD4 was pulled down using streptavidin beads. Bound proteins were eluted and analyzed by immunoblotting with the indicated antibodies. (G) SW480 OGG1 KO cells were untreated or treated with 2mMH 2 O 2 for 30 min followed by ChIP for control IgG, 8-OHdG, and CHD4 at the promoter CpG islands of eight representative genes and analyzed by real-time RT-PCR. Data are represented as mean ± SEM for triplicate experiments. (H) Cells were untreated or treated with 2 mM H 2 O 2 for 30 min. Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG at the promoter CpG islands of eight TSGs. Data are represented as mean ± SEM for triplicate experiments. (I) Cells were untreated or treated with 2mMH 2 O 2 for 30 min, and nascent RNA was labeled concurrently. Real-time RT-PCR data are presented as mean ± SEM of the treated over untreated values for triplicate experiments. (J) Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG or epigenetic silencing proteins at the promoter CpG islands of eight representative TSGs in fresh frozen human CRC tissues (n = 20) and normal colon epithelial tissues (n = 6). Data are represented as mean ± SEM. .

    Techniques Used: Purification, Incubation, Immunoprecipitation, Western Blot, Transfection, Labeling, Chromatin Immunoprecipitation, Quantitative RT-PCR

    36) Product Images from "Analysis of ?-globin Chromatin Micro-Environment Using a Novel 3C Variant, 4Cv"

    Article Title: Analysis of ?-globin Chromatin Micro-Environment Using a Novel 3C Variant, 4Cv

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013045

    Schematic representations of Chromosome Conformation Capture (3C) and Complete-genome 3C by Vectorette amplification (4Cv). A: 3C - The physical proximity between two DNA elements in cells is converted into a direct juxtaposition, which is detected by PCR using sequence-specific primers. B: The library of genomic interactions is digested with a second enzyme (NlaIII), vectorette linkers are added and the subset of interactions involving a specific sequence (e.g. β-globin, red line) is amplified. The resulting 4Cv library is analysed by sequencing cloned products or bands excised from a gel.
    Figure Legend Snippet: Schematic representations of Chromosome Conformation Capture (3C) and Complete-genome 3C by Vectorette amplification (4Cv). A: 3C - The physical proximity between two DNA elements in cells is converted into a direct juxtaposition, which is detected by PCR using sequence-specific primers. B: The library of genomic interactions is digested with a second enzyme (NlaIII), vectorette linkers are added and the subset of interactions involving a specific sequence (e.g. β-globin, red line) is amplified. The resulting 4Cv library is analysed by sequencing cloned products or bands excised from a gel.

    Techniques Used: Amplification, Polymerase Chain Reaction, Sequencing, Clone Assay

    37) Product Images from "APOBEC3A Functions as a Restriction Factor of Human Papillomavirus"

    Article Title: APOBEC3A Functions as a Restriction Factor of Human Papillomavirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.02383-14

    hA3A overexpression does not change A3A-type mutation signatures in HPV16 genomes. The HPV16 LCR (positions 7758 to 296) and the E2 region (positions 3098 to 3536) were amplified from purified virions produced with the overexpression of A3A or the vector by using barcoded MiSeq primers. The PCR products were gel purified and used as the input for PCR amplicon sequencing (Illumina MiSeq). The percentage of A3A-type mutation signatures was calculated by dividing the total number of A3A-specific dinucleotide (dark gray) or trinucleotide (light gray) motif transitions (Y represents any pyrimidine) by the total number of C > T and G > A nucleotide changes. The HPV16 W12 genome sequence was used as a reference for sequence alignment. Data are shown as percent means ± standard deviations.
    Figure Legend Snippet: hA3A overexpression does not change A3A-type mutation signatures in HPV16 genomes. The HPV16 LCR (positions 7758 to 296) and the E2 region (positions 3098 to 3536) were amplified from purified virions produced with the overexpression of A3A or the vector by using barcoded MiSeq primers. The PCR products were gel purified and used as the input for PCR amplicon sequencing (Illumina MiSeq). The percentage of A3A-type mutation signatures was calculated by dividing the total number of A3A-specific dinucleotide (dark gray) or trinucleotide (light gray) motif transitions (Y represents any pyrimidine) by the total number of C > T and G > A nucleotide changes. The HPV16 W12 genome sequence was used as a reference for sequence alignment. Data are shown as percent means ± standard deviations.

    Techniques Used: Over Expression, Mutagenesis, Amplification, Purification, Produced, Plasmid Preparation, Polymerase Chain Reaction, Sequencing

    38) Product Images from "Alteration of Forkhead Box O (Foxo4) Acetylation Mediates Apoptosis of Podocytes in Diabetes Mellitus"

    Article Title: Alteration of Forkhead Box O (Foxo4) Acetylation Mediates Apoptosis of Podocytes in Diabetes Mellitus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023566

    Binding of Foxo4 to the Bcl2l11 promoter. Conditionally immortalized podocytes were treated with either AGE-BSA (100 µg/ml) or BSA (100 µg/ml) as control and 30 mM of hyperglycemia (HG) or 30 mM of mannitol (Man) as osmotic control for 2 h. Binding of Foxo4 to a predicted forkhead binding elementer (FBE) in the promoter region of Bcl2l11 was characterized by chromatin immunoprecipiation (ChIP) using either an anti-Foxo4 antibody (Foxo4) or normal goal IgG (IgG). (A) Binding was assessed using primers flanking FBE (FBE for and FBE rev ) and immunoprecipitated DNA samples as PCR templates. (B) Primers flanking a region −2241 to −2043 upstream of the translational start site were used in a PCR reaction with Foxo4 immunoprecipitated DNA as templates to assess non-specific immunoprecipitation. Representative images of products of endpoint PCR resolved on agarose gels are shown. (C) Data of quantitative realtime PCR of ChIP DNA using primers flanking the FBE. Results from 3 independent sets of ChIPs. * p
    Figure Legend Snippet: Binding of Foxo4 to the Bcl2l11 promoter. Conditionally immortalized podocytes were treated with either AGE-BSA (100 µg/ml) or BSA (100 µg/ml) as control and 30 mM of hyperglycemia (HG) or 30 mM of mannitol (Man) as osmotic control for 2 h. Binding of Foxo4 to a predicted forkhead binding elementer (FBE) in the promoter region of Bcl2l11 was characterized by chromatin immunoprecipiation (ChIP) using either an anti-Foxo4 antibody (Foxo4) or normal goal IgG (IgG). (A) Binding was assessed using primers flanking FBE (FBE for and FBE rev ) and immunoprecipitated DNA samples as PCR templates. (B) Primers flanking a region −2241 to −2043 upstream of the translational start site were used in a PCR reaction with Foxo4 immunoprecipitated DNA as templates to assess non-specific immunoprecipitation. Representative images of products of endpoint PCR resolved on agarose gels are shown. (C) Data of quantitative realtime PCR of ChIP DNA using primers flanking the FBE. Results from 3 independent sets of ChIPs. * p

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction

    39) Product Images from "RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples"

    Article Title: RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh116

    Hybridization of a set of standard liver cDNA mixtures containing increasing amounts of bacterial LysA antisense cDNA. ( A ) LysA abundance varying from approximately 9 to 9100 copies LysA cDNA per cell equivalent are shown in this figure (left vertical
    Figure Legend Snippet: Hybridization of a set of standard liver cDNA mixtures containing increasing amounts of bacterial LysA antisense cDNA. ( A ) LysA abundance varying from approximately 9 to 9100 copies LysA cDNA per cell equivalent are shown in this figure (left vertical

    Techniques Used: Hybridization

    40) Product Images from "MicroRNA-21 contributes to renal cell carcinoma cell invasiveness and angiogenesis via the PDCD4/c-Jun (AP-1) signalling pathway"

    Article Title: MicroRNA-21 contributes to renal cell carcinoma cell invasiveness and angiogenesis via the PDCD4/c-Jun (AP-1) signalling pathway

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2019.4928

    Transcription of miR-21 is activated by p-c-Jun. (A) Chromatin immunoprecipitation analysis demonstrated that p-c-Jun binds specifically to the pri-miR-21 promoter region. PCR amplification of the region containing the p-c-Jun recognition sequence in the pri-miR-21 DNA. (B) Inhibition of miR-21 expression by a decrease in c-Jun expression in A498 and 786-O cells. Transfection of A498 and 786-O cells with mock, NC or c-Jun siRNA for 24 h. c-Jun and miR-21 expression levels were examined using reverse transcription-quantitative PCR analysis and normalized to GAPDH and U6 snRNA expression, respectively. Data are presented as the mean ± standard error of the mean. ** P
    Figure Legend Snippet: Transcription of miR-21 is activated by p-c-Jun. (A) Chromatin immunoprecipitation analysis demonstrated that p-c-Jun binds specifically to the pri-miR-21 promoter region. PCR amplification of the region containing the p-c-Jun recognition sequence in the pri-miR-21 DNA. (B) Inhibition of miR-21 expression by a decrease in c-Jun expression in A498 and 786-O cells. Transfection of A498 and 786-O cells with mock, NC or c-Jun siRNA for 24 h. c-Jun and miR-21 expression levels were examined using reverse transcription-quantitative PCR analysis and normalized to GAPDH and U6 snRNA expression, respectively. Data are presented as the mean ± standard error of the mean. ** P

    Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Sequencing, Inhibition, Expressing, Transfection, Real-time Polymerase Chain Reaction

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    Transfection:

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    Polymerase Chain Reaction:

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    Purification:

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    Electrophoresis:

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    Immunoprecipitation:

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    Expressing:

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    Qiagen pcr clean up kit
    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic <t>DNA</t> purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using <t>PCR.</t> b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses
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    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using PCR. b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses

    Journal: Epigenetics & Chromatin

    Article Title: Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination

    doi: 10.1186/s13072-018-0247-4

    Figure Lengend Snippet: TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using PCR. b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses

    Article Snippet: Immunoprecipitated bead–DNA complex was treated with proteinase K for 3 h at 50 °C in elution buffer and hydroxymethylated DNA purified by using the Qiagen PCR clean-up kit (Qiaquick).

    Techniques: Cell Culture, Purification, Immunoprecipitation, Polymerase Chain Reaction

    Modular engineering of the PE Sox1(+35) immediately upstream of the Gria1 -TSS. a, Strategy used to insert the PE Sox1(+35)TFBS and PE Sox1(+35)TFBS+CGI modules 380 bp upstream of the Gria1 TSS. The upper left panel shows a close-up view of the epigenomic and genetic features of the PE Sox1(+35) . The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. The lower left panel shows the two combinations of PE Sox1(+35) modules (i.e. PE Sox1(+35)TFBS and PE Sox1(+35)TFBS CGI) inserted 380 bp upstream of the Gria1 TSS. The right panel shows the TAD in which Gria1 is included (i.e. Gria1 -TAD) according to publically available Hi-C data 33 , 65 ; TAD boundaries are denoted with dotted lines; H3K27me3 ChIP-seq signals in mESC are shown in green 9 ; CGIs are indicated as green rectangles; CTCF binding sites 35 are indicated as black rectangles; the yellow triangle indicates the integration site of the PE Sox1(+35) modules, 380 bp upstream of the Gria1 TSS. b, For the identification of mESC clonal lines with the desired insertion of the different PE Sox1(+35) modules, primer pairs flanking the insertion borders (1+3 and 4+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for WT mESC or two mESC clonal lines with homozygous insertions of the different PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)TFBS+CGI ) 380 bp upstream of the Gria1 TSS are shown. c, Independent biological replicate for the data presented in Fig. 4f. The expression of Gria1 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) modules 380 bp upstream of the Gria1 TSS (TFBS (blue); TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ).

    Journal: bioRxiv

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes

    doi: 10.1101/2020.08.05.237768

    Figure Lengend Snippet: Modular engineering of the PE Sox1(+35) immediately upstream of the Gria1 -TSS. a, Strategy used to insert the PE Sox1(+35)TFBS and PE Sox1(+35)TFBS+CGI modules 380 bp upstream of the Gria1 TSS. The upper left panel shows a close-up view of the epigenomic and genetic features of the PE Sox1(+35) . The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. The lower left panel shows the two combinations of PE Sox1(+35) modules (i.e. PE Sox1(+35)TFBS and PE Sox1(+35)TFBS CGI) inserted 380 bp upstream of the Gria1 TSS. The right panel shows the TAD in which Gria1 is included (i.e. Gria1 -TAD) according to publically available Hi-C data 33 , 65 ; TAD boundaries are denoted with dotted lines; H3K27me3 ChIP-seq signals in mESC are shown in green 9 ; CGIs are indicated as green rectangles; CTCF binding sites 35 are indicated as black rectangles; the yellow triangle indicates the integration site of the PE Sox1(+35) modules, 380 bp upstream of the Gria1 TSS. b, For the identification of mESC clonal lines with the desired insertion of the different PE Sox1(+35) modules, primer pairs flanking the insertion borders (1+3 and 4+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for WT mESC or two mESC clonal lines with homozygous insertions of the different PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)TFBS+CGI ) 380 bp upstream of the Gria1 TSS are shown. c, Independent biological replicate for the data presented in Fig. 4f. The expression of Gria1 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) modules 380 bp upstream of the Gria1 TSS (TFBS (blue); TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ).

    Article Snippet: The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific).

    Techniques: Hi-C, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Standard Deviation

    Engineering of a PE Sox1(+35 ) construct with the TFBS module and an artificial CGI. a, Strategy used to insert the PE Sox1(+35)TFBS alone of together with an artificial CGI (aCGI; see Methods) into the Gata6 -TAD. The upper left panel shows a close-up view of the epigenomic and genetic features of the PE Sox1(+35) (Vert. Cons.= vertebrate PhastCons). The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. The lower left panel shows the two combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)TFBS+aCGI ) inserted into the Gata6 TAD. The right panel shows the TAD in which Gata6 is included (i.e. Gata6 -TAD) according to publically available Hi-C data 33 , 34 ; TAD boundaries are denoted with dotted lines; H3K27me3 ChIP-seq signals in mESC are shown in green 9 ; CGIs are indicated as green rectangles; CTCF binding sites 35 are indicated as black rectangles; the red triangle indicates the integration site of the PE Sox1(+35) modules, approximately 100 Kb downstream of Gata6 . b, For the identification of the PE Sox1(+35)TFBS+aCGI insertion, primer pairs flanking the insertion borders (1+3 and 4+2), amplifying potential duplications (4+3 and 4+4) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for two mESC clonal lines with homozygous insertions of PE Sox1(+35)TFBS+aCGI in the Gata6- TAD are shown. c, Independent biological replicate for the data presented in Fig. 2e. The expression of Gata6 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the PE Sox1(+35)TFBS (blue) or PE Sox1(+35)TFBS+aCGI (red) insertions. For the cells with the PE insertions, two different clonal cell lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two house-keeping genes ( Eef1a and Hprt ).

    Journal: bioRxiv

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes

    doi: 10.1101/2020.08.05.237768

    Figure Lengend Snippet: Engineering of a PE Sox1(+35 ) construct with the TFBS module and an artificial CGI. a, Strategy used to insert the PE Sox1(+35)TFBS alone of together with an artificial CGI (aCGI; see Methods) into the Gata6 -TAD. The upper left panel shows a close-up view of the epigenomic and genetic features of the PE Sox1(+35) (Vert. Cons.= vertebrate PhastCons). The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. The lower left panel shows the two combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)TFBS+aCGI ) inserted into the Gata6 TAD. The right panel shows the TAD in which Gata6 is included (i.e. Gata6 -TAD) according to publically available Hi-C data 33 , 34 ; TAD boundaries are denoted with dotted lines; H3K27me3 ChIP-seq signals in mESC are shown in green 9 ; CGIs are indicated as green rectangles; CTCF binding sites 35 are indicated as black rectangles; the red triangle indicates the integration site of the PE Sox1(+35) modules, approximately 100 Kb downstream of Gata6 . b, For the identification of the PE Sox1(+35)TFBS+aCGI insertion, primer pairs flanking the insertion borders (1+3 and 4+2), amplifying potential duplications (4+3 and 4+4) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for two mESC clonal lines with homozygous insertions of PE Sox1(+35)TFBS+aCGI in the Gata6- TAD are shown. c, Independent biological replicate for the data presented in Fig. 2e. The expression of Gata6 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the PE Sox1(+35)TFBS (blue) or PE Sox1(+35)TFBS+aCGI (red) insertions. For the cells with the PE insertions, two different clonal cell lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two house-keeping genes ( Eef1a and Hprt ).

    Article Snippet: The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific).

    Techniques: Construct, Hi-C, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Standard Deviation

    Modular engineering of the PE Sox1(+35) within the FoxA2 -TAD and of the PE Wnt8b(+21) within the Gata6 -TAD. a, Strategy used to insert the PE Sox1(+35) components into the Foxa2 -TAD. The upper left panel shows a close-up view of the epigenomic and genetic features of the PE Sox1(+35) (Vert. Cons.= vertebrate PhastCons). The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. The lower left panel shows the three combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) inserted into the FoxA2 -TAD. The right panel shows the TAD in which Foxa2 is included (i.e. Foxa2 -TAD) according to publically available Hi-C data 33 , 65 ; TAD boundaries are denoted with dotted lines; H3K27me3 ChIP-seq signals in mESC are shown in green 9 ; CGIs are indicated as green rectangles; the red triangle indicates the integration site of the PE Sox1(+35) modules, approximately 100 Kb downstream of Foxa2 . b, Strategy used to insert the PE Wnt8b(+21) components into the Gata6- TAD as described in (a). c-d, For identifying the successful insertion of the different PE Sox1(+35) (c) or PE Wnt8b(+21) (d) modules, primer pairs flanking the insertion borders (1+3 and 4+2; 1+5 and 6+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1; or 6+5, 5+2 and 6+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for two mESC clonal lines with homozygous insertions for each of the three different combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) or PE Wnt8b(+21) modules (i.e. (i) PE Wnt8b(+21)TFBS ; (ii) PE Wnt8b(+21)CGI ; (iii) PE Wnt8b(+21)TFBS+CGI ) in the Foxa2- TAD (c) or Gata6 -TAD (d), respectively, are shown. e-f, Independent biological replicate for the data shown in Fig. 2c (e) and Fig. 2d (f). The expression of Foxa2 (e), Gata6 (f), Sox1 (e) and Wnt8b (f) was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) (e) or Wnt8b(+21) (e) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal cell lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two house-keeping genes ( Eef1a and Hprt ).

    Journal: bioRxiv

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes

    doi: 10.1101/2020.08.05.237768

    Figure Lengend Snippet: Modular engineering of the PE Sox1(+35) within the FoxA2 -TAD and of the PE Wnt8b(+21) within the Gata6 -TAD. a, Strategy used to insert the PE Sox1(+35) components into the Foxa2 -TAD. The upper left panel shows a close-up view of the epigenomic and genetic features of the PE Sox1(+35) (Vert. Cons.= vertebrate PhastCons). The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. The lower left panel shows the three combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) inserted into the FoxA2 -TAD. The right panel shows the TAD in which Foxa2 is included (i.e. Foxa2 -TAD) according to publically available Hi-C data 33 , 65 ; TAD boundaries are denoted with dotted lines; H3K27me3 ChIP-seq signals in mESC are shown in green 9 ; CGIs are indicated as green rectangles; the red triangle indicates the integration site of the PE Sox1(+35) modules, approximately 100 Kb downstream of Foxa2 . b, Strategy used to insert the PE Wnt8b(+21) components into the Gata6- TAD as described in (a). c-d, For identifying the successful insertion of the different PE Sox1(+35) (c) or PE Wnt8b(+21) (d) modules, primer pairs flanking the insertion borders (1+3 and 4+2; 1+5 and 6+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1; or 6+5, 5+2 and 6+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for two mESC clonal lines with homozygous insertions for each of the three different combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) or PE Wnt8b(+21) modules (i.e. (i) PE Wnt8b(+21)TFBS ; (ii) PE Wnt8b(+21)CGI ; (iii) PE Wnt8b(+21)TFBS+CGI ) in the Foxa2- TAD (c) or Gata6 -TAD (d), respectively, are shown. e-f, Independent biological replicate for the data shown in Fig. 2c (e) and Fig. 2d (f). The expression of Foxa2 (e), Gata6 (f), Sox1 (e) and Wnt8b (f) was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) (e) or Wnt8b(+21) (e) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal cell lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two house-keeping genes ( Eef1a and Hprt ).

    Article Snippet: The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific).

    Techniques: Hi-C, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Standard Deviation

    PE Sox1(+35)CGI recruits PcG and contributes to the cis-regulatory function of PE Sox1(+35) . a, For the identification of the PE Sox1(+35)CGI deletion, primer pairs flanking each of the deletion breakpoints (1+3 and 4+2), located within the deleted region (5+6) or amplifying a large or small fragment depending on the absence or presence of the deletion (1+2) were used. The PCR results obtained for WT ESC and for two mESC clonal lines with homozygous deletions of the PE Sox1(+35)CGI ( PE Sox1(+35)CGI -/ ) are shown. b, H3K27me3 levels at PE Sox1(+35) were measured by ChIP-qPCR in WT mESC (grey), and in two PE Sox1(+35)CG I-/- mESCs clones using primers adjacent to the deleted region. ChIP-qPCR signals were normalized against two negative regions (Supplementary Data 1). Error bars correspond to standard deviations from technical triplicates. c, Independent biological replicate for the data presented in Fig. 1d. The expression of Sox1 was investigated by RT-qPCR in mESCs (left panel) and AntNPC (right panel) that were either WT (grey), homozygous for a deletion of the PE Sox1(+35) CGI ( PE Sox1 CGI -/- ; red) or homozygous for the complete PE Sox1(+35) deletion 9 ( PE Sox1 -/- ; black). Two different PE Sox1 CGI -/- mESC clones (circles and diamonds) and one PE Sox1 -/- clone were studied. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values of each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ).

    Journal: bioRxiv

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes

    doi: 10.1101/2020.08.05.237768

    Figure Lengend Snippet: PE Sox1(+35)CGI recruits PcG and contributes to the cis-regulatory function of PE Sox1(+35) . a, For the identification of the PE Sox1(+35)CGI deletion, primer pairs flanking each of the deletion breakpoints (1+3 and 4+2), located within the deleted region (5+6) or amplifying a large or small fragment depending on the absence or presence of the deletion (1+2) were used. The PCR results obtained for WT ESC and for two mESC clonal lines with homozygous deletions of the PE Sox1(+35)CGI ( PE Sox1(+35)CGI -/ ) are shown. b, H3K27me3 levels at PE Sox1(+35) were measured by ChIP-qPCR in WT mESC (grey), and in two PE Sox1(+35)CG I-/- mESCs clones using primers adjacent to the deleted region. ChIP-qPCR signals were normalized against two negative regions (Supplementary Data 1). Error bars correspond to standard deviations from technical triplicates. c, Independent biological replicate for the data presented in Fig. 1d. The expression of Sox1 was investigated by RT-qPCR in mESCs (left panel) and AntNPC (right panel) that were either WT (grey), homozygous for a deletion of the PE Sox1(+35) CGI ( PE Sox1 CGI -/- ; red) or homozygous for the complete PE Sox1(+35) deletion 9 ( PE Sox1 -/- ; black). Two different PE Sox1 CGI -/- mESC clones (circles and diamonds) and one PE Sox1 -/- clone were studied. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values of each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ).

    Article Snippet: The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific).

    Techniques: Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Clone Assay, Expressing, Quantitative RT-PCR, Standard Deviation

    Generation of mESC lines with structural variants within the Six3/Six2 locus. a, For the identification of mESC lines with the Six3/Six2 TAD boundary deletion, primer pairs flanking the deleted region (1+3 and 4+2), amplifying the deleted fragment (5+6) and amplifying a large or small fragment depending on the absence or presence of the deletion (1+2), respectively, were used. The PCR results obtained for two mESC clonal lines with 36Kb homozygous deletions ( del36 ) are shown. b, For the identification of mESC lines with the Six3/Six2 inversion, primer pairs flanking the inverted region (1+3, 4+2, 1+4 and 3+2) and amplifying potential duplications (4+3, 3+3 and 4+4) were used. The PCR results obtained for two mESC clonal lines with 110 Kb homozygous inversions ( inv110 ) are shown. c, 4C-seq experiments were performed using the PE Six3(-133) (upper panels) or the Six2 promoter (lower panels) as viewpoints in mESCs that were either WT (blue) or homozygous for the del36 deletion (red).

    Journal: bioRxiv

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes

    doi: 10.1101/2020.08.05.237768

    Figure Lengend Snippet: Generation of mESC lines with structural variants within the Six3/Six2 locus. a, For the identification of mESC lines with the Six3/Six2 TAD boundary deletion, primer pairs flanking the deleted region (1+3 and 4+2), amplifying the deleted fragment (5+6) and amplifying a large or small fragment depending on the absence or presence of the deletion (1+2), respectively, were used. The PCR results obtained for two mESC clonal lines with 36Kb homozygous deletions ( del36 ) are shown. b, For the identification of mESC lines with the Six3/Six2 inversion, primer pairs flanking the inverted region (1+3, 4+2, 1+4 and 3+2) and amplifying potential duplications (4+3, 3+3 and 4+4) were used. The PCR results obtained for two mESC clonal lines with 110 Kb homozygous inversions ( inv110 ) are shown. c, 4C-seq experiments were performed using the PE Six3(-133) (upper panels) or the Six2 promoter (lower panels) as viewpoints in mESCs that were either WT (blue) or homozygous for the del36 deletion (red).

    Article Snippet: The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific).

    Techniques: Polymerase Chain Reaction

    Epigenetic characterization of the PE Sox1(+35) modules engineered within the Gata6-TAD. a , Detailed view of the bisulfite sequencing data presented in Fig. 3a , in which mESC (Day0) and AntNPC (Day5) differentiated from cell lines with the PE Sox1(+35)TFBS (left panel) or PE Sox1(+35)TFBS+CGI modules (right panel) inserted in the Gata6- TAD were analyzed. DNA methylation levels were measured using a forward bisulfite primer upstream of the insertion site and a reverse primer inside the TFBS module (Methods). The circles in the plots correspond to individual CpG dinucleotides located within the TFBS module. Unmethylated CpGs are shown in white, methylated CpGs in black and not-covered CpGs are shown in gray. 10 alleles (rows) were analyzed for each differentiation stage and cell line. b , Chromatin accessibility at the endogenous PE Sox1(+35) , the Gata6 TAD insertion site (primer pairs P1 and P2) and the Gata6 promoter were measured by FAIRE-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (gray) or homozygous for the insertions of the different PE Sox1(+35) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). FAIRE-qPCR signals were normalized against two negative control regions (Supplementary Data 1). Error bars correspond to standard deviations from technical triplicates. The location of the primer pairs P1 and P2 around the Gata6- TAD insertion site is represented as red arrows in the diagram shown to the right. c , DNA methylation and nucleosome occupancy at the TFBS module were simultaneously analyzed by NOME-PCR in ESC lines with the PE Sox1(+35)TFBS (left panel) or PE Sox1(+35)TFBS+CGI modules (right panel) inserted in the Gata6- TAD. In the upper panels, the black and white circles represent methylated or unmethylated CpG sites, respectively. In the lower panels, the blue or white circles represent accessible or inaccessible GpC sites for the GpC methyltransferase, respectively. Red bars represent regions large enough to accommodate a nucleosome and that are considered as inaccessible. The dotted line represents the region where the TFBS sequence starts. The primers used to amplify the TFBS sequences are shown as red arrows in the schematic diagrams, with one of the primers being located within the inserted TFBS and the other one immediately outside. The grey shaded area represent a nucleosome depleted region. d , Scatter plots showing population-averaged nucleosome occupancy (red) and DNA methylation (black) levels within the TFBS sequence in cells with either the PE Sox1(+35)TFBS (left panel) or PE Sox1(+35)TFBS+CGI (right panel) modules inserted within the Gata6 -TAD. The grey shaded area represent a nucleosome depleted region.

    Journal: bioRxiv

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes

    doi: 10.1101/2020.08.05.237768

    Figure Lengend Snippet: Epigenetic characterization of the PE Sox1(+35) modules engineered within the Gata6-TAD. a , Detailed view of the bisulfite sequencing data presented in Fig. 3a , in which mESC (Day0) and AntNPC (Day5) differentiated from cell lines with the PE Sox1(+35)TFBS (left panel) or PE Sox1(+35)TFBS+CGI modules (right panel) inserted in the Gata6- TAD were analyzed. DNA methylation levels were measured using a forward bisulfite primer upstream of the insertion site and a reverse primer inside the TFBS module (Methods). The circles in the plots correspond to individual CpG dinucleotides located within the TFBS module. Unmethylated CpGs are shown in white, methylated CpGs in black and not-covered CpGs are shown in gray. 10 alleles (rows) were analyzed for each differentiation stage and cell line. b , Chromatin accessibility at the endogenous PE Sox1(+35) , the Gata6 TAD insertion site (primer pairs P1 and P2) and the Gata6 promoter were measured by FAIRE-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (gray) or homozygous for the insertions of the different PE Sox1(+35) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). FAIRE-qPCR signals were normalized against two negative control regions (Supplementary Data 1). Error bars correspond to standard deviations from technical triplicates. The location of the primer pairs P1 and P2 around the Gata6- TAD insertion site is represented as red arrows in the diagram shown to the right. c , DNA methylation and nucleosome occupancy at the TFBS module were simultaneously analyzed by NOME-PCR in ESC lines with the PE Sox1(+35)TFBS (left panel) or PE Sox1(+35)TFBS+CGI modules (right panel) inserted in the Gata6- TAD. In the upper panels, the black and white circles represent methylated or unmethylated CpG sites, respectively. In the lower panels, the blue or white circles represent accessible or inaccessible GpC sites for the GpC methyltransferase, respectively. Red bars represent regions large enough to accommodate a nucleosome and that are considered as inaccessible. The dotted line represents the region where the TFBS sequence starts. The primers used to amplify the TFBS sequences are shown as red arrows in the schematic diagrams, with one of the primers being located within the inserted TFBS and the other one immediately outside. The grey shaded area represent a nucleosome depleted region. d , Scatter plots showing population-averaged nucleosome occupancy (red) and DNA methylation (black) levels within the TFBS sequence in cells with either the PE Sox1(+35)TFBS (left panel) or PE Sox1(+35)TFBS+CGI (right panel) modules inserted within the Gata6 -TAD. The grey shaded area represent a nucleosome depleted region.

    Article Snippet: The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific).

    Techniques: Methylation Sequencing, DNA Methylation Assay, Methylation, Real-time Polymerase Chain Reaction, Negative Control, Polymerase Chain Reaction, Gel Permeation Chromatography, Sequencing

    Generation and characterization of cell lines with engineered PE Sox1(+35) modules within the Gria1-TAD . a, For the identification of mESC clonal lines with the desired insertion of the different PE Sox1(+35) modules, primer pairs flanking the insertion borders (1+3 and 4+2; 1+5 and 6+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1; or 6+5, 5+2 and 6+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for WT mESC or two mESC clonal lines with homozygous insertions of the different PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) in the Gria1- TAD are shown. b, Independent biological replicate for the data presented in Fig. 4b. The expression of Gria1 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) modules (TFBS (blue), CGI (yellow), TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ). c, Bisulfite sequencing analyses of ESC lines with the PE Sox1(+35) TFBS or PE Sox1(+35)TFBS+CGI modules inserted in the Gria1- TAD. DNA methylation levels were measured using a forward bisulfite primer upstream of the insertion site and a reverse primer inside the TFBS module (see Methods). The circles shown in the left plots correspond to individual CpG dinucleotides located within the TFBS module: unmethylated CpGs are shown in white, methylated CpGs in black and not-covered CpGs are shown in gray. 10 alleles (rows) were analyzed for each differentiation stage and cell line. The plot on the right summarizes the DNA methylation levels measured within the TFBS in the mESC lines containing the PE Sox1(+35)TFBS and PE Sox1(+35)TFBS+CGI mESC inserts within the Gria1 -TAD. d-e, RNAP2 (d), MED1 (d), H3K27me3 (e) and H2AK119ub (e) levels at the endogenous PE Sox1(+35) , the Gria1- TAD insertion site (primer pairs P1 and P2) and the Gria1 promoter were measured by ChIP-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (gray) or homozygous for the insertions of the different PE Sox1(+35) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). ChIP-qPCR signals were normalized against two negative control regions (Supplementary Data 1). Error bars correspond to standard deviations from technical triplicates. The location of the primers P1 and P2 around the Gria1- TAD insertion site is represented as red arrows in the diagram shown to the right.

    Journal: bioRxiv

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes

    doi: 10.1101/2020.08.05.237768

    Figure Lengend Snippet: Generation and characterization of cell lines with engineered PE Sox1(+35) modules within the Gria1-TAD . a, For the identification of mESC clonal lines with the desired insertion of the different PE Sox1(+35) modules, primer pairs flanking the insertion borders (1+3 and 4+2; 1+5 and 6+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1; or 6+5, 5+2 and 6+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for WT mESC or two mESC clonal lines with homozygous insertions of the different PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) in the Gria1- TAD are shown. b, Independent biological replicate for the data presented in Fig. 4b. The expression of Gria1 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) modules (TFBS (blue), CGI (yellow), TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ). c, Bisulfite sequencing analyses of ESC lines with the PE Sox1(+35) TFBS or PE Sox1(+35)TFBS+CGI modules inserted in the Gria1- TAD. DNA methylation levels were measured using a forward bisulfite primer upstream of the insertion site and a reverse primer inside the TFBS module (see Methods). The circles shown in the left plots correspond to individual CpG dinucleotides located within the TFBS module: unmethylated CpGs are shown in white, methylated CpGs in black and not-covered CpGs are shown in gray. 10 alleles (rows) were analyzed for each differentiation stage and cell line. The plot on the right summarizes the DNA methylation levels measured within the TFBS in the mESC lines containing the PE Sox1(+35)TFBS and PE Sox1(+35)TFBS+CGI mESC inserts within the Gria1 -TAD. d-e, RNAP2 (d), MED1 (d), H3K27me3 (e) and H2AK119ub (e) levels at the endogenous PE Sox1(+35) , the Gria1- TAD insertion site (primer pairs P1 and P2) and the Gria1 promoter were measured by ChIP-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (gray) or homozygous for the insertions of the different PE Sox1(+35) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). ChIP-qPCR signals were normalized against two negative control regions (Supplementary Data 1). Error bars correspond to standard deviations from technical triplicates. The location of the primers P1 and P2 around the Gria1- TAD insertion site is represented as red arrows in the diagram shown to the right.

    Article Snippet: The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific).

    Techniques: Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Standard Deviation, Methylation Sequencing, DNA Methylation Assay, Methylation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    Modular engineering of the PE Sox1(+35) within the Gata6 -TAD. a, Genome-browser view of the epigenomic and genomic features of two previously characterized PEs 9 (left: PE Six3-(133) ; Right: PE Lmx1b(+59) ) in which the oCGIs overlap with conserved sequences bound by P300 and, thus, likely to represent TFBS. The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. Vert. Cons.= vertebrate PhastCons. b, For the identification of the different PE Sox1(+35) module insertions, primer pairs flanking the insertion borders (1+3 and 4+2; 1+5 and 6+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1; or 6+5, 5+2 and 6+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for WT mESC and for two mESC clonal lines with homozygous insertions for each of the three different combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) inserted in the Gata6- TAD are shown. c, Independent biological replicate for the data presented in Fig. 2b. The expression of Gata6 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal cell lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ).

    Journal: bioRxiv

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes

    doi: 10.1101/2020.08.05.237768

    Figure Lengend Snippet: Modular engineering of the PE Sox1(+35) within the Gata6 -TAD. a, Genome-browser view of the epigenomic and genomic features of two previously characterized PEs 9 (left: PE Six3-(133) ; Right: PE Lmx1b(+59) ) in which the oCGIs overlap with conserved sequences bound by P300 and, thus, likely to represent TFBS. The represented CGIs correspond to those computationally defined in the UCSC browser according to the following criteria: GC content > 50%; Length > 200 bp; CpG Observed to expected ratio > 0.6. Vert. Cons.= vertebrate PhastCons. b, For the identification of the different PE Sox1(+35) module insertions, primer pairs flanking the insertion borders (1+3 and 4+2; 1+5 and 6+2; or 1+3 and 6+2), amplifying potential duplications (4+3, 3+2 and 4+1; or 6+5, 5+2 and 6+1) and amplifying a large or small fragment depending on the absence or presence of the insertion (1+2), respectively, were used. The PCR results obtained for WT mESC and for two mESC clonal lines with homozygous insertions for each of the three different combinations of PE Sox1(+35) modules (i.e. (i) PE Sox1(+35)TFBS ; (ii) PE Sox1(+35)CGI ; (iii) PE Sox1(+35)TFBS+CGI ) inserted in the Gata6- TAD are shown. c, Independent biological replicate for the data presented in Fig. 2b. The expression of Gata6 and Sox1 was measured by RT-qPCR in mESCs (left panels) and AntNPC (right panels) that were either WT (grey) or homozygous for the insertions of the different PE Sox1(+35) modules (i.e. TFBS (blue), CGI (yellow), TFBS+CGI (red)). For the cells with the PE module insertions, two different clonal cell lines (circles and diamonds) were studied in each case. For each cell line, two technical replicates of the AntNPC differentiation were performed. The plotted expression values for each clone correspond to the average and standard deviation (error bars) from three RT-qPCR technical replicates. Expression values were normalized to two housekeeping genes ( Eef1a and Hprt ).

    Article Snippet: The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific).

    Techniques: Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Standard Deviation

    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

    Journal: Scientific Reports

    Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

    doi: 10.1038/s41598-020-58586-3

    Figure Lengend Snippet: Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

    Article Snippet: Kit extractions We tested three different silica-column kits: Zymo ZR Viral DNA/RNA Kit (outdated protocol, D7021), Zymo Quick-DNA/RNA Kit (updated protocol, D7021), and the QIAquick PCR Purification Kit (28104, Qiagen).

    Techniques: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Purification

    Comparison of chromatin fragmentation by ultrasound alone or in combination with benzonase digestion. (A) Comparison of sonication efficiency at the L and H power outputs over time. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for various times (as indicated) in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The “Quick” lanes contain samples sonicated for 20 min and reverse cross-linked for 1 h at +60°C. (B) Coomassie Blue staining of protein fractions generated during the sonication time course shown in panel A. Note the absence of high molecular weight proteins after 20 min of ultrasound treatment. (C) Titration of benzonase (0.2U to 90U) to fragment chromatin solubilized by 2 min L-power sonication. Following the digest, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. See Material and Methods section for detailed reaction conditions. (D) Combination of brief sonication (2 min at L-power) and benzonase digestion (0.7U to 90U) preserves the integrity of large proteins.

    Journal: PLoS ONE

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

    doi: 10.1371/journal.pone.0148023

    Figure Lengend Snippet: Comparison of chromatin fragmentation by ultrasound alone or in combination with benzonase digestion. (A) Comparison of sonication efficiency at the L and H power outputs over time. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for various times (as indicated) in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The “Quick” lanes contain samples sonicated for 20 min and reverse cross-linked for 1 h at +60°C. (B) Coomassie Blue staining of protein fractions generated during the sonication time course shown in panel A. Note the absence of high molecular weight proteins after 20 min of ultrasound treatment. (C) Titration of benzonase (0.2U to 90U) to fragment chromatin solubilized by 2 min L-power sonication. Following the digest, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. See Material and Methods section for detailed reaction conditions. (D) Combination of brief sonication (2 min at L-power) and benzonase digestion (0.7U to 90U) preserves the integrity of large proteins.

    Article Snippet: In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104).

    Techniques: Sonication, Concentration Assay, Size-exclusion Chromatography, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Generated, Molecular Weight, Titration

    Systematic optimization of sonication conditions. Part 2. (A) The effect of sample position on sonication efficiency at low power setting. 500 μL cell suspensions were loaded into positions L1, L7, R1, R7, all other positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Left panel: Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. Right panel: Afterwards, the samples were sonicated for an additional 9 min and reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 . p -values for selected T-tests are shown on the graph. (B) The effect of pulse time on sonication efficiency. 500 μL cell suspensions were loaded into position R1; vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 2 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), with variable pulse times (as indicated), no rotation and no ice. Following sonication, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. (C) The effect of buffer composition on sonication efficiency. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 10 min in 1:4 ELB:H 2 O supplemented with varying concentrations of SDS and Triton X-100 (as indicated), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. (D) The effect of water level and sample volume on sonication efficiency. Cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. Analysis of variance p -values are shown on the graph for each sample volume.

    Journal: PLoS ONE

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

    doi: 10.1371/journal.pone.0148023

    Figure Lengend Snippet: Systematic optimization of sonication conditions. Part 2. (A) The effect of sample position on sonication efficiency at low power setting. 500 μL cell suspensions were loaded into positions L1, L7, R1, R7, all other positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Left panel: Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. Right panel: Afterwards, the samples were sonicated for an additional 9 min and reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 . p -values for selected T-tests are shown on the graph. (B) The effect of pulse time on sonication efficiency. 500 μL cell suspensions were loaded into position R1; vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 2 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), with variable pulse times (as indicated), no rotation and no ice. Following sonication, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. (C) The effect of buffer composition on sonication efficiency. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 10 min in 1:4 ELB:H 2 O supplemented with varying concentrations of SDS and Triton X-100 (as indicated), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. (D) The effect of water level and sample volume on sonication efficiency. Cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. Analysis of variance p -values are shown on the graph for each sample volume.

    Article Snippet: In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104).

    Techniques: Sonication, Concentration Assay, Size-exclusion Chromatography, Standard Deviation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Systematic optimization of sonication conditions. Part 1. (A) Schematic of the Bioruptor XL water bath with tube positions numbered from 1 to 12 in the left (L) and right (R) carousels; red arrows indicate the alignment marks for carousel assembly and positioning. (B) The effect of sample volume on sonication efficiency at low power setting. Cell suspensions of variable volume (100–700 μL, as indicated) were loaded into positions L3, L4, L5, L9, L10 and L11. Vacant L-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 8 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 , between all samples 1.4×10 −6 . p -values for selected T-tests are shown on the graph. (C) Reproducibility of sample sonication across positions L1–L12. Sonication was carried out for 40 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Samples were reverse cross-linked overnight and the resulting DNA was purified using the Qiagen PCR clean-up kit followed by 1.1% agarose gel analysis. (D) The effect of sample position and power setting on sonication efficiency. 500 μL cell suspensions were loaded into positions R10-R4 and vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 3.6×10 −9 (L-power) and 8.2×10 −10 (H-power). p -values for selected T-tests are shown on the graph.

    Journal: PLoS ONE

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

    doi: 10.1371/journal.pone.0148023

    Figure Lengend Snippet: Systematic optimization of sonication conditions. Part 1. (A) Schematic of the Bioruptor XL water bath with tube positions numbered from 1 to 12 in the left (L) and right (R) carousels; red arrows indicate the alignment marks for carousel assembly and positioning. (B) The effect of sample volume on sonication efficiency at low power setting. Cell suspensions of variable volume (100–700 μL, as indicated) were loaded into positions L3, L4, L5, L9, L10 and L11. Vacant L-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 8 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 , between all samples 1.4×10 −6 . p -values for selected T-tests are shown on the graph. (C) Reproducibility of sample sonication across positions L1–L12. Sonication was carried out for 40 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Samples were reverse cross-linked overnight and the resulting DNA was purified using the Qiagen PCR clean-up kit followed by 1.1% agarose gel analysis. (D) The effect of sample position and power setting on sonication efficiency. 500 μL cell suspensions were loaded into positions R10-R4 and vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 3.6×10 −9 (L-power) and 8.2×10 −10 (H-power). p -values for selected T-tests are shown on the graph.

    Article Snippet: In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104).

    Techniques: Sonication, Concentration Assay, Size-exclusion Chromatography, Standard Deviation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis