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Physical analysis of the alphoid tetO -HAC used for loading of tTS -containing cassettes. ( a ) Analysis of integrity of the alphoid tetO -HAC synthetic array after its transfer into HPRT-deficient HT1080 cells. Genomic <t>DNA</t> from the cells with the HAC was digested with SpeI endonuclease, separated by CHEF gel electrophoresis (range 10–70 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. Lane 1: the HAC with the loxP site in hamster CHO cells; lanes 2, 3, 4 and 5: four HAC clones with the loxP site in HPRT-deficient HT1080 cells; lane 6: the original HAC (clone AB2.218.21) generated in human cells ( 29 ). M1- Pulse Marker™ 0.1–200 kb (Sigma-Aldrich). ( b ) Mapping of the loxP site in a mega-base size alphoid DNA array. Genomic DNA from the cells possessing the original HAC was digested with PmeI endonuclease, separated by CHEF gel electrophoresis (range 200–1500 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. A single 1.1-Mb fragment was detected for the original HAC (left, lane 1). Two bands of 650 and 500 kb were detected for genomic DNA from cells with the HAC bearing an inserted NBS1 gene (right, lanes 1 and 2). The size of the fragments was determined by comparison with the DNA size standard , Saccharomyces cerevisiae chromosomes. Lane M2: Yeast Chromosome PFG Marker BioLabs. ( c ) <t>PCR</t> analysis of clones with insertion of X3.1-I-EGFP-I into the HAC confirming restoration of the full-length HPRT gene.
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1) Product Images from "Re-engineering an alphoidtetO-HAC-based vector to enable high-throughput analyses of gene function"

Article Title: Re-engineering an alphoidtetO-HAC-based vector to enable high-throughput analyses of gene function

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt205

Physical analysis of the alphoid tetO -HAC used for loading of tTS -containing cassettes. ( a ) Analysis of integrity of the alphoid tetO -HAC synthetic array after its transfer into HPRT-deficient HT1080 cells. Genomic DNA from the cells with the HAC was digested with SpeI endonuclease, separated by CHEF gel electrophoresis (range 10–70 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. Lane 1: the HAC with the loxP site in hamster CHO cells; lanes 2, 3, 4 and 5: four HAC clones with the loxP site in HPRT-deficient HT1080 cells; lane 6: the original HAC (clone AB2.218.21) generated in human cells ( 29 ). M1- Pulse Marker™ 0.1–200 kb (Sigma-Aldrich). ( b ) Mapping of the loxP site in a mega-base size alphoid DNA array. Genomic DNA from the cells possessing the original HAC was digested with PmeI endonuclease, separated by CHEF gel electrophoresis (range 200–1500 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. A single 1.1-Mb fragment was detected for the original HAC (left, lane 1). Two bands of 650 and 500 kb were detected for genomic DNA from cells with the HAC bearing an inserted NBS1 gene (right, lanes 1 and 2). The size of the fragments was determined by comparison with the DNA size standard , Saccharomyces cerevisiae chromosomes. Lane M2: Yeast Chromosome PFG Marker BioLabs. ( c ) PCR analysis of clones with insertion of X3.1-I-EGFP-I into the HAC confirming restoration of the full-length HPRT gene.
Figure Legend Snippet: Physical analysis of the alphoid tetO -HAC used for loading of tTS -containing cassettes. ( a ) Analysis of integrity of the alphoid tetO -HAC synthetic array after its transfer into HPRT-deficient HT1080 cells. Genomic DNA from the cells with the HAC was digested with SpeI endonuclease, separated by CHEF gel electrophoresis (range 10–70 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. Lane 1: the HAC with the loxP site in hamster CHO cells; lanes 2, 3, 4 and 5: four HAC clones with the loxP site in HPRT-deficient HT1080 cells; lane 6: the original HAC (clone AB2.218.21) generated in human cells ( 29 ). M1- Pulse Marker™ 0.1–200 kb (Sigma-Aldrich). ( b ) Mapping of the loxP site in a mega-base size alphoid DNA array. Genomic DNA from the cells possessing the original HAC was digested with PmeI endonuclease, separated by CHEF gel electrophoresis (range 200–1500 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. A single 1.1-Mb fragment was detected for the original HAC (left, lane 1). Two bands of 650 and 500 kb were detected for genomic DNA from cells with the HAC bearing an inserted NBS1 gene (right, lanes 1 and 2). The size of the fragments was determined by comparison with the DNA size standard , Saccharomyces cerevisiae chromosomes. Lane M2: Yeast Chromosome PFG Marker BioLabs. ( c ) PCR analysis of clones with insertion of X3.1-I-EGFP-I into the HAC confirming restoration of the full-length HPRT gene.

Techniques Used: HAC Assay, Nucleic Acid Electrophoresis, Clone Assay, Generated, Marker, DNA Array, Polymerase Chain Reaction

Doxycycline regulated expression of the tTS-EYFP transgene loaded into the HAC. ( a ) A diagram illustrating time-course analysis of tTS-EYFP expression in the cells grown in different media. Samples 1–11 correspond to cultures used for FACS analysis. The cultures marked by red stars were analyzed by ChIP (see below). ( b ) Relative mean EYFP fluorescence determined by FACS of cells carrying the tTS-EYFP cassette (construct #4) grown in different media. Sample 1 corresponds to control HT1080 cells without the EYFP transgene (fluorescence background). Sample 2 corresponds to the cells with the HAC carrying the tTS-EYFP cassette grown in HAT and dox + medium for 30 days. Sample 3 corresponds to the cells grown for 22 days in dox + medium without selection. Sample 4 corresponds to the cells grown for 22 days without doxycycline. Sample 5 corresponds to the cells grown for a month in dox − medium with BS selection. Sample 6 corresponds to the cells grown in bsr − dox − medium. Samples 7, 8 and 9 correspond to the cells grown in dox + BS + medium for 24 h, 6 and 12 days, correspondingly. Samples 10 and 11 correspond to the cells treated either TSA or SAHA in dox + medium. Error bars, SD ( n = 3). ( c ) Transcription of Hygro , TK, EGFP and HPRT genes from the HAC. The level of the transgene transcripts from cells cultured in dox + or dox − medium was analyzed by RT–PCR. The housekeeping gene, BRCA1 , was used as an internal control. Lane 1 (dox − ) and lane 2 (dox + ) correspond to transcripts for BRCA1 . Lane 3 (dox − ) and lane 4 (dox + ) correspond to transcripts for Hygro . Lane 5 (dox − ) and lane 7 (dox + ) correspond to transcripts for TK . Lane 6 (dox − ) and lane 8 (dox + ) correspond to transcripts for HPRT . Lane 9 (dox − ) and lane 10 (dox + ) correspond to transcripts for EGFP . Lane M- GeneRuler™ 1-kb DNA ladder. ( d ) ChIP analysis of H3K4me3 chromatin in the transgene cassette of the HAC in the presence and absence of doxycycline. The cell samples used for ChIP correspond to samples 2 and 6 in Figure 6 a and b. Enrichment is shown relative to the 5S rRNA control locus. Satellite 2 sequence, Sat2, corresponding endogenous pericentromeric repeats was included as a negative control. ( e ) ChIP analysis of CENP-A chromatin in the HAC in the presence and absence of doxycycline. Enrichment is shown relative to the chromosome 21 centromere.
Figure Legend Snippet: Doxycycline regulated expression of the tTS-EYFP transgene loaded into the HAC. ( a ) A diagram illustrating time-course analysis of tTS-EYFP expression in the cells grown in different media. Samples 1–11 correspond to cultures used for FACS analysis. The cultures marked by red stars were analyzed by ChIP (see below). ( b ) Relative mean EYFP fluorescence determined by FACS of cells carrying the tTS-EYFP cassette (construct #4) grown in different media. Sample 1 corresponds to control HT1080 cells without the EYFP transgene (fluorescence background). Sample 2 corresponds to the cells with the HAC carrying the tTS-EYFP cassette grown in HAT and dox + medium for 30 days. Sample 3 corresponds to the cells grown for 22 days in dox + medium without selection. Sample 4 corresponds to the cells grown for 22 days without doxycycline. Sample 5 corresponds to the cells grown for a month in dox − medium with BS selection. Sample 6 corresponds to the cells grown in bsr − dox − medium. Samples 7, 8 and 9 correspond to the cells grown in dox + BS + medium for 24 h, 6 and 12 days, correspondingly. Samples 10 and 11 correspond to the cells treated either TSA or SAHA in dox + medium. Error bars, SD ( n = 3). ( c ) Transcription of Hygro , TK, EGFP and HPRT genes from the HAC. The level of the transgene transcripts from cells cultured in dox + or dox − medium was analyzed by RT–PCR. The housekeeping gene, BRCA1 , was used as an internal control. Lane 1 (dox − ) and lane 2 (dox + ) correspond to transcripts for BRCA1 . Lane 3 (dox − ) and lane 4 (dox + ) correspond to transcripts for Hygro . Lane 5 (dox − ) and lane 7 (dox + ) correspond to transcripts for TK . Lane 6 (dox − ) and lane 8 (dox + ) correspond to transcripts for HPRT . Lane 9 (dox − ) and lane 10 (dox + ) correspond to transcripts for EGFP . Lane M- GeneRuler™ 1-kb DNA ladder. ( d ) ChIP analysis of H3K4me3 chromatin in the transgene cassette of the HAC in the presence and absence of doxycycline. The cell samples used for ChIP correspond to samples 2 and 6 in Figure 6 a and b. Enrichment is shown relative to the 5S rRNA control locus. Satellite 2 sequence, Sat2, corresponding endogenous pericentromeric repeats was included as a negative control. ( e ) ChIP analysis of CENP-A chromatin in the HAC in the presence and absence of doxycycline. Enrichment is shown relative to the chromosome 21 centromere.

Techniques Used: Expressing, HAC Assay, FACS, Chromatin Immunoprecipitation, Fluorescence, Construct, HAT Assay, Selection, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Sequencing, Negative Control

2) Product Images from "Activation of the AMPK-FOXO3 Pathway Reduces Fatty Acid-Induced Increase in Intracellular Reactive Oxygen Species by Upregulating Thioredoxin"

Article Title: Activation of the AMPK-FOXO3 Pathway Reduces Fatty Acid-Induced Increase in Intracellular Reactive Oxygen Species by Upregulating Thioredoxin

Journal: Diabetes

doi: 10.2337/db08-1512

AMPK promoted FOXO3 binding to the Trx promoter and formation of the FOXO3/p300 transcription complex in the Trx promoter. A : Depiction of FOXO binding sites in the Trx promoter. B : AICAR increased binding of FOXO3 to the Trx promoter. HAECs were treated with AICAR and palmitic acid (PA) for 24 h. FOXO3-DNA complexes were cross-linked by formaldehyde and immunoprecipitated with anti-FOXO3 antibody. Bound FOXO3 sites in the Trx promoter were detected by qPCR and normalized with input DNA. Relative DNA was compared and expressed as the percentage of the nontreatment control subjects. Representative blots and quantitative analysis from three independent experiments are shown. Data represent the means ± SE. * P
Figure Legend Snippet: AMPK promoted FOXO3 binding to the Trx promoter and formation of the FOXO3/p300 transcription complex in the Trx promoter. A : Depiction of FOXO binding sites in the Trx promoter. B : AICAR increased binding of FOXO3 to the Trx promoter. HAECs were treated with AICAR and palmitic acid (PA) for 24 h. FOXO3-DNA complexes were cross-linked by formaldehyde and immunoprecipitated with anti-FOXO3 antibody. Bound FOXO3 sites in the Trx promoter were detected by qPCR and normalized with input DNA. Relative DNA was compared and expressed as the percentage of the nontreatment control subjects. Representative blots and quantitative analysis from three independent experiments are shown. Data represent the means ± SE. * P

Techniques Used: Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

3) Product Images from "Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays"

Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

Journal: PLoS ONE

doi: 10.1371/journal.pone.0233085

ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.
Figure Legend Snippet: ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.

Techniques Used: Real-time Polymerase Chain Reaction, Migration, Concentration Assay

Comparison between ddPCR and qPCR quantitation results in low viral DNA range
Figure Legend Snippet: Comparison between ddPCR and qPCR quantitation results in low viral DNA range

Techniques Used: Real-time Polymerase Chain Reaction, Quantitation Assay

SIV ddPCR DNA assay characterization. (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance. DNA sample used was duodenum DNA from Rhesus macaque 313–08. (B) Total number of droplets generated during dropletization remains constant in the range of tissue DNA amount tested (1 million to 8 million cell equivalent input). Total droplet number for each sample after dropletization serves as an additional indicator of sample DNA input tolerance. Note that for each level of DNA input, only a fraction (~1.6%) of the droplets were counted for QC purpose by the Source machine. (C) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines [ 7 ]. According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (D) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument. (E) Instead of measuring 60 ddPCR replicates to obtain 95% confidence, we obtained an approximate estimation of the LoD using a lower number of ddPCR reaction replicates and required the false negative rate to be below 5% (i.e. all 10 replicates have to be positive).
Figure Legend Snippet: SIV ddPCR DNA assay characterization. (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance. DNA sample used was duodenum DNA from Rhesus macaque 313–08. (B) Total number of droplets generated during dropletization remains constant in the range of tissue DNA amount tested (1 million to 8 million cell equivalent input). Total droplet number for each sample after dropletization serves as an additional indicator of sample DNA input tolerance. Note that for each level of DNA input, only a fraction (~1.6%) of the droplets were counted for QC purpose by the Source machine. (C) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines [ 7 ]. According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (D) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument. (E) Instead of measuring 60 ddPCR replicates to obtain 95% confidence, we obtained an approximate estimation of the LoD using a lower number of ddPCR reaction replicates and required the false negative rate to be below 5% (i.e. all 10 replicates have to be positive).

Techniques Used: Generated, Real-time Polymerase Chain Reaction, Software

ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.
Figure Legend Snippet: ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.

Techniques Used: Real-time Polymerase Chain Reaction, Inhibition, Derivative Assay, Negative Control, Standard Deviation

Identifying an optimal ddPCR assay format and condition. Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV (on CCR5 background) spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311–08) infected with SIVmac239. (C) A summary of all other tested conditions (probe and mastermix) and the observed issue(s) associated with each condition.
Figure Legend Snippet: Identifying an optimal ddPCR assay format and condition. Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV (on CCR5 background) spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311–08) infected with SIVmac239. (C) A summary of all other tested conditions (probe and mastermix) and the observed issue(s) associated with each condition.

Techniques Used: Infection

SIV ddPCR DNA assay performance. (A) SIV ddPCR DNA assay detection of low single digit level SIV DNA input and linear dynamic range of the SIV ddPCR DNA assay. Different amount of SIV DNA template was spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. SIV DNA amount was quantified by direct ddPCR. (B) Quantification data of Fig 5A.
Figure Legend Snippet: SIV ddPCR DNA assay performance. (A) SIV ddPCR DNA assay detection of low single digit level SIV DNA input and linear dynamic range of the SIV ddPCR DNA assay. Different amount of SIV DNA template was spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. SIV DNA amount was quantified by direct ddPCR. (B) Quantification data of Fig 5A.

Techniques Used:

4) Product Images from "The Transcription Factor Rbf1 Is the Master Regulator for b-Mating Type Controlled Pathogenic Development in Ustilago maydis"

Article Title: The Transcription Factor Rbf1 Is the Master Regulator for b-Mating Type Controlled Pathogenic Development in Ustilago maydis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001035

Structure and expression of Rbf1. ( A ) Microarray analysis of b -dependent rbf1 expression after induction of compatible (AB31 and AB33) and incompatible (AB32 and AB34) combinations of bE and bW . Shown are the mean expression values of two biological replicates and the standard deviation (SD) ( B ) qRT-PCR analysis of rbf1 expression after induction of compatible (AB31) and incompatible (AB32) combinations of bE and bW . Samples were taken at the time-points indicated. qRT-PCR analysis was performed using the constitutively expressed ppi gene ( um03726 ) for normalization. Expression was calculated relative to the lowest expression value. Shown are mean values of two technical replicates. ( C ) Overview of primer binding sites in the rbf1 -promoter used for qChIP experiments and alignment of three putative b-binding sites ( bbs ) in the rbf1 promoter region to the bbs of lga2 and frb52 [10] , [11] . Nucleotide positions indicated are relative to the start codon. Nucleotides identical to the bbs in lga2 [10] and frb52 [11] are in bold. ( D ) qChIP analysis of bE1 binding to the rbf1 -promoter in strains AB31 and AB31bE1:3xHA 5h after induction of the bE1/bW2-heterodimer. AB31bE1:3xHA harbours a HA-tagged bE1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody. No significant enrichment was observed in control strain AB31. In AB31bE1:3xHA, the PCR-amplicon spanning bbs1 (bbs −1377 ) is significantly enriched (t-test) when compared to the amplicon spanning a control region (−2026) (p = 5.71 10 −5 ). As additional control, a region from the eIF2b gene ( um04869 ) was used. Given are the mean values of three technical replicates of three independent experiments each, and the standard deviation (SD). ( E ) Structure of the Rbf1 protein. The potential C2H2 zinc finger domain (aa 18 to 131) and a putative NLS (RHRR) (aa 95 to 98) within this domain are marked in dark grey and black, a glutamine rich sequence (aa 365 to 373) is marked in grey. The alignment shows the four C2H2 zinc finger domains; the conserved cysteine and histidine residues are in bold. ( F ) Subcellular localization of the Rbf1-3xeGFP fusion protein. Strain AB31 rbf1:3eGFP (UMS63) was induced in CM medium supplemented with 1% arabinose (CMA) for eight hours. The functional Rbf1-3xeGFP fusion protein localizes to the nucleus. Cells were stained with DAPI to visualize nuclei. Scale bar corresponds to 10 µm.
Figure Legend Snippet: Structure and expression of Rbf1. ( A ) Microarray analysis of b -dependent rbf1 expression after induction of compatible (AB31 and AB33) and incompatible (AB32 and AB34) combinations of bE and bW . Shown are the mean expression values of two biological replicates and the standard deviation (SD) ( B ) qRT-PCR analysis of rbf1 expression after induction of compatible (AB31) and incompatible (AB32) combinations of bE and bW . Samples were taken at the time-points indicated. qRT-PCR analysis was performed using the constitutively expressed ppi gene ( um03726 ) for normalization. Expression was calculated relative to the lowest expression value. Shown are mean values of two technical replicates. ( C ) Overview of primer binding sites in the rbf1 -promoter used for qChIP experiments and alignment of three putative b-binding sites ( bbs ) in the rbf1 promoter region to the bbs of lga2 and frb52 [10] , [11] . Nucleotide positions indicated are relative to the start codon. Nucleotides identical to the bbs in lga2 [10] and frb52 [11] are in bold. ( D ) qChIP analysis of bE1 binding to the rbf1 -promoter in strains AB31 and AB31bE1:3xHA 5h after induction of the bE1/bW2-heterodimer. AB31bE1:3xHA harbours a HA-tagged bE1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody. No significant enrichment was observed in control strain AB31. In AB31bE1:3xHA, the PCR-amplicon spanning bbs1 (bbs −1377 ) is significantly enriched (t-test) when compared to the amplicon spanning a control region (−2026) (p = 5.71 10 −5 ). As additional control, a region from the eIF2b gene ( um04869 ) was used. Given are the mean values of three technical replicates of three independent experiments each, and the standard deviation (SD). ( E ) Structure of the Rbf1 protein. The potential C2H2 zinc finger domain (aa 18 to 131) and a putative NLS (RHRR) (aa 95 to 98) within this domain are marked in dark grey and black, a glutamine rich sequence (aa 365 to 373) is marked in grey. The alignment shows the four C2H2 zinc finger domains; the conserved cysteine and histidine residues are in bold. ( F ) Subcellular localization of the Rbf1-3xeGFP fusion protein. Strain AB31 rbf1:3eGFP (UMS63) was induced in CM medium supplemented with 1% arabinose (CMA) for eight hours. The functional Rbf1-3xeGFP fusion protein localizes to the nucleus. Cells were stained with DAPI to visualize nuclei. Scale bar corresponds to 10 µm.

Techniques Used: Expressing, Microarray, Standard Deviation, Quantitative RT-PCR, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction, Amplification, T-Test, Sequencing, Functional Assay, Staining

Rbf1 binds to the promoter of the Rbf1-dependently expressed dik6 gene. (A) Overview of the dik6 promoter region investigated by qChIP in strain AB31 rbf1:3xHA . Shown are the positions of the amplicons used for qChIP (numbered from 1 to 10) and the promoter truncations and internal deletions assayed in the GFP-reporter assay. (B) qChIP analysis of Rbf1-binding to the dik6 -promoter in strains AB31 and AB31 rbf1:3xHA 5h after induction of the bE1/bW2-heterodimer in CMA. AB31 rbf1:3xHA expresses an HA-tagged Rbf1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody Start and end of the amplicons are given in nucleotides (nt) relative to the start codon of dik6 . The relative positions of the amplicons are given in (A). As additional control for qChIP, a region from the eIF2b gene ( um04869 ) was used. Given are the mean values of two technical replicates of three independent experiments each, and the standard deviation (SD). Significance of the difference to values obtained for the control-amplicon located within the dik6 open reading frame (amplicon 11) was calculated using Students t-test; the respective p-values are given for AB31 rbf1:3xHA . Highest enrichment was observed for amplicons 3, 4, 5 and 6. No significant enrichment was observed in control strain AB31. (C) The dik6 promoter fragments outlined in (A) were fused to GFP as a reporter and integrated in single copy into the ip -locus of U. maydis strain CP27 ( a2 Δb::P crg1 :rbf1 ). GFP-expression was visualized microscopically 5 hours after induction of rbf1 expression in CMA medium. GFP expression declined when the promoter was truncated from 816 bp to 638 bp, and was abolished when a 298 bp promoter fragment was used. Similarly, the internal deletion Δ3 led to reduced GFP expression, while no GFP signal was detectable in the Δ 5 deletion. Scale bar = 20 µm.
Figure Legend Snippet: Rbf1 binds to the promoter of the Rbf1-dependently expressed dik6 gene. (A) Overview of the dik6 promoter region investigated by qChIP in strain AB31 rbf1:3xHA . Shown are the positions of the amplicons used for qChIP (numbered from 1 to 10) and the promoter truncations and internal deletions assayed in the GFP-reporter assay. (B) qChIP analysis of Rbf1-binding to the dik6 -promoter in strains AB31 and AB31 rbf1:3xHA 5h after induction of the bE1/bW2-heterodimer in CMA. AB31 rbf1:3xHA expresses an HA-tagged Rbf1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody Start and end of the amplicons are given in nucleotides (nt) relative to the start codon of dik6 . The relative positions of the amplicons are given in (A). As additional control for qChIP, a region from the eIF2b gene ( um04869 ) was used. Given are the mean values of two technical replicates of three independent experiments each, and the standard deviation (SD). Significance of the difference to values obtained for the control-amplicon located within the dik6 open reading frame (amplicon 11) was calculated using Students t-test; the respective p-values are given for AB31 rbf1:3xHA . Highest enrichment was observed for amplicons 3, 4, 5 and 6. No significant enrichment was observed in control strain AB31. (C) The dik6 promoter fragments outlined in (A) were fused to GFP as a reporter and integrated in single copy into the ip -locus of U. maydis strain CP27 ( a2 Δb::P crg1 :rbf1 ). GFP-expression was visualized microscopically 5 hours after induction of rbf1 expression in CMA medium. GFP expression declined when the promoter was truncated from 816 bp to 638 bp, and was abolished when a 298 bp promoter fragment was used. Similarly, the internal deletion Δ3 led to reduced GFP expression, while no GFP signal was detectable in the Δ 5 deletion. Scale bar = 20 µm.

Techniques Used: Reporter Assay, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction, Standard Deviation, Amplification, Expressing

5) Product Images from "Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette"

Article Title: Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv124

Loading of the tTA VP64 - containing construct into the alphoid tetO -HAC propagated in human HPRT-minus HT1080 cells. ( a ) Diagram of the construct tetR-VP64-IRES-DsRed2 used in this study. The construct contains a 3′ HPRT sequence and loxP site, the tTA VP64 (tetR with the VP64 carrying four copies of VP16 activation domain) that is co-transcribed with the DsRed2 transgene under the same CAG promoter. The cHS4 insulator ( 54 ) flanks the expressing cassette from both sides. (A similar construct, tetR*-VP16-IRES-DsRed2, carrying one copy of the VP16 domain is described in Supplementary Figure S2). Both constructs were inserted into the loxP site of the alphoid tetO -HAC propagated in HPRT-deficient HT1080 cells. ( b ) Loading of the vector into the loxP site of the HAC is accompanied by reconstitution of the HPRT gene allowing cell selection on HAT medium. ( c ) A map of the resulting transgene cluster in the HAC containing TK and Hygro genes, the DsRed2 color marker and the reconstructed HPRT gene. Arrows indicate direction of transcription of the transgenes and 30–40 copies of BS incorporated in the alphoid tetO -HAC. ( d ) Lanes 1, 2 and 3 correspond to PCR products obtained with the genomic DNA isolated from HAC-containing clones of HT1080 cells using specific primers for the HPRT gene. The PCR products were sequenced and confirmed reconstitution of the HPRT gene. M-ladder marker. ( e – g ) FISH analysis of the HAC-containing HT1080 clone. Chromosomal DNA was counterstained with DAPI (blue) (e). The HAC was visualized using a BAC32–2-mer(tetO) probe (red) (f and g).
Figure Legend Snippet: Loading of the tTA VP64 - containing construct into the alphoid tetO -HAC propagated in human HPRT-minus HT1080 cells. ( a ) Diagram of the construct tetR-VP64-IRES-DsRed2 used in this study. The construct contains a 3′ HPRT sequence and loxP site, the tTA VP64 (tetR with the VP64 carrying four copies of VP16 activation domain) that is co-transcribed with the DsRed2 transgene under the same CAG promoter. The cHS4 insulator ( 54 ) flanks the expressing cassette from both sides. (A similar construct, tetR*-VP16-IRES-DsRed2, carrying one copy of the VP16 domain is described in Supplementary Figure S2). Both constructs were inserted into the loxP site of the alphoid tetO -HAC propagated in HPRT-deficient HT1080 cells. ( b ) Loading of the vector into the loxP site of the HAC is accompanied by reconstitution of the HPRT gene allowing cell selection on HAT medium. ( c ) A map of the resulting transgene cluster in the HAC containing TK and Hygro genes, the DsRed2 color marker and the reconstructed HPRT gene. Arrows indicate direction of transcription of the transgenes and 30–40 copies of BS incorporated in the alphoid tetO -HAC. ( d ) Lanes 1, 2 and 3 correspond to PCR products obtained with the genomic DNA isolated from HAC-containing clones of HT1080 cells using specific primers for the HPRT gene. The PCR products were sequenced and confirmed reconstitution of the HPRT gene. M-ladder marker. ( e – g ) FISH analysis of the HAC-containing HT1080 clone. Chromosomal DNA was counterstained with DAPI (blue) (e). The HAC was visualized using a BAC32–2-mer(tetO) probe (red) (f and g).

Techniques Used: Construct, HAC Assay, Sequencing, Activation Assay, Expressing, Plasmid Preparation, Selection, HAT Assay, Chromosome Transmission Fidelity Colony Color Assay, Polymerase Chain Reaction, Isolation, Clone Assay, Marker, Fluorescence In Situ Hybridization

Chromatin/transcription changes in the alphoid tetO -HAC kinetochore induced by tTA VP64 tethering. ( a and b ) De-repression of tetO-alpha-satellite DNA transcription in the HAC when cells are grown in the medium lacking doxycycline. Quantitative RT-PCR experiments for two clones, clone #1 and clone #2, showing that the transcripts of non-coding tetO-alpha-satellite DNA repeats in the alphoid tetO -HAC are significantly repressed in the cells growing in doxycycline-containing medium compared to that of the cells that have been grown in the medium lacking doxycycline. ( c and d ) Chromatin immunoprecipitation (ChIP) analysis of non-coding tetO-alpha-satellite DNA repeats in the alphoid tetO -HAC in two clones using antibodies against H3K4me3 in the cells growing in doxycycline-containing medium compared to that of the cells that have been grown in the medium lacking doxycycline after 1 and 3 days of culture. Data were normalized to the internal 5S rDNA controls. ( e ) ChIP analysis of CENP-A chromatin in the alphoid tetO -HAC in the clone #2 cultured in the presence and absence of doxycycline for different time intervals (3, 4, 8 and 12 days of culture). Enrichment is shown relative to the chromosome 21 centromere. Error bars indicate s.d. The significant differences were calculated using Student's two-tailed t -test.
Figure Legend Snippet: Chromatin/transcription changes in the alphoid tetO -HAC kinetochore induced by tTA VP64 tethering. ( a and b ) De-repression of tetO-alpha-satellite DNA transcription in the HAC when cells are grown in the medium lacking doxycycline. Quantitative RT-PCR experiments for two clones, clone #1 and clone #2, showing that the transcripts of non-coding tetO-alpha-satellite DNA repeats in the alphoid tetO -HAC are significantly repressed in the cells growing in doxycycline-containing medium compared to that of the cells that have been grown in the medium lacking doxycycline. ( c and d ) Chromatin immunoprecipitation (ChIP) analysis of non-coding tetO-alpha-satellite DNA repeats in the alphoid tetO -HAC in two clones using antibodies against H3K4me3 in the cells growing in doxycycline-containing medium compared to that of the cells that have been grown in the medium lacking doxycycline after 1 and 3 days of culture. Data were normalized to the internal 5S rDNA controls. ( e ) ChIP analysis of CENP-A chromatin in the alphoid tetO -HAC in the clone #2 cultured in the presence and absence of doxycycline for different time intervals (3, 4, 8 and 12 days of culture). Enrichment is shown relative to the chromosome 21 centromere. Error bars indicate s.d. The significant differences were calculated using Student's two-tailed t -test.

Techniques Used: HAC Assay, Quantitative RT-PCR, Clone Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

6) Product Images from "Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays"

Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

Journal: PLoS ONE

doi: 10.1371/journal.pone.0233085

ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.
Figure Legend Snippet: ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.

Techniques Used: Real-time Polymerase Chain Reaction, Migration, Concentration Assay

Comparison between ddPCR and qPCR quantitation results in low viral DNA range
Figure Legend Snippet: Comparison between ddPCR and qPCR quantitation results in low viral DNA range

Techniques Used: Real-time Polymerase Chain Reaction, Quantitation Assay

SIV ddPCR DNA assay characterization. (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance. DNA sample used was duodenum DNA from Rhesus macaque 313–08. (B) Total number of droplets generated during dropletization remains constant in the range of tissue DNA amount tested (1 million to 8 million cell equivalent input). Total droplet number for each sample after dropletization serves as an additional indicator of sample DNA input tolerance. Note that for each level of DNA input, only a fraction (~1.6%) of the droplets were counted for QC purpose by the Source machine. (C) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines [ 7 ]. According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (D) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument. (E) Instead of measuring 60 ddPCR replicates to obtain 95% confidence, we obtained an approximate estimation of the LoD using a lower number of ddPCR reaction replicates and required the false negative rate to be below 5% (i.e. all 10 replicates have to be positive).
Figure Legend Snippet: SIV ddPCR DNA assay characterization. (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance. DNA sample used was duodenum DNA from Rhesus macaque 313–08. (B) Total number of droplets generated during dropletization remains constant in the range of tissue DNA amount tested (1 million to 8 million cell equivalent input). Total droplet number for each sample after dropletization serves as an additional indicator of sample DNA input tolerance. Note that for each level of DNA input, only a fraction (~1.6%) of the droplets were counted for QC purpose by the Source machine. (C) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines [ 7 ]. According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (D) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument. (E) Instead of measuring 60 ddPCR replicates to obtain 95% confidence, we obtained an approximate estimation of the LoD using a lower number of ddPCR reaction replicates and required the false negative rate to be below 5% (i.e. all 10 replicates have to be positive).

Techniques Used: Generated, Real-time Polymerase Chain Reaction, Software

ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.
Figure Legend Snippet: ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.

Techniques Used: Real-time Polymerase Chain Reaction, Inhibition, Derivative Assay, Negative Control, Standard Deviation

Identifying an optimal ddPCR assay format and condition. Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV (on CCR5 background) spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311–08) infected with SIVmac239. (C) A summary of all other tested conditions (probe and mastermix) and the observed issue(s) associated with each condition.
Figure Legend Snippet: Identifying an optimal ddPCR assay format and condition. Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV (on CCR5 background) spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311–08) infected with SIVmac239. (C) A summary of all other tested conditions (probe and mastermix) and the observed issue(s) associated with each condition.

Techniques Used: Infection

SIV ddPCR DNA assay performance. (A) SIV ddPCR DNA assay detection of low single digit level SIV DNA input and linear dynamic range of the SIV ddPCR DNA assay. Different amount of SIV DNA template was spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. SIV DNA amount was quantified by direct ddPCR. (B) Quantification data of Fig 5A.
Figure Legend Snippet: SIV ddPCR DNA assay performance. (A) SIV ddPCR DNA assay detection of low single digit level SIV DNA input and linear dynamic range of the SIV ddPCR DNA assay. Different amount of SIV DNA template was spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. SIV DNA amount was quantified by direct ddPCR. (B) Quantification data of Fig 5A.

Techniques Used:

7) Product Images from "Comparing effects of CDK inhibition and E2F1/2 ablation on neuronal cell death pathways in vitro and after traumatic brain injury"

Article Title: Comparing effects of CDK inhibition and E2F1/2 ablation on neuronal cell death pathways in vitro and after traumatic brain injury

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1156-y

CR8 attenuates activation of the p53-linked pro-apoptotic pathways following etoposide-induced DNA damage at the mRNA level. Neurons were treated with 50 μm of etoposide ± 1 μm CR8. Neurons were collected 24 h after treatment. qPCR quantification of expression of a Noxa, p21, Puma, Apaf-1, and Mcl-1; b promotor region of Noxa and p21; c miR-711 and miR-23a in primary cortical neurons at different time points after treatment. Results of qPCR were normalized to a GAPDH expression; b input DNA; and c U6 snRNA. CR8 attenuates relative expression of PUMA, NOXA, and p21 following 50 μm etoposide treatment ( a ). No change in Apaf-1 relative to controls was observed until 24 h ( a ). Etoposide induced increases in occupancy of p53 in the promoter region of Noxa, and p21 was attenuated by etoposide + CR8 ( b ). n = 3/group for all groups. Etoposide and etoposide + CR8 increased expression of miR-711 and decreased miR-23a compared to control neurons ( c ). n = 6/group for all groups. Data represent mean ± SEM of one-way ANOVA and Tukey post hoc analysis, * p
Figure Legend Snippet: CR8 attenuates activation of the p53-linked pro-apoptotic pathways following etoposide-induced DNA damage at the mRNA level. Neurons were treated with 50 μm of etoposide ± 1 μm CR8. Neurons were collected 24 h after treatment. qPCR quantification of expression of a Noxa, p21, Puma, Apaf-1, and Mcl-1; b promotor region of Noxa and p21; c miR-711 and miR-23a in primary cortical neurons at different time points after treatment. Results of qPCR were normalized to a GAPDH expression; b input DNA; and c U6 snRNA. CR8 attenuates relative expression of PUMA, NOXA, and p21 following 50 μm etoposide treatment ( a ). No change in Apaf-1 relative to controls was observed until 24 h ( a ). Etoposide induced increases in occupancy of p53 in the promoter region of Noxa, and p21 was attenuated by etoposide + CR8 ( b ). n = 3/group for all groups. Etoposide and etoposide + CR8 increased expression of miR-711 and decreased miR-23a compared to control neurons ( c ). n = 6/group for all groups. Data represent mean ± SEM of one-way ANOVA and Tukey post hoc analysis, * p

Techniques Used: Activation Assay, Real-time Polymerase Chain Reaction, Expressing

8) Product Images from "Zinc knuckle of TAF1 is a DNA binding module critical for TFIID promoter occupancy"

Article Title: Zinc knuckle of TAF1 is a DNA binding module critical for TFIID promoter occupancy

Journal: Scientific Reports

doi: 10.1038/s41598-018-22879-5

TAF1 zinc knuckle binds DNA. ( A,B,C ) EMSA of TAF1 ZnA (aa 1234–1375) and three radiolabeled DNA fragments: IMD of super core promoter (position −6 to +38), cyclin D1 promoter (position −22 to +29, CD1P), and Random DNA sequence. ( D,E,F ) Bio-layer interferometry binding curves using biotinylated double-stranded DNA fragments described in above and the following ZnA protein concentrations: 3 μM, 1 μM, 333 nM, 111 nM, 37 nM, 12 nM. Raw data was plotted with GraphPAD Prism. K d was calculated from plotting steady-state binding levels against protein concentration.
Figure Legend Snippet: TAF1 zinc knuckle binds DNA. ( A,B,C ) EMSA of TAF1 ZnA (aa 1234–1375) and three radiolabeled DNA fragments: IMD of super core promoter (position −6 to +38), cyclin D1 promoter (position −22 to +29, CD1P), and Random DNA sequence. ( D,E,F ) Bio-layer interferometry binding curves using biotinylated double-stranded DNA fragments described in above and the following ZnA protein concentrations: 3 μM, 1 μM, 333 nM, 111 nM, 37 nM, 12 nM. Raw data was plotted with GraphPAD Prism. K d was calculated from plotting steady-state binding levels against protein concentration.

Techniques Used: Radial Immuno Diffusion, Sequencing, Binding Assay, Protein Concentration

Core Module and Key Residues in TAF1 Zinc Knuckle DNA Binding Domain. ( A ) TAF1 (aa 1234–1375) was incubated without (upper) and with (lower) IMD promoter DNA followed by digestion with increasing concentrations of protease. Digestion products were resolved by SDS-PAGE and detected by coomassie blue staining. Arrowheads indicate fragments stabilized by DNA. Quantification of protein fragments are provided in Supplemental Figure S3 . ( B ) EVfold map analysis of ZnA. Numbers represent amino acid residues of full-length TAF1. ( C ) Diagram of DNA stabilized regions of TAF1 with blue box indicating CCHC ZnK. DNA binding curves for ( D ) ZnC wild type, ( E ) ZnC cysteine mutant (C1285A and C1288A), ( F ) ZnC charge mutant (R1295A and K1298A), ( G ) ZnD wild type, ( H ) ZnD cysteine mutant, and ( I ) ZnD charge mutant. Raw data was plotted with GraphPAD Prism. K d was calculated by plotting steady-state binding levels against protein concentration and determining the concentration needed for half maximal binding.
Figure Legend Snippet: Core Module and Key Residues in TAF1 Zinc Knuckle DNA Binding Domain. ( A ) TAF1 (aa 1234–1375) was incubated without (upper) and with (lower) IMD promoter DNA followed by digestion with increasing concentrations of protease. Digestion products were resolved by SDS-PAGE and detected by coomassie blue staining. Arrowheads indicate fragments stabilized by DNA. Quantification of protein fragments are provided in Supplemental Figure S3 . ( B ) EVfold map analysis of ZnA. Numbers represent amino acid residues of full-length TAF1. ( C ) Diagram of DNA stabilized regions of TAF1 with blue box indicating CCHC ZnK. DNA binding curves for ( D ) ZnC wild type, ( E ) ZnC cysteine mutant (C1285A and C1288A), ( F ) ZnC charge mutant (R1295A and K1298A), ( G ) ZnD wild type, ( H ) ZnD cysteine mutant, and ( I ) ZnD charge mutant. Raw data was plotted with GraphPAD Prism. K d was calculated by plotting steady-state binding levels against protein concentration and determining the concentration needed for half maximal binding.

Techniques Used: Binding Assay, Incubation, Radial Immuno Diffusion, SDS Page, Staining, Mutagenesis, Protein Concentration, Concentration Assay

9) Product Images from "PRL-3 promotes telomere deprotection and chromosomal instability"

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx392

Silencing of PRL-3 promotes DDR and senescence. ( A ) Efficiencies of PRL-3 silencing in HCT116 (knockdown by two shRNAs using lentivirus system, left) and SW480 (knockout by CRISPR/Cas9 system, right) cells and its effects on indicated protein levels. WT, wild-type. KO, knockout. ( B ) Effects of PRL-3 silencing on phosphorylations of H2AX and CHK1. Samples treated with 20 μM etoposide (ETP) for 4 h were used as positive controls. ( C ) Effects of PRL-3 silencing on TIF formation. Indicated HCT116 cells were subjected to IF-FISH staining. Upper, representative staining. Arrows, colocalizations between γH2AX and telomere (TIFs). Scale bar, 5 μm. Lower, quantification of cells with ≥5 TIF. Mean ± SD of two independent experiments. n > 200 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 silencing on anaphase bridges (APB) and micronuclei (MN) formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. n > 1000 cells scored per sample for MN and n > 50 anaphase cells scored per sample for APB. Student's t -test. Representative images of APB (red arrow) and MN (white arrow) of HCT116 cells stained with DAPI were shown. ( E ) ChIP analysis of RAP1 and TRF2's binding to telomeric or Alu DNA in HCT116 and S480 cells silenced for PRL-3. Upper, representative blots after ChIP with indicated antibodies or IgG. Input, 2% DNA. Lower, quantification of relative OD. Relative OD was calculated by normalizing to that of input and relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( F ) PRL-3 silencing induced ROS-dependent cellular senescence and DNA damage response. Indica ted HCT116 cells were treated with NAC (10 mM), GSH (10 mM) or DMSO (1:1000) for 24 h. Part of cells were fixed and processed for β-galactosidase staining, others were analyzed by western blot. Upper, representative β-galactosidase staining of cells treated with DMSO. Middle, quantification of β-galactosidase positive cells. Mean ± SD of two independent experiments. n > 400 cells per single experiment. Student's t -test. Lower, western blot of γH2AX.
Figure Legend Snippet: Silencing of PRL-3 promotes DDR and senescence. ( A ) Efficiencies of PRL-3 silencing in HCT116 (knockdown by two shRNAs using lentivirus system, left) and SW480 (knockout by CRISPR/Cas9 system, right) cells and its effects on indicated protein levels. WT, wild-type. KO, knockout. ( B ) Effects of PRL-3 silencing on phosphorylations of H2AX and CHK1. Samples treated with 20 μM etoposide (ETP) for 4 h were used as positive controls. ( C ) Effects of PRL-3 silencing on TIF formation. Indicated HCT116 cells were subjected to IF-FISH staining. Upper, representative staining. Arrows, colocalizations between γH2AX and telomere (TIFs). Scale bar, 5 μm. Lower, quantification of cells with ≥5 TIF. Mean ± SD of two independent experiments. n > 200 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 silencing on anaphase bridges (APB) and micronuclei (MN) formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. n > 1000 cells scored per sample for MN and n > 50 anaphase cells scored per sample for APB. Student's t -test. Representative images of APB (red arrow) and MN (white arrow) of HCT116 cells stained with DAPI were shown. ( E ) ChIP analysis of RAP1 and TRF2's binding to telomeric or Alu DNA in HCT116 and S480 cells silenced for PRL-3. Upper, representative blots after ChIP with indicated antibodies or IgG. Input, 2% DNA. Lower, quantification of relative OD. Relative OD was calculated by normalizing to that of input and relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( F ) PRL-3 silencing induced ROS-dependent cellular senescence and DNA damage response. Indica ted HCT116 cells were treated with NAC (10 mM), GSH (10 mM) or DMSO (1:1000) for 24 h. Part of cells were fixed and processed for β-galactosidase staining, others were analyzed by western blot. Upper, representative β-galactosidase staining of cells treated with DMSO. Middle, quantification of β-galactosidase positive cells. Mean ± SD of two independent experiments. n > 400 cells per single experiment. Student's t -test. Lower, western blot of γH2AX.

Techniques Used: Knock-Out, CRISPR, Fluorescence In Situ Hybridization, Staining, Chromatin Immunoprecipitation, Binding Assay, Western Blot

RAP1 and TRF2-dependent recruitment of PRL-3 to telomere. ( A ) In situ PLA analysis of PRL-3's associations with RAP1 and TRF2. HCT116 cells were pre-extracted, fixed, inmunostained with indicated pairs of antibodies and probed with Duolink in situ PLA reagent. Binding foci were in red and dashed lines indicated outline of nucleus (determined by DAPI counter staining). Scale bar, 10 μm. ( B ) TRF2- and RAP1-dependent recruitment of PRL-3 to telomeric DNA in vitro . Purified myc-TRF2 (150 ng), His-RAP1 (120 ng), and His-PRL-3 (30 ng) were co-incubated with 1 μg biotin-labeled telomere (lanes 1–4) or Alu (lanes 5–8) probe as indicated and subjected to pull-down analysis with Streptavidin agarose. Precipitates were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( C and D ) TRF2 and RAP1-dependent recruitment of PRL-3 to telomere in cells. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h, pre-extracted, fixed and subjected to IF-FISH staining. (C) Representative PRL-3 association with telomere. Scale bar, 10 μm. Areas in white squares were enlarged. (D) Quantification of cells with ≥5 associations between PRL-3 foci and telomere. Mean ± SD of three independent experiments. n > 100 cells per single experiment. Student's t -test. ( E ) Knockdown efficiencies of RAP1 and TRF2. HCT116 cells were transfected with 50 nM siRNAs against RAP1 or TRF2 for 48 h. Lysates were analyzed by western blot with indicated antibodies. ( F ) ChIP analysis of PRL-3 binding to telomeric and Alu DNA. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h and processed for ChIP using anti-PRL-3 or pre-immune IgG. Upper, representative blots of hybridization with probe to telomere or Alu. Input, 2% DNA. Lower, quantification of relative optical densities (OD). Relative OD was calculated by normalizing to OD of Input and relative OD of control siRNA-transfected sample was set as 100%. Mean ± SD of three independent experiments. Student's t -test.
Figure Legend Snippet: RAP1 and TRF2-dependent recruitment of PRL-3 to telomere. ( A ) In situ PLA analysis of PRL-3's associations with RAP1 and TRF2. HCT116 cells were pre-extracted, fixed, inmunostained with indicated pairs of antibodies and probed with Duolink in situ PLA reagent. Binding foci were in red and dashed lines indicated outline of nucleus (determined by DAPI counter staining). Scale bar, 10 μm. ( B ) TRF2- and RAP1-dependent recruitment of PRL-3 to telomeric DNA in vitro . Purified myc-TRF2 (150 ng), His-RAP1 (120 ng), and His-PRL-3 (30 ng) were co-incubated with 1 μg biotin-labeled telomere (lanes 1–4) or Alu (lanes 5–8) probe as indicated and subjected to pull-down analysis with Streptavidin agarose. Precipitates were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( C and D ) TRF2 and RAP1-dependent recruitment of PRL-3 to telomere in cells. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h, pre-extracted, fixed and subjected to IF-FISH staining. (C) Representative PRL-3 association with telomere. Scale bar, 10 μm. Areas in white squares were enlarged. (D) Quantification of cells with ≥5 associations between PRL-3 foci and telomere. Mean ± SD of three independent experiments. n > 100 cells per single experiment. Student's t -test. ( E ) Knockdown efficiencies of RAP1 and TRF2. HCT116 cells were transfected with 50 nM siRNAs against RAP1 or TRF2 for 48 h. Lysates were analyzed by western blot with indicated antibodies. ( F ) ChIP analysis of PRL-3 binding to telomeric and Alu DNA. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h and processed for ChIP using anti-PRL-3 or pre-immune IgG. Upper, representative blots of hybridization with probe to telomere or Alu. Input, 2% DNA. Lower, quantification of relative optical densities (OD). Relative OD was calculated by normalizing to OD of Input and relative OD of control siRNA-transfected sample was set as 100%. Mean ± SD of three independent experiments. Student's t -test.

Techniques Used: In Situ, Proximity Ligation Assay, Binding Assay, Staining, In Vitro, Purification, Incubation, Labeling, Western Blot, Transfection, Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Hybridization

PRL-3 relocates RAP1 and TRF2 from telomeric DNA. ( A ) Effects of PRL-3 stable overexpression on the chromatin abundance of RAP1, TRF2 and TRF1. Nuclei from HCT116 cells were homogenized in buffer containing indicated concentrations of NaCl. Chromatin-enriched fractions were analyzed by western blot. Left, representative blots. Right, relative levels of TRF2, RAP1 and TRF1. Protein band were scanned and relative OD was calculated by normalizing to OD of H2B. The relative OD of sample prepared with 150 mM NaCl was set as 100%. Mean ± SD of three independent experiments. ANOVA. ( B ) Effects of PRL-3 stable overexpression on bindings of RAP1 and TRF2 to telomeric and Alu DNA. Indicated cells were crosslinked, immunoprecipitated with antibodies to RAP1, TRF2 or pre-immune IgG, and precipitated DNA was analyzed by ChIP. Upper, representative blots. Lower, quantification of relative OD, which was calculated by normalizing to that of Input. Relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on telomere associations of RAP1 and TRF2 in WI38 cells. Left, representative IF-FISH staining of telomere (red) and RAP1 or TRF2 (green). Arrows, foci of co-localization. Scale bar, 10 μm. Right, quantification of cells with ≥5 associations between RAP1 or TRF2 foci and telomere. Mean ± SD of two independent experiments. n > 80 cells per single experiment. Student's t -test. ( D ) EMSA analysis of PRL-3, RAP1 and TRF2's associations with telomeric DNA. Indicated concentrations of purified FLAG-TRF2, His-RAP1, myc-PRL-3 were co-incubated with Biotin-labeled telomere probe (20 nM). To induce super-shift, 0.1 μg anti-PRL-3 (lane 5), anti-TRF2 (lanes 6 and 18) and IgG (lane 7) were used. Note that anti-PRL-3 and anti-TRF2-induced super-shifts of Complex II partially co-migrated with Complex I (lanes 5 and 6).
Figure Legend Snippet: PRL-3 relocates RAP1 and TRF2 from telomeric DNA. ( A ) Effects of PRL-3 stable overexpression on the chromatin abundance of RAP1, TRF2 and TRF1. Nuclei from HCT116 cells were homogenized in buffer containing indicated concentrations of NaCl. Chromatin-enriched fractions were analyzed by western blot. Left, representative blots. Right, relative levels of TRF2, RAP1 and TRF1. Protein band were scanned and relative OD was calculated by normalizing to OD of H2B. The relative OD of sample prepared with 150 mM NaCl was set as 100%. Mean ± SD of three independent experiments. ANOVA. ( B ) Effects of PRL-3 stable overexpression on bindings of RAP1 and TRF2 to telomeric and Alu DNA. Indicated cells were crosslinked, immunoprecipitated with antibodies to RAP1, TRF2 or pre-immune IgG, and precipitated DNA was analyzed by ChIP. Upper, representative blots. Lower, quantification of relative OD, which was calculated by normalizing to that of Input. Relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on telomere associations of RAP1 and TRF2 in WI38 cells. Left, representative IF-FISH staining of telomere (red) and RAP1 or TRF2 (green). Arrows, foci of co-localization. Scale bar, 10 μm. Right, quantification of cells with ≥5 associations between RAP1 or TRF2 foci and telomere. Mean ± SD of two independent experiments. n > 80 cells per single experiment. Student's t -test. ( D ) EMSA analysis of PRL-3, RAP1 and TRF2's associations with telomeric DNA. Indicated concentrations of purified FLAG-TRF2, His-RAP1, myc-PRL-3 were co-incubated with Biotin-labeled telomere probe (20 nM). To induce super-shift, 0.1 μg anti-PRL-3 (lane 5), anti-TRF2 (lanes 6 and 18) and IgG (lane 7) were used. Note that anti-PRL-3 and anti-TRF2-induced super-shifts of Complex II partially co-migrated with Complex I (lanes 5 and 6).

Techniques Used: Over Expression, Western Blot, Immunoprecipitation, Chromatin Immunoprecipitation, Fluorescence In Situ Hybridization, Staining, Purification, Incubation, Labeling

10) Product Images from "Essential role of the iron-sulfur cluster binding domain of the primase regulatory subunit Pri2 in DNA replication initiation"

Article Title: Essential role of the iron-sulfur cluster binding domain of the primase regulatory subunit Pri2 in DNA replication initiation

Journal: Protein & Cell

doi: 10.1007/s13238-015-0134-8

Destabilized association between DNA polymerase α and early replication origins in the pri2 ( C336A , C417A , C474A ) mutant . Congenic wild-type ( POL1-Myc , PRI2-HA ) and mutant ( POL1-Myc , pri2 ( C336A , C417A , C474A ) -HA ) cells were synchronized in G 1 with α factor and released at 23°C. Cells were collected at the indicated time points for FACS (A) and ChIP (B) analyses as described in Fig. 3 . Pol1- and Pri2-associated chromatin was immunoprecipitated using anti-Myc and anti-HA monoclonal antibodies, respectively
Figure Legend Snippet: Destabilized association between DNA polymerase α and early replication origins in the pri2 ( C336A , C417A , C474A ) mutant . Congenic wild-type ( POL1-Myc , PRI2-HA ) and mutant ( POL1-Myc , pri2 ( C336A , C417A , C474A ) -HA ) cells were synchronized in G 1 with α factor and released at 23°C. Cells were collected at the indicated time points for FACS (A) and ChIP (B) analyses as described in Fig. 3 . Pol1- and Pri2-associated chromatin was immunoprecipitated using anti-Myc and anti-HA monoclonal antibodies, respectively

Techniques Used: Mutagenesis, FACS, Chromatin Immunoprecipitation, Immunoprecipitation

Impaired loading of both DNA primase subunits onto early replication origins in the pri2 ( C336A , C417A , C474A ) mutant . Congenic wild-type ( PRI1-Myc , PRI2-HA ) and mutant ( PRI1-Myc , pri2 ( C336A , C417A , C474A)-HA ) cells were synchronized in G 1 with α factor and released at 23°C. Cells were collected at the time points indicated. (A) FACS analysis indicates the DNA content of cells throughout the time course. (B) Chromatin-containing extracts were prepared from formaldehyde cross-linked cells collected at the indicated time points. Pri2-HA and Pri1-Myc were immunoprecipitated with anti-HA and anti-Myc monoclonal antibodies, respectively. The recovery efficiency of two early chromosomal replication origins, ARS305 and ARS607, in the immunoprecipitated material relative to the input material was determined by real-time PCR. Background was determined by calculating the amount of target DNA in the mock-IP sample relative to the Input sample (Beads). The results are an average of three independent experiments with standard deviations
Figure Legend Snippet: Impaired loading of both DNA primase subunits onto early replication origins in the pri2 ( C336A , C417A , C474A ) mutant . Congenic wild-type ( PRI1-Myc , PRI2-HA ) and mutant ( PRI1-Myc , pri2 ( C336A , C417A , C474A)-HA ) cells were synchronized in G 1 with α factor and released at 23°C. Cells were collected at the time points indicated. (A) FACS analysis indicates the DNA content of cells throughout the time course. (B) Chromatin-containing extracts were prepared from formaldehyde cross-linked cells collected at the indicated time points. Pri2-HA and Pri1-Myc were immunoprecipitated with anti-HA and anti-Myc monoclonal antibodies, respectively. The recovery efficiency of two early chromosomal replication origins, ARS305 and ARS607, in the immunoprecipitated material relative to the input material was determined by real-time PCR. Background was determined by calculating the amount of target DNA in the mock-IP sample relative to the Input sample (Beads). The results are an average of three independent experiments with standard deviations

Techniques Used: Mutagenesis, FACS, Immunoprecipitation, Real-time Polymerase Chain Reaction

11) Product Images from "Two Pif1 Family DNA Helicases Cooperate in Centromere Replication and Segregation in Saccharomyces cerevisiae"

Article Title: Two Pif1 Family DNA Helicases Cooperate in Centromere Replication and Segregation in Saccharomyces cerevisiae

Journal: Genetics

doi: 10.1534/genetics.118.301710

ScPif1 and Rrm3 binding to centromeres is cell cycle regulated. Cells, which were grown at 24° throughout the experiment, were arrested by incubation in α-factor and then released to proceed through the cell cycle. Samples were collected for ChIP-qPCR and FACS at indicated times ( T = 0, 15, 30, 45, 60, 75, 90, and 105 min). Immunoprecipitated DNA was purified and analyzed by qPCR. Data are presented as [(ChIP/Input) Target site /(ChIP/Input) YBL028C ]. Error bars are 1 SD from the average value of three independent experiments. (A) Rrm3 binding to CEN3 throughout a synchronous cell cycle in WT cells (red circles) or in pif1-m2 cells (purple diamonds). (B) ScPif1 binding to CEN3 throughout a synchronous cell cycle in WT cells (blue squares) or in rrm3 Δ cells (green triangles). (C) Same as (A) except Rrm3 binding is to CEN11. (D) Same as in (B) except that ScPif1 binding is to CEN11. (E) Same as (A) except Rrm3 binding is to CEN12. (F) Same as in (B) except that ScPif1 binding is to CEN12. ChIP, chromatin immunoprecipitation; qPCR, quantitative PCR; WT, wild-type.
Figure Legend Snippet: ScPif1 and Rrm3 binding to centromeres is cell cycle regulated. Cells, which were grown at 24° throughout the experiment, were arrested by incubation in α-factor and then released to proceed through the cell cycle. Samples were collected for ChIP-qPCR and FACS at indicated times ( T = 0, 15, 30, 45, 60, 75, 90, and 105 min). Immunoprecipitated DNA was purified and analyzed by qPCR. Data are presented as [(ChIP/Input) Target site /(ChIP/Input) YBL028C ]. Error bars are 1 SD from the average value of three independent experiments. (A) Rrm3 binding to CEN3 throughout a synchronous cell cycle in WT cells (red circles) or in pif1-m2 cells (purple diamonds). (B) ScPif1 binding to CEN3 throughout a synchronous cell cycle in WT cells (blue squares) or in rrm3 Δ cells (green triangles). (C) Same as (A) except Rrm3 binding is to CEN11. (D) Same as in (B) except that ScPif1 binding is to CEN11. (E) Same as (A) except Rrm3 binding is to CEN12. (F) Same as in (B) except that ScPif1 binding is to CEN12. ChIP, chromatin immunoprecipitation; qPCR, quantitative PCR; WT, wild-type.

Techniques Used: Binding Assay, Incubation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, FACS, Immunoprecipitation, Purification

12) Product Images from "Comparing effects of CDK inhibition and E2F1/2 ablation on neuronal cell death pathways in vitro and after traumatic brain injury"

Article Title: Comparing effects of CDK inhibition and E2F1/2 ablation on neuronal cell death pathways in vitro and after traumatic brain injury

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1156-y

CR8 attenuates activation of the p53-linked pro-apoptotic pathways following etoposide-induced DNA damage at the mRNA level. Neurons were treated with 50 μm of etoposide ± 1 μm CR8. Neurons were collected 24 h after treatment. qPCR quantification of expression of a Noxa, p21, Puma, Apaf-1, and Mcl-1; b promotor region of Noxa and p21; c miR-711 and miR-23a in primary cortical neurons at different time points after treatment. Results of qPCR were normalized to a GAPDH expression; b input DNA; and c U6 snRNA. CR8 attenuates relative expression of PUMA, NOXA, and p21 following 50 μm etoposide treatment ( a ). No change in Apaf-1 relative to controls was observed until 24 h ( a ). Etoposide induced increases in occupancy of p53 in the promoter region of Noxa, and p21 was attenuated by etoposide + CR8 ( b ). n = 3/group for all groups. Etoposide and etoposide + CR8 increased expression of miR-711 and decreased miR-23a compared to control neurons ( c ). n = 6/group for all groups. Data represent mean ± SEM of one-way ANOVA and Tukey post hoc analysis, * p
Figure Legend Snippet: CR8 attenuates activation of the p53-linked pro-apoptotic pathways following etoposide-induced DNA damage at the mRNA level. Neurons were treated with 50 μm of etoposide ± 1 μm CR8. Neurons were collected 24 h after treatment. qPCR quantification of expression of a Noxa, p21, Puma, Apaf-1, and Mcl-1; b promotor region of Noxa and p21; c miR-711 and miR-23a in primary cortical neurons at different time points after treatment. Results of qPCR were normalized to a GAPDH expression; b input DNA; and c U6 snRNA. CR8 attenuates relative expression of PUMA, NOXA, and p21 following 50 μm etoposide treatment ( a ). No change in Apaf-1 relative to controls was observed until 24 h ( a ). Etoposide induced increases in occupancy of p53 in the promoter region of Noxa, and p21 was attenuated by etoposide + CR8 ( b ). n = 3/group for all groups. Etoposide and etoposide + CR8 increased expression of miR-711 and decreased miR-23a compared to control neurons ( c ). n = 6/group for all groups. Data represent mean ± SEM of one-way ANOVA and Tukey post hoc analysis, * p

Techniques Used: Activation Assay, Real-time Polymerase Chain Reaction, Expressing

13) Product Images from "Deciphering the Molecular Mechanisms Sustaining the Estrogenic Activity of the Two Major Dietary Compounds Zearalenone and Apigenin in ER-Positive Breast Cancer Cell Lines"

Article Title: Deciphering the Molecular Mechanisms Sustaining the Estrogenic Activity of the Two Major Dietary Compounds Zearalenone and Apigenin in ER-Positive Breast Cancer Cell Lines

Journal: Nutrients

doi: 10.3390/nu11020237

ERα recruitment to chromatin at distinct ER binding sites in response to E2, zearalenone and apigenin. MCF-7 cells were treated with solvent (white) as a negative control, 10 −9 M E2 (blue) as a positive control, 10 −8 M zearalenone (red) or 10 −5 M apigenin (green) for 1 h. The recruitment of ERα to the GREB1 promoter ( A ), CUE domain containing 1 (CUEDC1) enhancer ( B ) and the two enhancers of x-box protein 1 (XBP1) ( C ) was assessed by chromatin immunoprecipitation followed by real-time PCR. For each binding site tested, the DNA sequence is indicated. The results are expressed in fold recruitment compared to control and are the means of four independent experiments. * indicates a p -value
Figure Legend Snippet: ERα recruitment to chromatin at distinct ER binding sites in response to E2, zearalenone and apigenin. MCF-7 cells were treated with solvent (white) as a negative control, 10 −9 M E2 (blue) as a positive control, 10 −8 M zearalenone (red) or 10 −5 M apigenin (green) for 1 h. The recruitment of ERα to the GREB1 promoter ( A ), CUE domain containing 1 (CUEDC1) enhancer ( B ) and the two enhancers of x-box protein 1 (XBP1) ( C ) was assessed by chromatin immunoprecipitation followed by real-time PCR. For each binding site tested, the DNA sequence is indicated. The results are expressed in fold recruitment compared to control and are the means of four independent experiments. * indicates a p -value

Techniques Used: Binding Assay, Negative Control, Positive Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Sequencing

14) Product Images from "Potential roles of microRNA-29a in the molecular pathophysiology of T-cell acute lymphoblastic leukemia"

Article Title: Potential roles of microRNA-29a in the molecular pathophysiology of T-cell acute lymphoblastic leukemia

Journal: Cancer Science

doi: 10.1111/cas.12766

Effect of microRNA-29a (miR-29a) on promoter methylation and expression of selected genes. The T-cell acute lymphoblastic leukemia (T-ALL) cell lines Jurkat and Molt-4 were transfected with miR-29a synthetic mimics (PM29a) and a corresponding control unspecific miR molecule (PMC) and, 48 h later, DNA and RNA was collected and evaluated. (a) Methylation status was evaluated by immunoprecipitation of methylated DNA (meDIP) followed by quantitative PCR (using primers directed to a CpG island region encompassing the transcription start site). For each gene, the enrichment was calculated by initially normalizing the CT values of the immunoprecipitated DNA sample by the CT values of the Input DNA sample, then calculating the enrichment relative to the enrichment observed for a negative control region (a region with very few CpGs from the IG5 gene). Selected genes, included those previously reported by Kuang et al . 24 as methylated in Jurkat and Molt-4 cells (including GFPT2 , GNA14 , SALL1 ), positive control regions from H19ICR , BRDT , and TSH2B genes (methylated in normal somatic cells), and negative control regions from HIST1H3B and UBE2B genes (unmethylated in normal somatic cells) and from the LHX4 gene (reported by Kuang et al . as unmethylated in Jurkat and Molt-4 cells). Results are the mean of two meDIP experiments carried out with a pool of four independently transfected samples. (b) Transcript levels of selected targets were evaluated by real-time quantitative PCR. Statistical significance was evaluated by a two-tailed non-paired t -test ( n = 4). * P
Figure Legend Snippet: Effect of microRNA-29a (miR-29a) on promoter methylation and expression of selected genes. The T-cell acute lymphoblastic leukemia (T-ALL) cell lines Jurkat and Molt-4 were transfected with miR-29a synthetic mimics (PM29a) and a corresponding control unspecific miR molecule (PMC) and, 48 h later, DNA and RNA was collected and evaluated. (a) Methylation status was evaluated by immunoprecipitation of methylated DNA (meDIP) followed by quantitative PCR (using primers directed to a CpG island region encompassing the transcription start site). For each gene, the enrichment was calculated by initially normalizing the CT values of the immunoprecipitated DNA sample by the CT values of the Input DNA sample, then calculating the enrichment relative to the enrichment observed for a negative control region (a region with very few CpGs from the IG5 gene). Selected genes, included those previously reported by Kuang et al . 24 as methylated in Jurkat and Molt-4 cells (including GFPT2 , GNA14 , SALL1 ), positive control regions from H19ICR , BRDT , and TSH2B genes (methylated in normal somatic cells), and negative control regions from HIST1H3B and UBE2B genes (unmethylated in normal somatic cells) and from the LHX4 gene (reported by Kuang et al . as unmethylated in Jurkat and Molt-4 cells). Results are the mean of two meDIP experiments carried out with a pool of four independently transfected samples. (b) Transcript levels of selected targets were evaluated by real-time quantitative PCR. Statistical significance was evaluated by a two-tailed non-paired t -test ( n = 4). * P

Techniques Used: Methylation, Expressing, Transfection, Immunoprecipitation, Methylated DNA Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Two Tailed Test

Methylated DNA immunoprecipitation qPCR analysis
Figure Legend Snippet: Methylated DNA immunoprecipitation qPCR analysis

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

15) Product Images from "A potential diagnostic marker for ovarian cancer: Involvement of the histone acetyltransferase, human males absent on the first"

Article Title: A potential diagnostic marker for ovarian cancer: Involvement of the histone acetyltransferase, human males absent on the first

Journal: Oncology Letters

doi: 10.3892/ol.2013.1380

A reduction in hMOF mRNA levels is observed in human ovarian cancer. (A) PCR analysis of 47 clinical ovarian cancer tissues. Total RNA was isolated from the tissues using TRIzol. The PCR assay was performed to detect the mRNA expression levels of hMOF, CA9, VEGF, HIF1α and hSTC1 in clinical ovarian cancer and normal ovarian tissues. The PCR products were then separated by electrophoresis on a 2% agarose gel. The DNA fragments were visualized and photographed under ultraviolet light with ethidium bromide. The mRNA levels from 37 ovarian cancer tissues were compared with corresponding contralateral ovarian normal tissues. However, 10 clinical ovarian cancer tissues were missing contralateral ovarian normal tissues and were compared with non-corresponding normal ovarian tissues. (B) Summarization of the PCR results. The 100% stacked column charts were used to compare the case numbers of differentially-expressed mRNAs in the ovarian cancer tissues. The total case numbers of differentially-expressed mRNAs (increased, decreased and no change) in the ovarian cancer tissues is equal to 100%. (C) Statistical analysis of quantified mRNA levels between the ovarian cancer and normal tissues. The mRNA expression signals shown in (A) were quantified by densitometry using Quantity One Basic Software. The significant difference is expressed as * P
Figure Legend Snippet: A reduction in hMOF mRNA levels is observed in human ovarian cancer. (A) PCR analysis of 47 clinical ovarian cancer tissues. Total RNA was isolated from the tissues using TRIzol. The PCR assay was performed to detect the mRNA expression levels of hMOF, CA9, VEGF, HIF1α and hSTC1 in clinical ovarian cancer and normal ovarian tissues. The PCR products were then separated by electrophoresis on a 2% agarose gel. The DNA fragments were visualized and photographed under ultraviolet light with ethidium bromide. The mRNA levels from 37 ovarian cancer tissues were compared with corresponding contralateral ovarian normal tissues. However, 10 clinical ovarian cancer tissues were missing contralateral ovarian normal tissues and were compared with non-corresponding normal ovarian tissues. (B) Summarization of the PCR results. The 100% stacked column charts were used to compare the case numbers of differentially-expressed mRNAs in the ovarian cancer tissues. The total case numbers of differentially-expressed mRNAs (increased, decreased and no change) in the ovarian cancer tissues is equal to 100%. (C) Statistical analysis of quantified mRNA levels between the ovarian cancer and normal tissues. The mRNA expression signals shown in (A) were quantified by densitometry using Quantity One Basic Software. The significant difference is expressed as * P

Techniques Used: Polymerase Chain Reaction, Isolation, Expressing, Electrophoresis, Agarose Gel Electrophoresis, Software

16) Product Images from "Crosstalk between NSL Histone Acetyltransferase and MLL/SET Complexes: NSL Complex Functions in Promoting Histone H3K4 Di-Methylation Activity by MLL/SET Complexes"

Article Title: Crosstalk between NSL Histone Acetyltransferase and MLL/SET Complexes: NSL Complex Functions in Promoting Histone H3K4 Di-Methylation Activity by MLL/SET Complexes

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003940

NSL complex is essential for targeting of RbBP5-MLL complex to ANKRD2. A. Five primer sets in the ANKRD2 locus used for amplifying ChIP'd DNA. B. Distribution of hMOF on the ANKRD2 locus. ChIP assays were performed using hMOF antibody. ChIP'd DNA was analyzed by qPCR. Bar graph shows the ratios of ChIP'd DNA signals (normalized to input) to IgG (also normalized to input; right panel). Error bars represent the standard error of the mean of 3 independent experiments. Significant. C. Enrichment of histone H3K4me and H4K16ac on the ANKRD2 promoter region (−0.25 kb). ChIP assays were performed using H3K4me1/me2/me3 or H4K16ac antibodies. ChIP'd DNA was amplified by PCR (top). Quantified PCR signals (Quantity One software) were analyzed by t -test (bottom). Error bars indicate SE and the significant difference is expressed as **p
Figure Legend Snippet: NSL complex is essential for targeting of RbBP5-MLL complex to ANKRD2. A. Five primer sets in the ANKRD2 locus used for amplifying ChIP'd DNA. B. Distribution of hMOF on the ANKRD2 locus. ChIP assays were performed using hMOF antibody. ChIP'd DNA was analyzed by qPCR. Bar graph shows the ratios of ChIP'd DNA signals (normalized to input) to IgG (also normalized to input; right panel). Error bars represent the standard error of the mean of 3 independent experiments. Significant. C. Enrichment of histone H3K4me and H4K16ac on the ANKRD2 promoter region (−0.25 kb). ChIP assays were performed using H3K4me1/me2/me3 or H4K16ac antibodies. ChIP'd DNA was amplified by PCR (top). Quantified PCR signals (Quantity One software) were analyzed by t -test (bottom). Error bars indicate SE and the significant difference is expressed as **p

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

17) Product Images from "T-cell receptor α enhancer is inactivated in αβ T lymphocytes"

Article Title: T-cell receptor α enhancer is inactivated in αβ T lymphocytes

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

doi: 10.1073/pnas.1406551112

E2A is not present at the Eα enhanceosome assembled in αβ T lymphocytes. ( A ) Diagram depicts the location of the Tα1-Tα4 regions as well as the described TF binding sites. ( B and C ) For CTCF q ChIPs, chromatin was prepared from sorted DP and T lymphocytes from RxDO11.10 mice. For all other TF ChIPs, DP thymocyte chromatin was prepared from EαKI Rxßtg thymocytes and T lymphocyte chromatin was prepared from EαKI βtg T lymphocytes. Immunoprecipitation was performed with the indicated control (c) or specific Abs (Ab). DNA purified from the input and Ab-bound fractions was used as a template for qPCR to evaluate the presence of Eα ( B ) and Eαi ( C ). Data represent the mean ± SEM of duplicate qPCR from three independent experiments. Two-tailed Student’s t tests were used to determine the statistical significance between the values of the qChIPs from DP thymocytes vs. T lymphocytes (* P
Figure Legend Snippet: E2A is not present at the Eα enhanceosome assembled in αβ T lymphocytes. ( A ) Diagram depicts the location of the Tα1-Tα4 regions as well as the described TF binding sites. ( B and C ) For CTCF q ChIPs, chromatin was prepared from sorted DP and T lymphocytes from RxDO11.10 mice. For all other TF ChIPs, DP thymocyte chromatin was prepared from EαKI Rxßtg thymocytes and T lymphocyte chromatin was prepared from EαKI βtg T lymphocytes. Immunoprecipitation was performed with the indicated control (c) or specific Abs (Ab). DNA purified from the input and Ab-bound fractions was used as a template for qPCR to evaluate the presence of Eα ( B ) and Eαi ( C ). Data represent the mean ± SEM of duplicate qPCR from three independent experiments. Two-tailed Student’s t tests were used to determine the statistical significance between the values of the qChIPs from DP thymocytes vs. T lymphocytes (* P

Techniques Used: Binding Assay, Mouse Assay, Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction, Two Tailed Test

18) Product Images from "Zinc knuckle of TAF1 is a DNA binding module critical for TFIID promoter occupancy"

Article Title: Zinc knuckle of TAF1 is a DNA binding module critical for TFIID promoter occupancy

Journal: Scientific Reports

doi: 10.1038/s41598-018-22879-5

TAF1 zinc knuckle binds DNA. ( A,B,C ) EMSA of TAF1 ZnA (aa 1234–1375) and three radiolabeled DNA fragments: IMD of super core promoter (position −6 to +38), cyclin D1 promoter (position −22 to +29, CD1P), and Random DNA sequence. ( D,E,F ) Bio-layer interferometry binding curves using biotinylated double-stranded DNA fragments described in above and the following ZnA protein concentrations: 3 μM, 1 μM, 333 nM, 111 nM, 37 nM, 12 nM. Raw data was plotted with GraphPAD Prism. K d was calculated from plotting steady-state binding levels against protein concentration.
Figure Legend Snippet: TAF1 zinc knuckle binds DNA. ( A,B,C ) EMSA of TAF1 ZnA (aa 1234–1375) and three radiolabeled DNA fragments: IMD of super core promoter (position −6 to +38), cyclin D1 promoter (position −22 to +29, CD1P), and Random DNA sequence. ( D,E,F ) Bio-layer interferometry binding curves using biotinylated double-stranded DNA fragments described in above and the following ZnA protein concentrations: 3 μM, 1 μM, 333 nM, 111 nM, 37 nM, 12 nM. Raw data was plotted with GraphPAD Prism. K d was calculated from plotting steady-state binding levels against protein concentration.

Techniques Used: Radial Immuno Diffusion, Sequencing, Binding Assay, Protein Concentration

Core Module and Key Residues in TAF1 Zinc Knuckle DNA Binding Domain. ( A . ( B ) EVfold map analysis of ZnA. Numbers represent amino acid residues of full-length TAF1. ( C ) Diagram of DNA stabilized regions of TAF1 with blue box indicating CCHC ZnK. DNA binding curves for ( D ) ZnC wild type, ( E ) ZnC cysteine mutant (C1285A and C1288A), ( F ) ZnC charge mutant (R1295A and K1298A), ( G ) ZnD wild type, ( H ) ZnD cysteine mutant, and ( I ) ZnD charge mutant. Raw data was plotted with GraphPAD Prism. K d was calculated by plotting steady-state binding levels against protein concentration and determining the concentration needed for half maximal binding.
Figure Legend Snippet: Core Module and Key Residues in TAF1 Zinc Knuckle DNA Binding Domain. ( A . ( B ) EVfold map analysis of ZnA. Numbers represent amino acid residues of full-length TAF1. ( C ) Diagram of DNA stabilized regions of TAF1 with blue box indicating CCHC ZnK. DNA binding curves for ( D ) ZnC wild type, ( E ) ZnC cysteine mutant (C1285A and C1288A), ( F ) ZnC charge mutant (R1295A and K1298A), ( G ) ZnD wild type, ( H ) ZnD cysteine mutant, and ( I ) ZnD charge mutant. Raw data was plotted with GraphPAD Prism. K d was calculated by plotting steady-state binding levels against protein concentration and determining the concentration needed for half maximal binding.

Techniques Used: Binding Assay, Mutagenesis, Protein Concentration, Concentration Assay

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Article Snippet: .. Immunoprecipitated DNA and input DNA were analyzed by qPCR with SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad). ..

Article Title: Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette
Article Snippet: .. The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real time PCR detection system (Bio-Rad) and iQ SYBR Green Supermix (Bio-Rad). ..

Article Title: Re-engineering an alphoidtetO-HAC-based vector to enable high-throughput analyses of gene function
Article Snippet: .. The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real-time PCR detection system (Bio-Rad, USA) and iQ SYBR Green Supermix (Bio-Rad, USA). ..

Amplification:

Article Title: Zinc knuckle of TAF1 is a DNA binding module critical for TFIID promoter occupancy
Article Snippet: .. Fifty nanograms of input DNA and 2 μL of purified TAF1 bound DNA was amplified with SsoFast EvaGreen Supermix (Bio-Rad) using primers spanning the promoters of cyclin D1 and cyclin A2 (Supplemental Table ). .. Quantitative PCR was performed on the Applied Biosystems 7500Fast Real-Time PCR system and the data expressed as percent input.

Quantitative RT-PCR:

Article Title: Activation of the AMPK-FOXO3 Pathway Reduces Fatty Acid-Induced Increase in Intracellular Reactive Oxygen Species by Upregulating Thioredoxin
Article Snippet: .. The immunoprecipitated DNA and the input DNA were quantified with the qRT-PCR detection system (Bio-Rad). ..

Purification:

Article Title: Zinc knuckle of TAF1 is a DNA binding module critical for TFIID promoter occupancy
Article Snippet: .. Fifty nanograms of input DNA and 2 μL of purified TAF1 bound DNA was amplified with SsoFast EvaGreen Supermix (Bio-Rad) using primers spanning the promoters of cyclin D1 and cyclin A2 (Supplemental Table ). .. Quantitative PCR was performed on the Applied Biosystems 7500Fast Real-Time PCR system and the data expressed as percent input.

SYBR Green Assay:

Article Title: Comparing effects of CDK inhibition and E2F1/2 ablation on neuronal cell death pathways in vitro and after traumatic brain injury
Article Snippet: .. Immunoprecipitated DNA and input DNA were analyzed by qPCR with SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad). ..

Article Title: Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette
Article Snippet: .. The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real time PCR detection system (Bio-Rad) and iQ SYBR Green Supermix (Bio-Rad). ..

Article Title: Re-engineering an alphoidtetO-HAC-based vector to enable high-throughput analyses of gene function
Article Snippet: .. The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real-time PCR detection system (Bio-Rad, USA) and iQ SYBR Green Supermix (Bio-Rad, USA). ..

Immunoprecipitation:

Article Title: Comparing effects of CDK inhibition and E2F1/2 ablation on neuronal cell death pathways in vitro and after traumatic brain injury
Article Snippet: .. Immunoprecipitated DNA and input DNA were analyzed by qPCR with SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad). ..

Article Title: Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette
Article Snippet: .. The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real time PCR detection system (Bio-Rad) and iQ SYBR Green Supermix (Bio-Rad). ..

Article Title: Re-engineering an alphoidtetO-HAC-based vector to enable high-throughput analyses of gene function
Article Snippet: .. The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real-time PCR detection system (Bio-Rad, USA) and iQ SYBR Green Supermix (Bio-Rad, USA). ..

Article Title: Activation of the AMPK-FOXO3 Pathway Reduces Fatty Acid-Induced Increase in Intracellular Reactive Oxygen Species by Upregulating Thioredoxin
Article Snippet: .. The immunoprecipitated DNA and the input DNA were quantified with the qRT-PCR detection system (Bio-Rad). ..

Inhibition:

Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays
Article Snippet: .. In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s). .. These assays offer potential valuable tools for evaluating the treatment strategies aimed at reducing the latent reservoir and curing viral infection.

Software:

Article Title: The Transcription Factor Rbf1 Is the Master Regulator for b-Mating Type Controlled Pathogenic Development in Ustilago maydis
Article Snippet: .. Amplicons were normalized to input DNA using the Bio-Rad IQ5 software. .. DNA array and data analysis Custom-designed Affymetrix chips were used for DNA-array analysis.

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    Bio-Rad input dna
    Physical analysis of the alphoid tetO -HAC used for loading of tTS -containing cassettes. ( a ) Analysis of integrity of the alphoid tetO -HAC synthetic array after its transfer into HPRT-deficient HT1080 cells. Genomic <t>DNA</t> from the cells with the HAC was digested with SpeI endonuclease, separated by CHEF gel electrophoresis (range 10–70 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. Lane 1: the HAC with the loxP site in hamster CHO cells; lanes 2, 3, 4 and 5: four HAC clones with the loxP site in HPRT-deficient HT1080 cells; lane 6: the original HAC (clone AB2.218.21) generated in human cells ( 29 ). M1- Pulse Marker™ 0.1–200 kb (Sigma-Aldrich). ( b ) Mapping of the loxP site in a mega-base size alphoid DNA array. Genomic DNA from the cells possessing the original HAC was digested with PmeI endonuclease, separated by CHEF gel electrophoresis (range 200–1500 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. A single 1.1-Mb fragment was detected for the original HAC (left, lane 1). Two bands of 650 and 500 kb were detected for genomic DNA from cells with the HAC bearing an inserted NBS1 gene (right, lanes 1 and 2). The size of the fragments was determined by comparison with the DNA size standard , Saccharomyces cerevisiae chromosomes. Lane M2: Yeast Chromosome PFG Marker BioLabs. ( c ) <t>PCR</t> analysis of clones with insertion of X3.1-I-EGFP-I into the HAC confirming restoration of the full-length HPRT gene.
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    Physical analysis of the alphoid tetO -HAC used for loading of tTS -containing cassettes. ( a ) Analysis of integrity of the alphoid tetO -HAC synthetic array after its transfer into HPRT-deficient HT1080 cells. Genomic DNA from the cells with the HAC was digested with SpeI endonuclease, separated by CHEF gel electrophoresis (range 10–70 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. Lane 1: the HAC with the loxP site in hamster CHO cells; lanes 2, 3, 4 and 5: four HAC clones with the loxP site in HPRT-deficient HT1080 cells; lane 6: the original HAC (clone AB2.218.21) generated in human cells ( 29 ). M1- Pulse Marker™ 0.1–200 kb (Sigma-Aldrich). ( b ) Mapping of the loxP site in a mega-base size alphoid DNA array. Genomic DNA from the cells possessing the original HAC was digested with PmeI endonuclease, separated by CHEF gel electrophoresis (range 200–1500 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. A single 1.1-Mb fragment was detected for the original HAC (left, lane 1). Two bands of 650 and 500 kb were detected for genomic DNA from cells with the HAC bearing an inserted NBS1 gene (right, lanes 1 and 2). The size of the fragments was determined by comparison with the DNA size standard , Saccharomyces cerevisiae chromosomes. Lane M2: Yeast Chromosome PFG Marker BioLabs. ( c ) PCR analysis of clones with insertion of X3.1-I-EGFP-I into the HAC confirming restoration of the full-length HPRT gene.

    Journal: Nucleic Acids Research

    Article Title: Re-engineering an alphoidtetO-HAC-based vector to enable high-throughput analyses of gene function

    doi: 10.1093/nar/gkt205

    Figure Lengend Snippet: Physical analysis of the alphoid tetO -HAC used for loading of tTS -containing cassettes. ( a ) Analysis of integrity of the alphoid tetO -HAC synthetic array after its transfer into HPRT-deficient HT1080 cells. Genomic DNA from the cells with the HAC was digested with SpeI endonuclease, separated by CHEF gel electrophoresis (range 10–70 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. Lane 1: the HAC with the loxP site in hamster CHO cells; lanes 2, 3, 4 and 5: four HAC clones with the loxP site in HPRT-deficient HT1080 cells; lane 6: the original HAC (clone AB2.218.21) generated in human cells ( 29 ). M1- Pulse Marker™ 0.1–200 kb (Sigma-Aldrich). ( b ) Mapping of the loxP site in a mega-base size alphoid DNA array. Genomic DNA from the cells possessing the original HAC was digested with PmeI endonuclease, separated by CHEF gel electrophoresis (range 200–1500 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. A single 1.1-Mb fragment was detected for the original HAC (left, lane 1). Two bands of 650 and 500 kb were detected for genomic DNA from cells with the HAC bearing an inserted NBS1 gene (right, lanes 1 and 2). The size of the fragments was determined by comparison with the DNA size standard , Saccharomyces cerevisiae chromosomes. Lane M2: Yeast Chromosome PFG Marker BioLabs. ( c ) PCR analysis of clones with insertion of X3.1-I-EGFP-I into the HAC confirming restoration of the full-length HPRT gene.

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real-time PCR detection system (Bio-Rad, USA) and iQ SYBR Green Supermix (Bio-Rad, USA).

    Techniques: HAC Assay, Nucleic Acid Electrophoresis, Clone Assay, Generated, Marker, DNA Array, Polymerase Chain Reaction

    Doxycycline regulated expression of the tTS-EYFP transgene loaded into the HAC. ( a ) A diagram illustrating time-course analysis of tTS-EYFP expression in the cells grown in different media. Samples 1–11 correspond to cultures used for FACS analysis. The cultures marked by red stars were analyzed by ChIP (see below). ( b ) Relative mean EYFP fluorescence determined by FACS of cells carrying the tTS-EYFP cassette (construct #4) grown in different media. Sample 1 corresponds to control HT1080 cells without the EYFP transgene (fluorescence background). Sample 2 corresponds to the cells with the HAC carrying the tTS-EYFP cassette grown in HAT and dox + medium for 30 days. Sample 3 corresponds to the cells grown for 22 days in dox + medium without selection. Sample 4 corresponds to the cells grown for 22 days without doxycycline. Sample 5 corresponds to the cells grown for a month in dox − medium with BS selection. Sample 6 corresponds to the cells grown in bsr − dox − medium. Samples 7, 8 and 9 correspond to the cells grown in dox + BS + medium for 24 h, 6 and 12 days, correspondingly. Samples 10 and 11 correspond to the cells treated either TSA or SAHA in dox + medium. Error bars, SD ( n = 3). ( c ) Transcription of Hygro , TK, EGFP and HPRT genes from the HAC. The level of the transgene transcripts from cells cultured in dox + or dox − medium was analyzed by RT–PCR. The housekeeping gene, BRCA1 , was used as an internal control. Lane 1 (dox − ) and lane 2 (dox + ) correspond to transcripts for BRCA1 . Lane 3 (dox − ) and lane 4 (dox + ) correspond to transcripts for Hygro . Lane 5 (dox − ) and lane 7 (dox + ) correspond to transcripts for TK . Lane 6 (dox − ) and lane 8 (dox + ) correspond to transcripts for HPRT . Lane 9 (dox − ) and lane 10 (dox + ) correspond to transcripts for EGFP . Lane M- GeneRuler™ 1-kb DNA ladder. ( d ) ChIP analysis of H3K4me3 chromatin in the transgene cassette of the HAC in the presence and absence of doxycycline. The cell samples used for ChIP correspond to samples 2 and 6 in Figure 6 a and b. Enrichment is shown relative to the 5S rRNA control locus. Satellite 2 sequence, Sat2, corresponding endogenous pericentromeric repeats was included as a negative control. ( e ) ChIP analysis of CENP-A chromatin in the HAC in the presence and absence of doxycycline. Enrichment is shown relative to the chromosome 21 centromere.

    Journal: Nucleic Acids Research

    Article Title: Re-engineering an alphoidtetO-HAC-based vector to enable high-throughput analyses of gene function

    doi: 10.1093/nar/gkt205

    Figure Lengend Snippet: Doxycycline regulated expression of the tTS-EYFP transgene loaded into the HAC. ( a ) A diagram illustrating time-course analysis of tTS-EYFP expression in the cells grown in different media. Samples 1–11 correspond to cultures used for FACS analysis. The cultures marked by red stars were analyzed by ChIP (see below). ( b ) Relative mean EYFP fluorescence determined by FACS of cells carrying the tTS-EYFP cassette (construct #4) grown in different media. Sample 1 corresponds to control HT1080 cells without the EYFP transgene (fluorescence background). Sample 2 corresponds to the cells with the HAC carrying the tTS-EYFP cassette grown in HAT and dox + medium for 30 days. Sample 3 corresponds to the cells grown for 22 days in dox + medium without selection. Sample 4 corresponds to the cells grown for 22 days without doxycycline. Sample 5 corresponds to the cells grown for a month in dox − medium with BS selection. Sample 6 corresponds to the cells grown in bsr − dox − medium. Samples 7, 8 and 9 correspond to the cells grown in dox + BS + medium for 24 h, 6 and 12 days, correspondingly. Samples 10 and 11 correspond to the cells treated either TSA or SAHA in dox + medium. Error bars, SD ( n = 3). ( c ) Transcription of Hygro , TK, EGFP and HPRT genes from the HAC. The level of the transgene transcripts from cells cultured in dox + or dox − medium was analyzed by RT–PCR. The housekeeping gene, BRCA1 , was used as an internal control. Lane 1 (dox − ) and lane 2 (dox + ) correspond to transcripts for BRCA1 . Lane 3 (dox − ) and lane 4 (dox + ) correspond to transcripts for Hygro . Lane 5 (dox − ) and lane 7 (dox + ) correspond to transcripts for TK . Lane 6 (dox − ) and lane 8 (dox + ) correspond to transcripts for HPRT . Lane 9 (dox − ) and lane 10 (dox + ) correspond to transcripts for EGFP . Lane M- GeneRuler™ 1-kb DNA ladder. ( d ) ChIP analysis of H3K4me3 chromatin in the transgene cassette of the HAC in the presence and absence of doxycycline. The cell samples used for ChIP correspond to samples 2 and 6 in Figure 6 a and b. Enrichment is shown relative to the 5S rRNA control locus. Satellite 2 sequence, Sat2, corresponding endogenous pericentromeric repeats was included as a negative control. ( e ) ChIP analysis of CENP-A chromatin in the HAC in the presence and absence of doxycycline. Enrichment is shown relative to the chromosome 21 centromere.

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real-time PCR detection system (Bio-Rad, USA) and iQ SYBR Green Supermix (Bio-Rad, USA).

    Techniques: Expressing, HAC Assay, FACS, Chromatin Immunoprecipitation, Fluorescence, Construct, HAT Assay, Selection, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Sequencing, Negative Control

    AMPK promoted FOXO3 binding to the Trx promoter and formation of the FOXO3/p300 transcription complex in the Trx promoter. A : Depiction of FOXO binding sites in the Trx promoter. B : AICAR increased binding of FOXO3 to the Trx promoter. HAECs were treated with AICAR and palmitic acid (PA) for 24 h. FOXO3-DNA complexes were cross-linked by formaldehyde and immunoprecipitated with anti-FOXO3 antibody. Bound FOXO3 sites in the Trx promoter were detected by qPCR and normalized with input DNA. Relative DNA was compared and expressed as the percentage of the nontreatment control subjects. Representative blots and quantitative analysis from three independent experiments are shown. Data represent the means ± SE. * P

    Journal: Diabetes

    Article Title: Activation of the AMPK-FOXO3 Pathway Reduces Fatty Acid-Induced Increase in Intracellular Reactive Oxygen Species by Upregulating Thioredoxin

    doi: 10.2337/db08-1512

    Figure Lengend Snippet: AMPK promoted FOXO3 binding to the Trx promoter and formation of the FOXO3/p300 transcription complex in the Trx promoter. A : Depiction of FOXO binding sites in the Trx promoter. B : AICAR increased binding of FOXO3 to the Trx promoter. HAECs were treated with AICAR and palmitic acid (PA) for 24 h. FOXO3-DNA complexes were cross-linked by formaldehyde and immunoprecipitated with anti-FOXO3 antibody. Bound FOXO3 sites in the Trx promoter were detected by qPCR and normalized with input DNA. Relative DNA was compared and expressed as the percentage of the nontreatment control subjects. Representative blots and quantitative analysis from three independent experiments are shown. Data represent the means ± SE. * P

    Article Snippet: The immunoprecipitated DNA and the input DNA were quantified with the qRT-PCR detection system (Bio-Rad).

    Techniques: Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

    ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.

    Article Snippet: In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

    Techniques: Real-time Polymerase Chain Reaction, Migration, Concentration Assay

    Comparison between ddPCR and qPCR quantitation results in low viral DNA range

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: Comparison between ddPCR and qPCR quantitation results in low viral DNA range

    Article Snippet: In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

    Techniques: Real-time Polymerase Chain Reaction, Quantitation Assay

    SIV ddPCR DNA assay characterization. (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance. DNA sample used was duodenum DNA from Rhesus macaque 313–08. (B) Total number of droplets generated during dropletization remains constant in the range of tissue DNA amount tested (1 million to 8 million cell equivalent input). Total droplet number for each sample after dropletization serves as an additional indicator of sample DNA input tolerance. Note that for each level of DNA input, only a fraction (~1.6%) of the droplets were counted for QC purpose by the Source machine. (C) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines [ 7 ]. According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (D) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument. (E) Instead of measuring 60 ddPCR replicates to obtain 95% confidence, we obtained an approximate estimation of the LoD using a lower number of ddPCR reaction replicates and required the false negative rate to be below 5% (i.e. all 10 replicates have to be positive).

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: SIV ddPCR DNA assay characterization. (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance. DNA sample used was duodenum DNA from Rhesus macaque 313–08. (B) Total number of droplets generated during dropletization remains constant in the range of tissue DNA amount tested (1 million to 8 million cell equivalent input). Total droplet number for each sample after dropletization serves as an additional indicator of sample DNA input tolerance. Note that for each level of DNA input, only a fraction (~1.6%) of the droplets were counted for QC purpose by the Source machine. (C) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines [ 7 ]. According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (D) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument. (E) Instead of measuring 60 ddPCR replicates to obtain 95% confidence, we obtained an approximate estimation of the LoD using a lower number of ddPCR reaction replicates and required the false negative rate to be below 5% (i.e. all 10 replicates have to be positive).

    Article Snippet: In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

    Techniques: Generated, Real-time Polymerase Chain Reaction, Software

    ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.

    Article Snippet: In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

    Techniques: Real-time Polymerase Chain Reaction, Inhibition, Derivative Assay, Negative Control, Standard Deviation

    Identifying an optimal ddPCR assay format and condition. Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV (on CCR5 background) spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311–08) infected with SIVmac239. (C) A summary of all other tested conditions (probe and mastermix) and the observed issue(s) associated with each condition.

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: Identifying an optimal ddPCR assay format and condition. Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV (on CCR5 background) spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311–08) infected with SIVmac239. (C) A summary of all other tested conditions (probe and mastermix) and the observed issue(s) associated with each condition.

    Article Snippet: In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

    Techniques: Infection

    SIV ddPCR DNA assay performance. (A) SIV ddPCR DNA assay detection of low single digit level SIV DNA input and linear dynamic range of the SIV ddPCR DNA assay. Different amount of SIV DNA template was spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. SIV DNA amount was quantified by direct ddPCR. (B) Quantification data of Fig 5A.

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: SIV ddPCR DNA assay performance. (A) SIV ddPCR DNA assay detection of low single digit level SIV DNA input and linear dynamic range of the SIV ddPCR DNA assay. Different amount of SIV DNA template was spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. SIV DNA amount was quantified by direct ddPCR. (B) Quantification data of Fig 5A.

    Article Snippet: In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

    Techniques:

    Structure and expression of Rbf1. ( A ) Microarray analysis of b -dependent rbf1 expression after induction of compatible (AB31 and AB33) and incompatible (AB32 and AB34) combinations of bE and bW . Shown are the mean expression values of two biological replicates and the standard deviation (SD) ( B ) qRT-PCR analysis of rbf1 expression after induction of compatible (AB31) and incompatible (AB32) combinations of bE and bW . Samples were taken at the time-points indicated. qRT-PCR analysis was performed using the constitutively expressed ppi gene ( um03726 ) for normalization. Expression was calculated relative to the lowest expression value. Shown are mean values of two technical replicates. ( C ) Overview of primer binding sites in the rbf1 -promoter used for qChIP experiments and alignment of three putative b-binding sites ( bbs ) in the rbf1 promoter region to the bbs of lga2 and frb52 [10] , [11] . Nucleotide positions indicated are relative to the start codon. Nucleotides identical to the bbs in lga2 [10] and frb52 [11] are in bold. ( D ) qChIP analysis of bE1 binding to the rbf1 -promoter in strains AB31 and AB31bE1:3xHA 5h after induction of the bE1/bW2-heterodimer. AB31bE1:3xHA harbours a HA-tagged bE1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody. No significant enrichment was observed in control strain AB31. In AB31bE1:3xHA, the PCR-amplicon spanning bbs1 (bbs −1377 ) is significantly enriched (t-test) when compared to the amplicon spanning a control region (−2026) (p = 5.71 10 −5 ). As additional control, a region from the eIF2b gene ( um04869 ) was used. Given are the mean values of three technical replicates of three independent experiments each, and the standard deviation (SD). ( E ) Structure of the Rbf1 protein. The potential C2H2 zinc finger domain (aa 18 to 131) and a putative NLS (RHRR) (aa 95 to 98) within this domain are marked in dark grey and black, a glutamine rich sequence (aa 365 to 373) is marked in grey. The alignment shows the four C2H2 zinc finger domains; the conserved cysteine and histidine residues are in bold. ( F ) Subcellular localization of the Rbf1-3xeGFP fusion protein. Strain AB31 rbf1:3eGFP (UMS63) was induced in CM medium supplemented with 1% arabinose (CMA) for eight hours. The functional Rbf1-3xeGFP fusion protein localizes to the nucleus. Cells were stained with DAPI to visualize nuclei. Scale bar corresponds to 10 µm.

    Journal: PLoS Pathogens

    Article Title: The Transcription Factor Rbf1 Is the Master Regulator for b-Mating Type Controlled Pathogenic Development in Ustilago maydis

    doi: 10.1371/journal.ppat.1001035

    Figure Lengend Snippet: Structure and expression of Rbf1. ( A ) Microarray analysis of b -dependent rbf1 expression after induction of compatible (AB31 and AB33) and incompatible (AB32 and AB34) combinations of bE and bW . Shown are the mean expression values of two biological replicates and the standard deviation (SD) ( B ) qRT-PCR analysis of rbf1 expression after induction of compatible (AB31) and incompatible (AB32) combinations of bE and bW . Samples were taken at the time-points indicated. qRT-PCR analysis was performed using the constitutively expressed ppi gene ( um03726 ) for normalization. Expression was calculated relative to the lowest expression value. Shown are mean values of two technical replicates. ( C ) Overview of primer binding sites in the rbf1 -promoter used for qChIP experiments and alignment of three putative b-binding sites ( bbs ) in the rbf1 promoter region to the bbs of lga2 and frb52 [10] , [11] . Nucleotide positions indicated are relative to the start codon. Nucleotides identical to the bbs in lga2 [10] and frb52 [11] are in bold. ( D ) qChIP analysis of bE1 binding to the rbf1 -promoter in strains AB31 and AB31bE1:3xHA 5h after induction of the bE1/bW2-heterodimer. AB31bE1:3xHA harbours a HA-tagged bE1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody. No significant enrichment was observed in control strain AB31. In AB31bE1:3xHA, the PCR-amplicon spanning bbs1 (bbs −1377 ) is significantly enriched (t-test) when compared to the amplicon spanning a control region (−2026) (p = 5.71 10 −5 ). As additional control, a region from the eIF2b gene ( um04869 ) was used. Given are the mean values of three technical replicates of three independent experiments each, and the standard deviation (SD). ( E ) Structure of the Rbf1 protein. The potential C2H2 zinc finger domain (aa 18 to 131) and a putative NLS (RHRR) (aa 95 to 98) within this domain are marked in dark grey and black, a glutamine rich sequence (aa 365 to 373) is marked in grey. The alignment shows the four C2H2 zinc finger domains; the conserved cysteine and histidine residues are in bold. ( F ) Subcellular localization of the Rbf1-3xeGFP fusion protein. Strain AB31 rbf1:3eGFP (UMS63) was induced in CM medium supplemented with 1% arabinose (CMA) for eight hours. The functional Rbf1-3xeGFP fusion protein localizes to the nucleus. Cells were stained with DAPI to visualize nuclei. Scale bar corresponds to 10 µm.

    Article Snippet: Amplicons were normalized to input DNA using the Bio-Rad IQ5 software.

    Techniques: Expressing, Microarray, Standard Deviation, Quantitative RT-PCR, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction, Amplification, T-Test, Sequencing, Functional Assay, Staining

    Rbf1 binds to the promoter of the Rbf1-dependently expressed dik6 gene. (A) Overview of the dik6 promoter region investigated by qChIP in strain AB31 rbf1:3xHA . Shown are the positions of the amplicons used for qChIP (numbered from 1 to 10) and the promoter truncations and internal deletions assayed in the GFP-reporter assay. (B) qChIP analysis of Rbf1-binding to the dik6 -promoter in strains AB31 and AB31 rbf1:3xHA 5h after induction of the bE1/bW2-heterodimer in CMA. AB31 rbf1:3xHA expresses an HA-tagged Rbf1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody Start and end of the amplicons are given in nucleotides (nt) relative to the start codon of dik6 . The relative positions of the amplicons are given in (A). As additional control for qChIP, a region from the eIF2b gene ( um04869 ) was used. Given are the mean values of two technical replicates of three independent experiments each, and the standard deviation (SD). Significance of the difference to values obtained for the control-amplicon located within the dik6 open reading frame (amplicon 11) was calculated using Students t-test; the respective p-values are given for AB31 rbf1:3xHA . Highest enrichment was observed for amplicons 3, 4, 5 and 6. No significant enrichment was observed in control strain AB31. (C) The dik6 promoter fragments outlined in (A) were fused to GFP as a reporter and integrated in single copy into the ip -locus of U. maydis strain CP27 ( a2 Δb::P crg1 :rbf1 ). GFP-expression was visualized microscopically 5 hours after induction of rbf1 expression in CMA medium. GFP expression declined when the promoter was truncated from 816 bp to 638 bp, and was abolished when a 298 bp promoter fragment was used. Similarly, the internal deletion Δ3 led to reduced GFP expression, while no GFP signal was detectable in the Δ 5 deletion. Scale bar = 20 µm.

    Journal: PLoS Pathogens

    Article Title: The Transcription Factor Rbf1 Is the Master Regulator for b-Mating Type Controlled Pathogenic Development in Ustilago maydis

    doi: 10.1371/journal.ppat.1001035

    Figure Lengend Snippet: Rbf1 binds to the promoter of the Rbf1-dependently expressed dik6 gene. (A) Overview of the dik6 promoter region investigated by qChIP in strain AB31 rbf1:3xHA . Shown are the positions of the amplicons used for qChIP (numbered from 1 to 10) and the promoter truncations and internal deletions assayed in the GFP-reporter assay. (B) qChIP analysis of Rbf1-binding to the dik6 -promoter in strains AB31 and AB31 rbf1:3xHA 5h after induction of the bE1/bW2-heterodimer in CMA. AB31 rbf1:3xHA expresses an HA-tagged Rbf1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody Start and end of the amplicons are given in nucleotides (nt) relative to the start codon of dik6 . The relative positions of the amplicons are given in (A). As additional control for qChIP, a region from the eIF2b gene ( um04869 ) was used. Given are the mean values of two technical replicates of three independent experiments each, and the standard deviation (SD). Significance of the difference to values obtained for the control-amplicon located within the dik6 open reading frame (amplicon 11) was calculated using Students t-test; the respective p-values are given for AB31 rbf1:3xHA . Highest enrichment was observed for amplicons 3, 4, 5 and 6. No significant enrichment was observed in control strain AB31. (C) The dik6 promoter fragments outlined in (A) were fused to GFP as a reporter and integrated in single copy into the ip -locus of U. maydis strain CP27 ( a2 Δb::P crg1 :rbf1 ). GFP-expression was visualized microscopically 5 hours after induction of rbf1 expression in CMA medium. GFP expression declined when the promoter was truncated from 816 bp to 638 bp, and was abolished when a 298 bp promoter fragment was used. Similarly, the internal deletion Δ3 led to reduced GFP expression, while no GFP signal was detectable in the Δ 5 deletion. Scale bar = 20 µm.

    Article Snippet: Amplicons were normalized to input DNA using the Bio-Rad IQ5 software.

    Techniques: Reporter Assay, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction, Standard Deviation, Amplification, Expressing